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WO1991005605A1 - Marqueurs en cyclodextrine pour essais immunitaires et analyses biochimiques - Google Patents

Marqueurs en cyclodextrine pour essais immunitaires et analyses biochimiques Download PDF

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Publication number
WO1991005605A1
WO1991005605A1 PCT/US1990/005759 US9005759W WO9105605A1 WO 1991005605 A1 WO1991005605 A1 WO 1991005605A1 US 9005759 W US9005759 W US 9005759W WO 9105605 A1 WO9105605 A1 WO 9105605A1
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WO
WIPO (PCT)
Prior art keywords
label
analyte
labels
cyclodextrin
tracer
Prior art date
Application number
PCT/US1990/005759
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English (en)
Inventor
Kenneth M. Kosak
Original Assignee
Kosak Kenneth M
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Publication date
Application filed by Kosak Kenneth M filed Critical Kosak Kenneth M
Publication of WO1991005605A1 publication Critical patent/WO1991005605A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • This invention relates to compositions and ernods for measuring organic and biochemical substances using nonradioactive, cyclodext ⁇ n labelec tracers in a " ligand Dinding reaction, or im unoassay.
  • This invention provides cyclodextrin label compositions that provi ⁇ e a coupling group on the cyclo ⁇ dextrin molecule, and include a fluorescent, chemiluminescent or catalytic substance. Said labels are used for labeling various substances to provided tracers for use in immunoassays and ether ligand binding assays.
  • cyclodextrin complexes provide potentially higher solubility and sig ⁇ nal efficiency in aqueous solutions; o. cyclodextrin complexes provi ⁇ e potentially longer shelf life;
  • cyclodextrin labels provide convenient coupling groups for one-step coupling to various substances without derivatizmg them;
  • luminescent labels detected in a "black” background avoid prcolems of fluorescence and allow for more sensitivity; — —
  • cyclodextrin assay methods are readi y automated for high volume immunoassays and chromatography.
  • this invention is intended for use in assays employing photomultipliers or cnarge-coupled device (CCDj cameras, with computerized data collection and reduction. It is also suitable for photo ⁇ iode ⁇ etection (eg. Aizawa, M., et al , Anal. Lett. 17(B7j, 555-564. 1984), or photographic detection and recording. Still another suitable use of this invention is in continuous flow and/or solid phase ⁇ etection systems, (eg. Van Zoonen, P., et a , Anal. Chim. Acta.
  • an object of tnis invention is to provide reagents, containers and other components in the form of a mer ⁇ cantile kit, to carry out the methods of this invention.
  • this invention can oe use ⁇ with a variety of test formats and ⁇ etection systems.
  • luminometers such as portable or automated, " tube luminometers” ana otner automated instruments that read 96 well, microtiter plates can be used.
  • this invention provides advantages of speed, sensitivity, simplicity and unexpected versatility previously unknown or suggested in the art of immunoassay.
  • Ligand A ligand is defined as a selectively bindable material, that has a selective (or specific), affinity for another substance. Said ligand s bound by a usually, but not necessarily, larger specific binding body or 'partner", or “receptor”, in a ligand binding reaction or assay. Such lig ⁇ and binding reactions are well known and include several cifferent tyoes, which serve to differentiate between various ligand measuring methods in the art.
  • Examples of different selective affinity bincing reactions are oetween antigen ana antibody, biotins ana avidins, lectins ana glycoproteins, recep ⁇ tors and normones, substrates or cofactors and enzymes, and oetween restric ⁇ tion enzymes and nucleic aci ⁇ s, among others.
  • Ligands are also capaole of oeing bound to non-bio logical types of binding substances such as chelators, cavitands, resins and surfactants.
  • a ligand When applied to the immunoassays of this invention, a ligand is limited to an antigen, or hapten tnat is capable of being bound Dy, or tc, its cor ⁇ responding antioody or fraction thereof. Under certain conditions, tms invention is also applicable to other ligand binding reactions, especially between certain biological substances and their specific receptors. In tne case of an enzyme, a ligand would be the substrate or tne coenzy e.
  • Other substances that are capable of being bound as ligands by organic or biolog ⁇ ical substances are proteins, oncoprotems, histones, enzymes, enzyme frac ⁇ tions and derivatives, hormones, vitamins, steroids, prostaglandins, poly ⁇ peptides.
  • Ligands also include various substances witn selective affinity for ligators that are produced through recombinant DNA, genetic and molecular engineering. Except when stated otherwise, lig ⁇ ands of this invention also include ⁇ e the Vigands as defined by .E. Rubenstem, et al , U.S. Pat. No. 3,817,837 (1974).
  • a ligator is defined as a sD ⁇ cific binding body or "partner cr "receptor”, that is usually, but not necessarily, larger than the ligand it can bind to. For the purposes of this invention, it is a specific substance or material or chemical or ' reactant" that is capable of selective affinity binding with a specific ligand in a ligand binding reaction.
  • a ligator can be a protein such as an antibody, a nonprotein binding body or a "specific reactor. '
  • a ligator When applied to the immunoassays of this invention, a ligator is limited to an antibody, which is defined to include all classes of antibodies, mono ⁇ clonal antibodies, chimer z antibodies and fractions, fragments and derivat ⁇ ives tnereof. Under certain conditions, this invention is also aoplicable to using other substances as ligators. For instance, other ligators include naturally occurring receptors, ana cell membrane and nuclear derivatives, that bind specifically to hormones, vitamins, drugs, antibiotics, cancer marKers, genetic markers, viruses, and histocompatibility markers. Anotner group of ligators includes any RNA and DNA binding oroteins. - A -
  • ligators also include enzymes, plasma proteins, avidins, strep- tavidms, chalones, cavitands, thyrogiobulin, intrinsic factor, globulins, chelators, surfactants, organometallic substances, staphylococcal protein A, protein G, ribosomes, bacteriophages, cytochromes, lectins, certain resins, and organic polymers.
  • Ligators also include various substances such as any proteins with selective affinity for ligands that are pro ⁇ uced through recombinant DNA, genetic and molecular engineering.
  • nucleic Acid A nucleic acid is defined as any nucleic acid sequence from any source that is suitable for use in this invention. Said nucleic acid include ⁇ es all types of RNA, all types of DNA, oligonucleotides and other genetic materials including synthetic nucleic acid polymers. Also included are DNA and/or RNA fragments, and derivatives from any tissue, cells, nuclei, chromosomes, cytoplasm, mitochondria, ribosomes, and other cellular sources. Also included are modified and derivatized nucleic acid sequences including those that are coupled to or associated with other substances such as proteins, lectins, histones, polypeptides, carbohydrates, lioids, resins, steroids, hormones and enzymes.
  • nucleic acid may be pretreated by well known methods Defore use in this invention.
  • nucleic acid may be extracted, purified, amplified, denatured by various means, immobilized, and/or suitably derivatized as needed.
  • analyte is defined as any specific substance being tested for, including any ligands, ligators and nucleic acids described herein. Especially preferred in this invention are ligands that are antigens or nap- tens. Antigens are defined as substances that antibodies will bind to through their Fab receptors.
  • Antigens can be divided into two general groups based on their size ana binding properties, which poses certain limitations on which methods are used for their detection.
  • the smaller antigens which usually includes nap- tens, have only one Dindmg site or one epitope (monoepitopic), to which an antibody can bind.
  • Said monoepitopic antigens are generally less suitaole for detection in sandwich-type assays.
  • monoepitopic antigens include molecules witn a molecular weight of approxi ⁇ mately 500 or less.
  • the larger antigens, whicn usually includes most proteins, may have two or more binding sites (polyepitopic), and generally are suitable in a wide variety of assays.
  • Antigens that are preferred in this invention are aflatoxins; alphafetoproteins, CEA, any drugs including anticancer drugs, antifunga " ; ⁇ rugs, antiviral drugs, cardiac drugs, neurological drugs, and drugs of abuse; antibiotics; bioactive pepti ⁇ es; steroids: steroid normones; polypep- tide hormones; interferons; interleukins; narcotics; nucleic acids; pesti ⁇ cides; prostaglandins; viral antigens including those from any DNA anc RNA viruses, AIDS, HIV and nepatitis viruses, adenoviruses, alpnaviruses, arenaviruses, coronaviruses, flaviviruses, herpesviruses, oncornaviruses, papovaviruses, parvoviruses, picornaviruses, poxviruses, reoviruses, rhab- doviruses, rhinoviruses, togaviruses and vir
  • analytes are certain antigenic antibodies that can func ⁇ tion as antigens in a ligand binding reaction.
  • an antibody from one animal eg. human
  • antibodies derived from the immunized animal sucn as monoclonal antibodies are used in vitro to detect the presence of analogous antibodies to those of the first animal species.
  • useful antigenic antibodies to test for are antibodies specific for any dis ⁇ eases, including AIDS, HIV, hepatitis, herpes, clamydia, syphilis, gono ⁇ rrhea, influenza, ana antinuclear antibodies, among others.
  • Luminescence Luminescence is defined as the product of a luminescent reaction.
  • a luminescent reaction for purposes of this invention, is defined as nonradioactive, electromagnetic radiation or light produced by some means of electronic excitation or ionization of molecules or atoms, in the absence of an incident light source. Specifically, this includes phot ⁇ ons emitted through a chemical or biochemical reaction such as oxidation, peroxide cleavage or ionization. However, it would exclude certain physical energy sources for electronic excitation that require an incident light source for photon emission.
  • fluorescent Although certain methods that use an incident light source (eg. delayed fluorescence), have been called “luminescent” in the prior art, by definit ⁇ ion in this invention, those are defined as fluorescent methods. Said fluorescent methods are generally used to solve different problems from tne luminescent art. Therefore, fluorescence, pnosphorescence, apoluminescence and radioactive photon emission are define ⁇ as different arts and are excluded from the luminescent definition.
  • chemiluminescent (CL) substances are inorganic and organic substances that are "activated' to generate light during an irreversible chemical change or decomposition.
  • Chemiluminescent substances can be readily activated by various inorganic oxidizers and do not require enzymes or other proteins to produce luminesc ⁇ ence.
  • certain CL substances can also be activated directly or indirectly through enzymatic reactions. For instance, certain enzymes can generate peroxide as a by-product, which subsequently reacts with the CL substance. Or, certain enzymes can destabilize a CL suostance by cleaving off a phosphate or ester group, and promoting decomposition of the CL subs ⁇ tance.
  • a CL substance, or luminescer can be coupled to and/or complexed within a cyclodextrin label that is detected through activation of the CL substance and measurement of the light produced. Also, under appropriate conditions, CL substances can be used to activate fluorophores through energy transfer.
  • CL substances that would be useful in this invention include a number of compounds such as luminols, isoluminols, aminooutylethyl- isoluminols (ABED, aminobutylethyl-naphthalene-isoluminols (ABEN) ana any other cyclic or acyclic hydrazides.
  • dioxetanes including 3-phospnate-9H-xanthene-9-ylidmeadamantanes, tert-but ldimethyl- siloxyl-substituted and/or adamantyl-substituted dioxetane ⁇ , and other diox- etane and dioxetanone derivatives and precursors, 2,4,5-t ⁇ phenyl ⁇ midazones (lophmes), acridines, acridine and acridinium esters and salts, including derivatives and precursors, indole-3-pyruvic acid, aryl Grignard reagents, riboflavin, lucigenins, 9,10-bis(phenylethynyl)anthracenes (BPEA), 9,10-b ⁇ s- (phenylethynyl)naphthacenes (BPEN), luciferins, phthalazme diones,
  • CL substances that would be useful in this invention have been disclosed in parent application, PCT/US 90/04375 and priority US application SN 418,843, those disclosures are incorporated nerein by refer ⁇ ence.
  • Biolummescence Biolummescence is light pro ⁇ uced by a bioluminescent (BL) reaction using certain light generating proteins or protein containing substances that can be extracted from various bioluminescent organisms. Examples of BL substances are luciferases and photoproteins.
  • Coupling The preparations and components of this invention are synthe ⁇ sized by coupling labels, ligands, ligators, nucleic aci ⁇ s, support mater ⁇ ials and other substances in various combinations as described below. Said coupling can be through noncovalent, "attractive” binding or through coval ⁇ ent, electron-pair bonds.
  • Functional Group A functional group is defined here as a potentially reactive site on a substance where one or more atoms are available for cov ⁇ alent coupling to some other substance. Some substances have functional groups as part of their structure such as those provided by amino acid residues on certain proteins. Other substances may require chemical "activation" of their functional groups to produce aldehydes, ketones or other useful groups. Also, functional groups can be added to various sub ⁇ stances through derivatization or substitution reactions.
  • Examples of functional groups are aldehydes, allyls, amines, amides, azides, carboxyls, carbonyls, epoxys, ethynyls, hydroxyls, ketones, metals, nitrenes, phosphates, propargyls, sulfhydryls, sulfonyls, thioethers, phenolic hydroxyls, indoles, bromines, chlorines, iodines, and others.
  • the prior art has shown that most, if not all of these functional groups can be incorporated into or added to cyclodextrins, ligands, ligators, nucleic acids and support materials if not already present.
  • a coupling agent is defined as a chemical substance or energy that produces and/or reacts with functional groups on a target substance so that covalent coupling or conjugation can occur with the target substance. Because of the stability of covalent coupling, this is often the preferred method.
  • the appropriate coupling agent is used to provide tne necessary active functional group or to react with said functional group.
  • energy type coupling agents are ultraviolet (u.v. , visible and radioactive radiation that can promote coupling or crosslin ing of certain substances.
  • photochemical coupling agents disclosed in U.S. Pat. No. 4,737,454, among others.
  • enzymes that produce covalent coupling such as nucleic acid polymerases and ligases, among others.
  • the coupling agent When the coupling agent is a chemical substance, it can provi ⁇ e the linkage for synthesizing the preparations and components of this invention.
  • Covalent coupling or conjugation can be done through functional groups using coupling agents such as glutaraldehyde, formaldehyde, cyanogen bromide, azides, p-benzoqumone, succinic anhydrides, carbodiimides, maleimides, epi- chlorohydrin, periodic acid, ethyl chloroformate, dipyridyl disulphide and polyaldehy ⁇ es.
  • coupling agents such as glutaraldehyde, formaldehyde, cyanogen bromide, azides, p-benzoqumone, succinic anhydrides, carbodiimides, maleimides, epi- chlorohydrin, periodic acid, ethyl chloroformate, dipyridyl disulphide and polyaldehy ⁇ es.
  • coupling agents useful in this invention are: bifunc- tional imidoesters such as dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP;, dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobis-propionimidate (DTBP), and 2-iminothio ane (Traut's reagent); bifunctional NHS esters such as disuccinimidyl suberate (DSS), bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), disuccinimidyl ,N,N !
  • DSAH disuccinimidyl tartarate
  • DST disuccinimidyl tartarate
  • DSP dithiobis- (succinimidyl propionate)
  • EGS ethylene glycol bis(succinimidyl suc- cinate)
  • ANB-NOS N-5-azido-2-nitrobenzoyloxysuccin- imide
  • p-azidophenacyl oromide p-azi ⁇ ophenylglyoxal
  • FNPA N-hydroxysuccinimidyl-4-azidobenzoate
  • MCS metnyl-4-azidobenzo- imidate
  • MABI metnyl-4-azidobenzo- imidate
  • Certain coupling agents may be less suitable than others due to adverse modification of the cyclodextrins, nucleic acids, ligands, ligators, and components of this invention being coupled. In these cases, routine precau ⁇ tions by one skilled in the art of covalent coupling can be taken to prevent such difficulties.
  • Intermediate Coupling Substance In addition to covalently coupling directly through functional groups, it is also useful to include an interme ⁇ diate substance or intermediate".
  • Said intermediate can function as a "spacer” (eg. "spacer arm " of O'Carra, supra), between the materials being covalently coupled to overcome steric hindrance of subsequent binding reac ⁇ tions.
  • Said intermediate substance may also provide the advantage cf ad ⁇ i- tional coupling sites and thereby increase the amount of label coupled to a ligand or ligator, and/or introduce certain other ⁇ esirable properties, such as more efficient energy transfer.
  • they are coupled to components of this invention by using the appropriate func- tional groups ana coupling agents. Then, the desired ligand or ligator or other substance, is coupled to the available sites on the intermediate subs ⁇ tance and is thereby coupled indirectly to said components of this inven ⁇ tion.
  • intermediate coupling substances are proteins, polypep ⁇ tides, polyamino acids, glycoproteins, lipoproteins, enzymes, nucleic acid polymers, oligonucleotides, DNA, RNA, carbohydrates, amino sugars, glucosam- ines, polysaccha ⁇ des, amino polysaccharides, polygluta ic acids, polylysines, poly(allylamines), nylons, polyacrylamides, lipids, gly- colipids, antibiotics, amphotericins, nystatins, and suitable synthetic polymers, resins and surfactants, as well as suitable derivatives of said substances. Also included are the polymers disclosed in U.S. Pat. Nc. 4,645,646.
  • a coupling group is defined in this invention as a com ⁇ ponent of a CD label that readily couples to the substance to be labeled. Said coupling group is generally targeted for coupling to certain types of functional groups (eg. amines, sulfhydryls, etc.), available on the subs ⁇ tance to be labeled so that further derivatization is not required.
  • a coupling group can include all or part of any coupling agent that has been incorporated into a label, and can include intermediate substances, and spacers as described previously.
  • said spacer is a substance of 4 or more atoms in length and can include aliphatic, aromatic and hetero- cyclic structures.
  • Preferred coupling groups are those used in coupling agents including N-hydroxysuccinimides, imidoesters, maleimides, phenyl azides, and azidos (nitrenes), among others.
  • a suitable enzyme in close proximity to a cyclodextrin label is able to produce a product that could activate a fluorescent or luminesc ⁇ ent substance inside the cyclodextrin label.
  • a fluorophore or CL com ⁇ pound, coupled in close proximity to a cyclodextrin molecule, is able to form an inclusion complex with said cyclodextrin, and/or participate in an energy transfer reaction.
  • Cyclodextrins A cyclodextrin (CD), is an oligosaccharide composed of glucose monomers coupled together to form a conical, hollow molecule with a hydrophobic interior or cavity.
  • Said cyclodextrins (CD's), of this inven ⁇ tion can be any suitable cyclodextrin, including alpha-, beta-, and gamma- cyclodextrins, and their combinations, analogs, isomers, and derivatives. Also included are altered forms, such as crown ether-like compounds prepared by Kandra, L., et al , J. Indus. Phenom.
  • a cyclodextrin (CD) label is defined herein as any CD of suitable size that is capable of complexing or combining with one or more molecules to form an "inclusion complex", or "inclusion compound' , anc wherein said CD also has available one or more suitable coupling groups, defined previously, for coupling to a substance to be labeled.
  • references to a CD "complex”, means an inclusion com ⁇ plex.
  • Said inclusion complex is defined herein as said CD, functioning as a "host” molecule, combined with one or more "guest” molecules that are cont ⁇ ained or bound, wholly or partially, within the hydrophobic cavity of said CD.
  • said CD labels disclosed herein can include more than one host CD molecule, each corr.Dlexec to different parts of the same guest molecule.
  • a useful CD label is composed of said host CD molecules coupled together through suitable linkages or spacers, so that each can complex with tne same guest, forming a duplex, triplex, etc.
  • type of coupling group, and the method of activation a variety of labels with unique properties are possible.
  • CD fluorcphore label is defined herein as a CD label wherein said guest molecule is any suitable fluorophore described herein, including any suitable fluorescent, phosphorescent, or scmtillator substance, or organic dye.
  • a CD chemiluminescent (CL), label is defined herein as a CD label wherein said guest molecule is any suitable CL substance previously described, combined with said CD to form a light emitting inclusion complex.
  • a new form of cyclodextrin CL label comprises said inclusion complex wherein said CL substance is any polyaromat c hydraz ⁇ ide CL compound, disclosed in the copending patent application SN 363,081, filed June 8, 1989, previously described.
  • a CD catalyst label is defined herein as a CD label wherein said CD host functions as an "artificial enzyme", and certain guest molecules can function as a chemical substrate.
  • said chemical substrate comes in contact with said CD catalyst label under appro ⁇ priate conditions, it is modified to produce a detectable signal directly or indirectly (eg. Ikeda, VanEtten, Hirai, or Tabushi , below).
  • said signal can be due to the production of a colorimetric or fluorimetric or CL substance (product).
  • said CD catalyst labels can be synthesized to bind specific substrates and catalyze specific reactions.
  • said CD catalyst label requires derivatives that provide a "recognition site " and one or more "catalytic groups " on said CD aoel.
  • said recognition site and catalytic groups can be pro ⁇ vided through one or several derivatives, as needed.
  • Said recognition site generally involves the hydrophobic cavity of the CD molecule, and provides a means for specifically binding and/or orienting the substrate of interest with the CD molecule.
  • Said catalytic groups are generally organic and/or inorganic chemical residues, functional groups and ionic species that provide a suitable chemi ⁇ cal environment for promoting the catalytic reaction.
  • Said catalytic groups can be any known chemical residue or species that provides the desired cata ⁇ lytic reaction, including carboxylates, imidazoles, histamines, nydroxyls, amines, amides, aldehydes, ketones, phosphates, sulfhydryls, halogens, ammo acids, nucleic acids, chelators, and any suitable substances that involve aliphatic and/or aromatic carbon, nitrogen, oxygen, phosphorous, sulfur, anc metals.
  • Suitable catalytic groups useful in this invention can be found in the art of de ⁇ vatizi ⁇ g CD's anc de ⁇ vatizing or genetic engineering" of antibodies for use as enzymes. Suitable references are: M.L. Bender, or I. Tabushi , or E. Baldwin, or P.G. Schultz, (below), among others.
  • an improved CD catalyst label can be synthesized wherein said recognition site and/or catalytic groups is provided or augmented through the use of one or more suitable captured guests, described herein, that interact (eg. binding, alignment and/or excimer formation), with the substrate being catalyzed.
  • said captured guest is preferably coupled to said CD molecule by a suitable spacer group to allow interaction with said substrate, and can be any suitable aliphatic, aromatic, or netero- cyclic compound, including any suitable inclusion compounds, fluorophores, scintillators and CL substances described herein.
  • CD catalyst labels eg. size, affinity, reactivity, etc.
  • several labels can be used to label different substances so that their presence is detectable in the same solution.
  • each CD catalyst label could be distinguished from the others by the produc ⁇ tion of a different product which produced a correspondingly different sig ⁇ nal, such as a different color.
  • Suitable CD catalyst label reactions include hydrolysis (eg. of any suitable ester or amide containing substrates . , oxidation, dephosphorylation, acid-base catalysis, formylation, dichloromethylation, carboxy!ation, rearrangement, substitution, allylation, and halogenation, among others.
  • said catalyst CD labels are prepared so that they meet the requirements of (1) the catalytic action is suitably more efficient than background or noncatalytic reactions, (2) the catalyst CD label is suitably detectable through measurement of a catalytic product that is colorimet ⁇ c, fluorometric or chemiluminescent, (3) they have at least one suitable coupl ⁇ ing group for coupling to the substance to be labeled, and (4) the labels ⁇ c not significantly impair specific binding reactions.
  • Said CD catalyst labels can be coupled to a variety of substances, such as ligands, antigens, antibodies, nucleotides, nucleic acids, and liposomes, as well as to a van- ety of support materials including magnetic particles for use and retrieval in assays and chemical processes.
  • An improved CD catalyst label comprises the direct or indirect coupling of any suitable antenna substance described herein, to said CD molecule, for collection of light energy that is transferred to the CD catalyst and thereby accelerates the desired reaction.
  • a CD activator label (energy transfer label), is any CD label described herein, wherein one or more suitable "energy producing" substances are coupled to the CD surface.
  • Said energy producing substances can include suitable CL substances, BL substances, per- oxyoxalates, dioxetanes, dioxetanones, oxalate-type esters, catalysts, enz ⁇ ymes, including activators described herein (eg. oxidases and peroxidases), and other substances, that under suitable conditions, can chemically gener ⁇ ate peroxides, activator products, or ionization and/or electronic excita ⁇ tion energy which can be transferred to guest molecules inside said complex.
  • activators described herein eg. oxidases and peroxidases
  • a plurality of CD molecules or CD labels can be coupled to an intermediate substance that is an activator, such as an enzyme (eg. glu ⁇ cose oxidase, horseradish peroxidase, esterases, or alkaline phosphatase), which also includes a coupling group for coupling to a substance to be labeled.
  • an activator such as an enzyme (eg. glu ⁇ cose oxidase, horseradish peroxidase, esterases, or alkaline phosphatase), which also includes a coupling group for coupling to a substance to be labeled.
  • Yet another label composition provides a plurality of said CD activator labels, that can be readily coupled to any suitable substance, in numbers that are greater than the number of available sites on said substance as described previously for multiple CD labels.
  • Cyclodextrin Tracer A cyclodextrin (CD) tracer is a component of this invention composed of a specific binding substance such as a igand, lig ⁇ ator, or nucleic acid, among others, that has been coupled to, or associated with, the surface of one or more of any CD molecules, derivatives, or labels described herein.
  • said specific binding substance is not an inclu ⁇ sion complex with said CD.
  • said CD tracer maintains specific binding properties that are functionally identical or homologous to those of said ligand or ligator before coupling.
  • said label coupled to said CD tracer is not adversely inhibited from generating a detectable signal.
  • a CD tracer in a competitive binding assay, a CD tracer is preferred that has specific binding properties that are functionally identical or homologous to any analyte (eg. antigen or ligand), it is intended to compete with. Also, in a sandwich type assay, a CD tracer is preferred that nas specific binding properties that are functionally identical or homologous to any ligator (eg. antibody or receptor), that the tracer contains.
  • any ligator eg. antibody or receptor
  • Said CD tracer can be composed of a specific binding substance coupled to a fluorescent or luminescence CD label, detected by activating the label and measuring the light produced.
  • a CD tracer can also be said specific binding substance coupled to said CD catalyst label.
  • Cyclodextrin tracers composed of CD labels coupled to avidins and strep- tavidins are useful for subsequent noncovalent coupling to any suitable biotinylated substance.
  • CD labeled antibody tracers can be non- covalently coupled to another antibody, or to a nucleic acid or other suit ⁇ able substance that has the appropriate antigenic properties.
  • Another use ⁇ ful CD tracer comprises protein A, protein G, or any suitable lectin that has been covalently or noncovalently coupled to a CD label.
  • Peroxyoxalates are defined in this invention as organic substances that contain as part of their structure, an oxalate derivative that can decompose in the presence of H2O2 or other peroxides and thereby activate or electronically excite a fluorophore or fluorescer to emit light.
  • peroxyoxalates useful in this invention are; oxalyl chloride, bis(3-trifluoromethyl-4-nitrophenyl)oxalate (TFMNPO), bis(2,4- dinitrophenyDoxalate (DNPO), bis(2,4,6-trichlorophenyl)oxalate (TCPO), bis(2,4,5-trichloro-6-carbopentoxyphenyl)oxalate (CPPO), 4,4'- ⁇ oxalylbis[(trifluoromethylsulfonyl)imino]ethylene ⁇ bis[4-methylmorpholinium trifluoromethane sulfonate] (METQ), among others, including analogs, precur ⁇ sors and derivatives such as mixed anhydrides, amides, sulfona ides, and N- trifyloxamides of oxalates.
  • TPMNPO bis(3-trifluoromethyl-4-nitrophenyl)oxalate
  • fluorophores are defined as any suitable compound including any fluorescent, phosphorescent, scintillator substance and/or dye, and aromatic hydroxylated compound described herein, that can form a light emitting or absorbing inclusion complex with a CD of this invention.
  • Scintillators are fluorophores that include light emitting compounds generally used in scintillation counting systems and as laser dyes. Under suitable condi ⁇ tions, they are useful as fluorescent acceptors, and in energy transfer sys ⁇ tems with CD's and other preparations described herein.
  • Useful scintil ⁇ lators can be found under "Laser Dyes", p. 877-880, Sigma Catalogue, 1989, Sigma Chem. Co., St. Louis, MO.; and "Kodak Laser Dyes", Public. JJ-169, Eastman Kodak Co., NY (1987).
  • fluoro- pnores are defined in this invention as not themselves cne i luminescent. Therefore, fluorophores of this invention do not undergo an irreversible chemical decomposition during light emission.
  • fluorophores useful in this invention are: 1-amino-4- chloro-naphthalenes, 1-aminopyrenes, 1-fluorenecarboxylic acids, 1-pyrene- butyric acids, 2-aminoanthracenes, 2-armnofluorenes, 2-am ⁇ no-3-chloro-7- nitro-9-fluorenones, 2-amino-9-fluorenones, 2-p-toluidinylnaphthalene sulfo- nates (TNS), 3-aminofluoranthenes, 4-chloro-7-nitrobenzo-2-oxa-1 ,3-diazole (NBD chloride), 4-fluoro-7-nitrobenzofurans (NBD-F), 6-aminochrysenes, 7- amino-4-methycoumarins, 8-anili-1-naphthalene sulfonates (ANS), 8-anilo-1- naphthalenes, 9-(cargol
  • the fluorophore and luminescent labels of this invention emit light after electronic excitation or " activation”.
  • CL substances can be activated directly by exposure to suitable oxidizers and/or catalysts in suitable buffer to maintain the appropriate pH. Fluorophores of this inven ⁇ tion are activated through some outside energy source.
  • Said outside energy source can be physical, such as radioactive, or an incident light source (fluorescence or phosphorescence), an electrical current (electrolumines ⁇ cence), sonication (tribolu inescence), or heat (ther oluminescence), or said energy source can be a chemical reaction (energy transfer luminesc ⁇ ence).
  • CL activation is defined as any suitable chemical reaction capable cf activating fluorophores and CL substances.
  • Fluorophores are CL activated by any suitable chemical reaction, including those involving acridinium esters, various hydrazides, enzymes, dioxetanes, dioxetanones, oxalates, and peroxyoxalates.
  • other suit able activation reactions that are applicable to this invention are reviewed by Cilento, G., et al, Photochem. Photobiol. 48, 361-368 (1988).
  • a variety cf known activation methods can be used in this invention, such as by suitable modification of the flow injection method of Mahant, V.K., et al , Anal. Chim. Acta 145, 203-206 (1983), among others.
  • fluorophores are CL activated through energy transfer from peroxide decomposition of various peroxyoxalates.
  • CL activation using peroxyoxalates and similar CL substances involves the use of a perox ⁇ ide such as H2O2.
  • H2O2 can be added directly to a reac ⁇ tion mixture of CD fluorophore label and peroxyoxalate, in suitable buffer solution such as phosphate buffer.
  • H2O2 can be generated by enzymes such as glucose oxidases, xanthine oxidases and galactose oxidases, among others, with suitable substrate.
  • Activation can also include energy transfer from any suitable "energized" substance wherein said energy is first generated by a suitable physical source to produce said energized substance, which is then exposed to said fluorophore.
  • fluorophores of this invention can convert some of this energy to visible light. Examples of this approach are disclosed by Tseng et al , U.S. Pat. Nos. 4,338,213 and 4,407,743; Arthen et al, U.S. Pat. No. 4,401,585; Kamhi et al , U.S. Pat No. 4,404,513; Fren- zel, U.S. Pat. No. 4,433,060 and Berthold et al , U.S. Pat No. 4,435,509. Others in the prior art have used chemically coupled CL substances that are capable of energy transfer.
  • an oxidizing substance such as peroxide is used.
  • H2O2 can be added directly to a reaction mixture of CD chemiluminescent label in suitable buffer solution.
  • H2O2 can be gener ⁇ ated by enzymes such as glucose oxidases, xanthine oxidases ana galactose oxidases, among others, with suitable substrate.
  • enzymes such as glucose oxidases, xanthine oxidases ana galactose oxidases, among others, with suitable substrate.
  • combinations of these enzymes, catalysts or apoenzymes that eventually produces peroxides such as H2O2 can be used.
  • a peroxide such as H2O2.
  • suitable buffer solution preferably of pH above 7.5.
  • enzymes such as glucose oxidases, xanthine oxidases and galactose oxidases, to generate H2O2, with suitable adjustments in pH.
  • the CL reaction is also catalyzed through the addition of a heme, hematin, or heme containing substance.
  • hydrazide catalysts are oxidizing enzymes such as peroxidases, icroperoxidases, or combinations of these enzymes, catalysts or cofactors.
  • transition metal salts such as cobalt(II), copper(II), and ferricyanides may be used.
  • any CL activation reactions that employ H2O2 a variety of suitable peroxides can be substituted, under suitable conditions.
  • perox ⁇ ides that may be substituted include t-butyl hydroperoxides, benzoyl perox ⁇ ides, linoleic hydroperoxides, cholesterol hydroperoxides, peroxylau ⁇ c acid, and cumene hydroperoxides, among others.
  • Other examples are ⁇ isclosec by Cathcart, R. , et al, Anal. Biochem. 134, 111 (1983), disclosures tnerein are incorporated herein by reference.
  • analyte is determined through its effect on the binding of a tracer which may mimic the binding behavior of said analyte, or bind to it, or demonstrated some other binding response. Therefore, by determining the binding behavior of said tracer, the presence or absence of said analyte is inferred. Said binding behavior of said tracer is determined by "selec ⁇ tively" detecting how much tracer is bound and/or not bound. Depending on the type of assay and relationship between analyte and tracer, selective detection may only require measurement of the bound tracer, or, both the bound versus unbound tracer.
  • CD tracers are detected or measured by activating the label coupled to the tracer, to produce a detectable signal, such as light emis ⁇ sion.
  • a homogeneous assay is defined as selectively determining the presence of bound (versus unbound), label on a tracer after said tracer has selectively bound (directly or indirectly), to an analyte, without the need for washing away or separating the unbound fraction.
  • Said selective determination or detec ⁇ tion is accomplished by measurement of a signal producing reaction (eg. lum ⁇ inescent or catalytic), that is substantially dependent on the close proximity of an activator and said label.
  • a signal producing reaction eg. lum ⁇ inescent or catalytic
  • said selective binding and luminescent reaction can be started during a specific binding reaction, by addition of a suitable labeled tracer in the presence of suitable substrates and scaven ⁇ gers, as needed.
  • said specific binding reaction can be com ⁇ pleted with appropriate washes, and then the labeled tracer with suitable substrate is added in suitable buffer solution.
  • additional washing steps can be employed wherein substrate is finally a ⁇ ded to generate a signal such as luminescence.
  • this invention pro ⁇ vides the user with the option, after addition of the labeled component, of eliminating washing steps before selective detection of said label.
  • a captor is a component of this invention composed of any substance or material, and/or ligan ⁇ or lig ⁇ ator that binds to the analyte so that a CD tracer (eg. CD labeled anti ⁇ body) can also bind to the analyte (eg. to form an antigen-antibody com ⁇ plex), and usually, but not necessarily, form a so called "sandwich" con ⁇ figuration.
  • a sandwich arrangement means that the analyte is bound between a captor substance and one or more ligands, ligators and/or CD tracers.
  • Said captor can be an immunosorbent composed of any suitable support mater ⁇ ial coupled to any suitable antibody or fragment thereof.
  • Said captor can be composed of various materials including glasses, plastics, celluloses, papers, resins, latexes, polymers, proteins, carbohydrates, gels, fibers, ceramics, salts, including hydroxylapatites, and metals or various combina ⁇ tions of these, that bind to the analyte or a complex containing the ana ⁇ lyte.
  • Said captor can be composed of various materials with any suitable ligand or ligator coupled to it.
  • Said captor can be immobilized material in various suitable configura ⁇ tions such as the inside surface of any vessel, microtiter well, or tubing or the surface of a bead, rod, sheet, disk, matrix, or membrane, or combina ⁇ tions of these, such as filter entrapped particles, etc.
  • the captor may also be any ligand or ligator coupled to said immobilized material.
  • a captor can also be mobile, wherein said captor is a soluble or par ⁇ tially soluble or colloidal or insolubilized particle, fragment, filament, liposome, or bead.
  • Said mobile captor is composed of various glasses, plastics, celluloses, papers, resins, latexes, polymers, proteins, carbohy ⁇ drates, gels, fibers, ceramics or metals or various combinations of these.
  • a useful mobile captor comprises any suitable ferrous or magnetic particles or microspheres (eg. Ithakissios, D.S., Clin. Chim. Acta 84(1-2), 69-84, 1978, and European patent applic. 88 301 839.2, 1988), that are suit- ably prepared with specific binding properties. These captors are useful for various magnetic manipulations such as dispensing, transferring, washing and separating bound substances from free, during an assay procedure.
  • the captor may also be any ligand or ligator coupled to said mobile captor.
  • a captor can have a variety of other substances coupled or associated with it. For instance, under suitable conditions, a captor can have any BL or CL activators, ligands, ligators, components of this inven ⁇ tion and intermediate coupling substances, coupled to it.
  • an activator is defined as a component of this invention com ⁇ posed of one, or a combination of, catalysts, enzymes, apoenzymes, enzyme derivatives or precursors, coenzymes, cofactors and substrates that is capable of generating products or substrates that are used directly or indirectly to promote a reaction that produces a detectable signal such as luminescence, fluorescence or color absorbance.
  • an activator is not itself luminescent, but is a substance that promotes a luminescent or CL activation reaction described previously.
  • a “catalytic activator” is employed.
  • Said catalytic activator comprises any suitable substance, (eg. enzyme), that is capable of generating products or substr ⁇ ates that are used directly or indirectly in the catalytic reaction of a CD catalyst label.
  • an activator is covalently or noncovalently coupled to, or in close proximity with a captor specific for the analyte.
  • a captor specific for the analyte.
  • the luminescence tracer can be activated by the activator in the presence of substrate, to produce light.
  • an activator is coupled to a suitable tracer, (eg. CD labeled antibody, avidin or streptavidin), where the activator is brought in close proximity to a captor or a luminescent substance, through a specific binding reaction such as in said sandwich con ⁇ figuration.
  • the coupled activator can be protected from excessive loss of activity during preparation, storage and assay procedures.
  • enzymatic activators eg. glucose oxidase
  • Other known protective methods include protecting the enzyme active site with excess substrate or rever ⁇ sibly blocking the site and unblocking it when needed.
  • activator enzymes such as oxidases
  • the desired activator enzymes can be synthesized or modified to be more resistant tc inactivation through known protein engineering methods.
  • buffer solutions and substrates needed for a particular assay will depend on the activator, such as an enzyme or combination of enz ⁇ ymes, being used. Also, the desired activating product, such as H2O2, needed to produce a luminescent reaction with the label being used, will determine the type of substrates used. These biochemical and luminescent requirements are well known and can be determined by those skilled in the art.
  • a scavenger is a compon ⁇ ent of this invention composed of any substance or material that competi ⁇ tively consumes, blocks or inhibits one or more of the products of an acti ⁇ vator.
  • a scavenger When used, a scavenger must not significantly catalyze a signal producing reaction and is usually included in the bulk solution, matrix, media or support member of a homogeneous assay.
  • a scavenger reduces the availability of one or more activator products to the CD tracer and thereby inhibits or reduces the rate of a luminescent or catalytic reac ⁇ tion outside of the immediate vicinity of the activator, where specific binding occurs.
  • substances that can serve as scavengers are various organic substances and enzymes that consume or degrade peroxides (eg. H2O2).
  • Exam ⁇ ples are catalases, among others, which can compete for H2O2 used in various CL reactions.
  • enzymes that consume coenzymes such as ATPases, apyrases, reductases and hexokinases, oxidoreductases, dehydro- genases and the like.
  • hexokinase with glucose as substrate can be an ATP scavenger that competes with firefly luciferase in solution.
  • Other suitable examples can be found in, "Methods in Enzymology", 133, p.198-248, (1986), wherein the disclosed enzymes could be used in soluble or immobilized form depending on the application.
  • a carrier or “carrier mem ⁇ ber” is any substance, matrix, medium or surface capable of being impreg ⁇ nated or coated with specific binding and activating reagents.
  • said carrier is any bibulous material, polymer layers, porous or nonporous material, or combinations of these, that will suitably entrap, contain, adhere or couple the necessary assay components and reagents of test devices described below. Disclosures of carrier materials and synthesis methods for use in luminescent immunoassays are presented by Greenquist, A.C., et al, ir, U.S. Pat. No's. 4,668,619 (1987), and 4,442,204 (1984), said materials and methods are hereby incorporated into this invention by reference.
  • compositions of this invention it will be understood by those skilled in the art of organic synthesis methods, that there are certain limitations and conditions as to what compositions will comprise a suitable label. Said limitations include types and numbers of derivatives used, steric properties, fluorescent properties of fluoro ⁇ phores or scintillators, excitation energy transfer requirements, oxidation requirements in aqueous solution, stability at room temperature and solubility, among others.
  • Said limitations include types and numbers of derivatives used, steric properties, fluorescent properties of fluoro ⁇ phores or scintillators, excitation energy transfer requirements, oxidation requirements in aqueous solution, stability at room temperature and solubility, among others.
  • cyclodextrins there are limitations as to which inclusion compounds, fluorophores and CL compounds can be used to form inclusion complexes with certain CD's.
  • the purpose is to provide a CD label that can be readily coupled to a variety of ligands, ligators, antibodies, antigens, and nucleic acids. Improvements provide for coupling a plurality of CD labels in numbers that are greater than the number of available sites on the labeled molecule.
  • the general approach is; (1) to produce or modify, as needed, one or more functional or coupling groups on the outside of said CD molecule, (2) combine under appropriate conditions, said CD and a CL subs ⁇ tance, scintillator or fluorophore to synthesize said inclusion complex, or a suitable chemical substrate to produce a detectable signal.
  • said CD molecule may be suitably derivatized to include other useful substances and/or chemical groups (eg. capping, antenna, activator, and catalytic substances), to perform a particular function.
  • said derivatization can be done before addition of a suitable coupling group, or afterward, using suitable protection and deprotection methods as needed.
  • Said coupling group is located on the outside of said ⁇ erivatized CD tc allow coupling to any desired substance and not substantially interfere (chemically or sterically), with the intended function of said CD.
  • said coupling group is located a suitable distance from other derivatives and/or substances on said CD molecule, such as through a spacer, and/or is located at the opposite end of said CD molecule.
  • CD's are composed of carbohydrates, they can be suitably derivat ⁇ ized and coupled through well known procedures used for other carbohydrates, especially through available hydroxyl groups. For instance, vicinal hydroxyl groups on the CD can be appropriately oxidized to produce aldehydes. In addition, any coupling group can be suitably added through well known methods while preserving the CD structure and complexing proper ⁇ ties.
  • Examples are: amidation, esterification, acylation, N-alkylation, allylation, ethynylation, oxidation, halogenation, hydrolysis, reactions with anhydrides, or hydrazines and other amines, including the formation of acetals, aldehydes, amides, imides, carbonyls, esters, isopropyli ⁇ enes, nitrenes, osazones, oximes, propargyls, sulfonates, sulfonyls, sulfcnami ⁇ es, nitrates, carbonates, metal salts, hydrazones, glycosones, mercaptals, and suitable combinations of these.
  • Said coupling groups are then available for the coupling of one or more CD molecules to a bifunctional reagent and/or to an appropriate ligand, ligator or nucleic acid. Coupling can be done before forming said inclusion complex or afterward.
  • CD's inclusion compounds, CL substances, catal ⁇ ytic groups, scintillators and fluorophores, including chemical methods for modifying and/or derivatizing CD's that are useful in this invention are described in the following references incorporated herein by reference:
  • the purpose is to provide new CD derivatives and labels that are; (1) water soluble, (2) form complexes with CL substances, scintillators, fluoro ⁇ phores or substrates, (3) can be readily coupled to the desired substance, and (4) have the needed "spacer", to overcome steric interference after coupling to the labeled substance.
  • Suitable coupling agents for preparing CD labels of tnis invention can oe a variety of reagents previously described, including well known crosslinkers such as epichlorohydrin, isocyanates, and formaldehyde, used to polymerize CD's.
  • Other suitable cross!inkers are various epoxy compounds including propylene oxide, 1,2-diethoxyethane, 1 ,2,7,8-diepoxyoctane, 2,3- epoxy-1-propanol (glycidol), 2,3-epoxy-1 ,4-butanedio! , (eg. Gramera, or Case, or Johnson, or Parmerter, supra).
  • acrylic esters such as m-nitrophenyl acrylates, and hexamethylenedia ine and p-xylylenediamine complexes (eg. Furue, or Harada, or Hatano, or Ogata, supra)
  • aldehydes ketones
  • alkyl halides acyl halides
  • silicon ha!ides eg. Buckler, supra
  • isothiocyanates eg. Buckler, supra
  • Derivatizing is defined as the chemical modification of a CD through addition of any functional or coupling group and/or other substance.
  • Capping is defined herein as coupl ⁇ ing any suitable chemical substance to two or more sites on the CD molecule so that said substance spans the area between the coupled sites.
  • one or more said chemical substances are used, wherein each one spans across one of the end openings of the CD molecule and thereby influences the passage of other molecules into or out of the CD molecule.
  • the CD's used herein can be suitably complexe ⁇ with one or more guest molecules and/or derivatized and/or capped before or after their incorpor*.- tion into the labels of this invention.
  • said derivatizing and/or capping can be a done to produce CD's with the desired substances coupled to specific locations on the CD molecule.
  • the host CD's of this invention are preferably derivatized (eg. methylated), and/or capped by various means to increase host-guest affinity, provided that a suitable functional or coupling group is also provided for coupling to the substances to be labeled.
  • said capping subs ⁇ tance is coupled at the primary or secondary "end" of the CD molecule, form ⁇ ing a bridge across either (or both) openmg(s), that includes suitable hydrophobic groups in said capping substance.
  • Said capping substances can be coupled directly to available hydroxyls on the CD, or they can be coupled to suitable functional groups such as; diamino (or triamino), compounds to iodinated CD, or azido compounds to sulfonylated hydroxyls, and/or through "spacers" added to the CD.
  • Suitable capping substances are 6-methyla ino- deoxy and 6-methylamino-6-deoxy derivatives transformed to the corresponding N-formyl compounds, imidazoles, m,m'-benzophenqne-disulfonyl chloride, p,p'- stilbene-disulfonyl chloride, diphenylmethane-p,p'-disulfonyl chloride, terephthaloyl chloride, dianhydrides such as 3,3' ,4,4'-benzophenonetetra- carboxylic dianhydride and 3,4,9,10-perylenetetracarboxylic anhydride, amino compounds such as basic fuchsins, bismark browns, N,N'-bis(3-aminophenyl- 3,4,9,10-perylenetetracarboxylic diimide, 1 ,4-bis(3-aminopropyDpiperizine, direct yellow, azido compounds such as 2,6-bis
  • C. Protected Cyclodextrins Primary and/or secondary hydroxyl groups (or derivatives), can be selectively protected and deprotected using known methods during derivatizing and/or capping procedures, to provide selective coupling at the primary or secondary end of the CD molecule, as desired. For instance, formation of protective esters (eg. benzoates using ben ⁇ oyl chloride), and selective cleavage (deprotection), of primary esters using anhydrous alcoholysis (eg. Boyer, supra), provides mostly primary hydroxyls for derivatization. After derivatization and/or coupling the primary hydr ⁇ oxyls, the secondary hydroxyls can be deprotected for additional derivatiza ⁇ tion, coupling and/or capping.
  • protective esters eg. benzoates using ben ⁇ oyl chloride
  • selective cleavage deprotection
  • secondary hydroxyls can be deprotected for additional derivatiza ⁇ tion, coupling and/or capping
  • N-hydroxysuccinimidyl Cyclodextrin (NHS-CD) Labels This is a method for synthesizing new CD labels (including multiple CD labels, below), wherein an N-hydroxysuccinimidyl (NHS), coupling group is included in the label composition to provide for labeling any suitable substance with an available amino group, in a single step.
  • the substance to be labeled can be a suitable protein, including antibodies and avidins or streptavidin, or ligands, or nucleic acids.
  • CD (10 gm) is combined with a suitable sulfonylating reagent (20 gm), such as p- toluenesulfonyl (tosyl) chloride, mesitylenesulfonyl chloride or naphthal- enesulfonyl chloride, among others, in anhydrous pyridine, for 3-5 Hrs at room temperature (RT).
  • a suitable sulfonylating reagent such as p- toluenesulfonyl (tosyl) chloride, mesitylenesulfonyl chloride or naphthal- enesulfonyl chloride, among others, in anhydrous pyridine, for 3-5 Hrs at room temperature (RT).
  • Dialdehyde CD is prepared by oxidation of CD using known methods (eg. Royer or Kobayashi , supra), with sodium metaperiodate. A more selective procedure is to oxidize the CD with an oxidizing enzyme (eg. glucose oxidase), in suitable buffer solution (eg. 0.2 M phosphate saline, pH 5-7).
  • an oxidizing enzyme eg. glucose oxidase
  • suitable buffer solution eg. 0.2 M phosphate saline, pH 5-7.
  • Said sulfonylated CD is suitably iodinated so that it will couple to primary amino groups, using known methods (eg. Ikeda or Iwakura, supra).
  • 10 gm of sulfony ⁇ lated CD is combined with 12 gm of Nal on 200 ml of methanol, and mix at 70 "C for 48-60 Hrs.
  • the iodinated CD product is collected by precipitation with acetone and purified by column chromatography.
  • Said iodinated CD or said Dial-CD is coupled through the amino group to a suitable amino-carboxylic acid to provide the desired length of spacer.
  • suitable amino-carboxylic acids are; 4-aminobutyric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminocaprylic acid, 12-aminododecanoic acid, and other aliphatic, or aromatic, or heterocyclic carboxylic acids with an available amino group for coupling.
  • Coupling to iodinated CD is done in a suitable solvent such as dimethyl ⁇ formamide (DMF), mixing 10 gm of iodinated CD with a molar excess of amino- carboxylic acid (eg. 10 gm of 6-aminohexanoic acid), at 100 °C for 24 Hrs.
  • a suitable solvent such as dimethyl ⁇ formamide (DMF)
  • amino- carboxylic acid eg. 10 gm of 6-aminohexanoic acid
  • the product, Acid-CD is concentrated and purified by column chromatography.
  • Coupling to said Dial-CD is done by reductive alkylation.
  • a suitable buffer eg. 0.1 M borate, pH 7.5-8.5
  • 0.1-0.5 M triethanolamine 10 gm of dial CD is mixed with a molar excess of amino-carboxylic acid (eg. 10 gm of 12-aminodecanoic acid), at RT for 1-2 Hrs.
  • the Schiff's base coupling is stabilized by suitable reduction with NaBH 4 (eg. 0.1-1 mg/ml), for 1-12 Hrs.
  • the product, CD-carboxylic acid is concentrated and purified by column chromatography and dried for other reactions as needed.
  • a CD (or multiple CD, below), is suitably protected and/or deprotected as needed and a sulfonylated CD is prepared as described previously.
  • a ino groups can be introduced into CD by reaction of said sulfonylated CD with azide compounds including hydrazine, and 2,6-bis(4-azidobenzylidene)-4- methylcyclohexanone (eg. Ikeda, supra), or coupling to diamines as describee by Kawaguchi, or Matsui, supra.
  • a "monoamino" CD wherein one amino group has Deen coupled, can be prepared through known methods, including limited or steri- cally determined monosulfonylation, and/or by specific protection and deprc- tection schemes.
  • Said sulfonylated CD is suitably iodinated ⁇ s described previously.
  • Said iodinated CD is coupled through an amino group to a suitable diamino substance.
  • suitable diamino substances are; 1,4- diaminobutane, 1 ,6-diaminohexane, 1 ,7-diaminoheptane, 1 ,8-diaminooctane,
  • Coupling is done in a suitable solvent such as DMF, mixing 10 gm cf iodinated CD with a molar excess of the diamino substance (eg. 10-20 gm of 1 ,6-diaminohexane), at 100 °C for 24 Hrs.
  • the product, Amino-CD is con ⁇ centrated and purified by column chromatography.
  • said diamino substances of various chain lengths can be suitably derivatized before coupling. For instance, they can be "half protected” as trifluoroacetamidoalkanes at one cf the ammo ends, as described by Guil- ford, H. , et a! , Biochem. Soc. Trans. 3, 438 (1975), before coupling, and then suitably deprotected such as by hydrolysis or alcoholysis. Yet another suitable method involves the coupling of THP-protected a ino-al ynes, previ ⁇ ously described, to said iodinated CD, and subsequent deprotection as needed.
  • NHS-CD derivatives can be prepared by coupling NHS esters directly to said Amino-CD's.
  • said NHS ester is a bifunctional NHS coupling agent with a suitable spacer.
  • Suitable NHS coupling agents for use in this invention have been previously described, including DSS, bis(sulfosuccinimidyl)suberate (BS 3 ), DSP, DTSSP, SPDP, BS0C0ES, DSAH, DST, and EGS, among others.
  • Sulfhvdryl-Cyclodextrin (SH-CD) Derivatives A sulfhy ⁇ dryl group is added to said Amino-CD, suitably prepared as described previ ⁇ ously, by coupling the appropriate thiolating agent to the available am o group. For instance, thiolation of available amino groups can be done by known methods using S-acetylmercaptosuccinic anhydride (SAMSA), (eg. Klotz, Rector, or Lui , supra), SIAB, or 2-iminothiolane (eg. Traut, supra). The sulfhydryl can be exposed through disulfide cleavage.
  • SAMSA S-acetylmercaptosuccinic anhydride
  • SIAB 2-iminothiolane
  • Sulfhydryls can also be introduced through reaction of available hydrox ⁇ yls with a suitable epoxy compound.
  • a suitable epoxy compound For instance, epichlorohydrin or a suitable epoxy crosslinker previously described, is coupled to CD (preferably immobilized by a cleavable coupling agent), wherein free epoxy groups are produced. Free epoxy groups are then reacted with sodium thiosulfate to give thiosulphate esters (eg. Carlsson, supra). Said thiosulphate esters are subsequently reduced to sulfhydryls with dithio- threitol.
  • Sulfhydryls can be used for disulfide coupling to other available sulfhy ⁇ dryls on the desired substance to be labeled such as antibodies, or avidins, or streptavidin, or ligands, or nucleic acids.
  • Said available sulfhydryls may be native, or introduced by thiolation of said substance before coupl ⁇ ing.
  • said sulfhydryl containing CD label is coupled to any maleimide derivative of protein, ligand, nucleic acid or biotin, (eg. bio- tin-maleimide) or iodoacetyl derivatives such as N-iodoacetyl-N'-biotmyl- hexylenediamine.
  • the aleimido-cyclodextrin (Mal-CD), label of this invention is suitable for coupling to native or introduced sulfhydryls on the desired substance to be labeled in a single step.
  • a maleimido group is added to the Amino-CD, suitably prepared as descr- ibed previously, by coupling a suitable hetero-bifunctional coupling agent to the available amino group.
  • Said hetero-bifunctional coupling agent con ⁇ sists of a suitable spacer with a maleimide group at one end and an NHS ester at the other end. Examples are previously described and include MBS, SMCC, SMPB, SPDP, among others.
  • the reaction is carried out so that the NHS ester couples to the available amino group on the CD, leaving the maleimide group free for subsequent coupling to an available sulfhydryl on the subs ⁇ tance to be labeled.
  • Iodo-Cyclodextrin (Iodo-CD) labels can be prepared for coupling to sulfhydryl groups.
  • NHS esters of iodoacids can be coupled to said Amino-CD's.
  • Suitable iodoacids for use in this invention are iodopropionic acid, iodobutyric acid, iodohexanoic acid, iodohippuric acid, 3-iodotyrosine, among others.
  • the appropriate Io ⁇ o-NHS ester is prepared by known methods (eg. Rector, supra).
  • iodopropionic acid and N-hydroxy- succinimide are mixed, with suitable carbodiimide, in anhydrous dioxane at RT for 1-2 Hrs, the precipitate removed by filtration, and the NHS iodoprop ⁇ ionic acid ester is collected in the filtrate. Said NHS iodopropionic acid ester is then coupled to the Amino-CD.
  • Biotinylated Cyclodextrin Biotinylated CD can be pro ⁇ substituted by a variety of known biotinylation methods suitably modified for use with CD's. For instance, combining said Amino-CD derivative with a known N- hydroxysuccinimide derivative of biotin in appropriate buffer such as 0.1 M phosphate, pH 8.0, reacting for up to 1 hour at room temperature.
  • appropriate buffer such as 0.1 M phosphate, pH 8.0
  • biotin derivatives examples include, biotin-N-hydroxysuccinimide, biotinamidocaproate N-hydroxysuccinimide ester or sulfosuccinimidyl 2- (biotinamino)ethyl-l ,3'-dithiopropionate, among others.
  • any CD label of this invention can be coupled to biotin or a suit ⁇ able derivative thereof, through any suitable coupling group on said label.
  • biocytin can be coupled through the available amino group to any NHS-CD label described herein.
  • thiolated biotin can be coupled to any Mal-CD label.
  • Said biotinylated CD can be used to couple a plurality of CD labels tc any other biotinylated compound. For instance, by combining dilute solu ⁇ tions of said biotinylated CD with avidin or streptavidin in the appropriate molar ratio, 1, 2 or 3 biotinylated CD molecules will couple to the avidin or streptavidin and produce a complex with one or more biotin-biding sites still available. Then, said complex is added to the biotinylated compound to be labeled, and allowed to couple through the remaining biotin-binding site.
  • n a multiple of 1 or more, the general structure is:
  • Photochemical Cyclodextrin (Photo-CD) Labels A Photo ⁇ chemical CD label is a CD label (including multiple CD labels, below), cont ⁇ aining a photoactive coupling group in its composition, for coupling said label to any suitable substance. Typically, the photoactive group is an aryl azide which upon exposure to light, generates a highly reactive nitrene coupling group. Photo-CD labels are synthesized by several methods.
  • said Amino-CD derivatives previously described can be der ⁇ ivatized with a suitable bifunctional coupling agent that will couple to amino groups at one end and provide a photoactive group at the other end.
  • a suitable bifunctional coupling agent that will couple to amino groups at one end and provide a photoactive group at the other end.
  • Some examples are NHS-ASA, ANB-NOS, APG, EADB, HSAB, MABI, SANPAH, SADP, SAND, Sulfo-SADP, Sulfo-SANPAH, and SASD, (all available from Pierce Chemi ⁇ cal Co., IL.), including carbenes and nitrenes disclosed by Knowles, J.R., Ace. Chem. Res. 5, 155-160, (1972), among others.
  • a suitable procedure is to combine, in a dark environment, said Amino-CD (0.5-1.0 gm) in phosphate buffered saline (PB), pH 8.5, or a suitable anhy ⁇ drous solvent such as N,N dimethylformamide (DMF), with NHS-ASA (1-2 gm), and let react 1 to 2 hours at 0 "C.
  • the photo-CD label product is purified by column chromatography, dried, and stored in the dark until used for coupling. Coupling is initiated by mixing the photo-CD label with the subs ⁇ tance to be labeled, in suitable solvent, and irradiating with u.v. light for 5-20 minutes at RT to induce crosslinking.
  • said sulfhydryl derivatized CD labels can be derivatized with a suitable bifunctional coupling agent that will couple to sulfhydryl groups at one end and provide a photoactive group (eg. phenyl azide), at the other end.
  • a suitable bifunctional coupling agent that will couple to sulfhydryl groups at one end and provide a photoactive group (eg. phenyl azide), at the other end.
  • a photoactive group eg. phenyl azide
  • CD Labels with Different Colored Guests A major advantage cf the CD labels of this invention is the ease with which dif ⁇ ferent guest molecules can be comolexed with said CD labels to produce easily distinguishable labels.
  • the distinguishing feature is the different light emission wavelengths (or peak emission, or "color”), obtained from different guest molecules when they are activated or electronically excited.
  • Said emission can be generated by electronically exciting the guest mol ⁇ ecule through various means such as by chemical reaction (eg. energy trans ⁇ fer), by an incident light source (eg. fluorescence and phosphorescence), electrically (eg. electroluminescence), or heat (eg. thermolurmnescence.. Also, through the choice of appropriate CL substance as a guest, various colors of chemiluminescent emission are possible.
  • chemical reaction eg. energy trans ⁇ fer
  • an incident light source eg. fluorescence and phosphorescence
  • electrically eg. electroluminescence
  • heat eg. thermolurmnescence.
  • various colors of chemiluminescent emission are possible.
  • said guest molecules are efficient emitters that form high affinity inclusion complexes with said CD label host, generally through a wide choice of fluorophores with a suitable shape and size compatible with the CD molecules used. It is possible to produce CD labels with guests that have a diversity of size and shape and color, yet are contained in tne same sized CD host molecule. In applications where it is desirable to have several different colored guests that also have similar chemical and physi ⁇ cal characteristics, the choice of guests can be derived from the same chem ⁇ ically related group.
  • any scintillator or fluorophore described previously or a substance containing any aromatic nucleus including acridines, anthracenes, benzenes, biphenyls, biphenylenes, fluorenes, fluoresceins, naphthacenes, naphthalenes, pentacenes, pentalenes, perylenes, phenanthrenes, among others, can have its emission wavelength altered or "shifted" by coupling or derivatizing with one or more specific groups.
  • Said wavelength-altering group can be any suitable substance including hydrogens, oxygens, nitrogens, sulfurs, halogens, metals, methyls, ethyls, toluyls, and any suitable func ⁇ tional group, among others.
  • Suitable guest molecules are; fluorescein dyes such as 9-(carboxyethyl)-3-hydroxy-6-oxo-6H-xanthenes (eg. SF-505, SF-51 , SF-519, and SF-526, of Prober, supra), acridines, anthracenes, naphthalenes, and any suitable fluorophores previously described, among others.
  • fluorescein dyes such as 9-(carboxyethyl)-3-hydroxy-6-oxo-6H-xanthenes (eg. SF-505, SF-51 , SF-519, and SF-526, of Prober, supra), acridines, anthracenes, naphthalenes, and any suitable fluorophores previously described, among others.
  • ⁇ captured guest is any (one or more), guest molecules or inclusion compounds that are captured by, or coupled in close proximity tc, the CD host so that tney can ⁇ not separate by the normal processes of diffusion. Said capturing is accom ⁇ plished through physical entrapment by the CD host, or by covalent coupling of the guest in the immediate vicinity of the CD host.
  • one type cf physical entrapment is done by capping both ends cf the host CD, thereby entrapping the guest molecule.
  • Another useful method is to couple or polymerize two or more host CD's together so that said guest molecule is entrapped between them (eg. "duplex cyclodextrin" of Tabushi, supra), and wherein the outside ends are too small and/or are capped to prevent escape of the guest.
  • Said guest is captured by covalent coupling when the guest is coupled by- various suitable means to the CD host or to an intermediate substance in close proximity.
  • said coupling is done after the guest enters the host CD.
  • said guest can be coupled through a suitable spacer of suffic ⁇ ient length (eg. 6 or more carbons), to allow the guest to enter the host CD after coupling (eg. Ueno, supra).
  • guest molecule to use will depend on compatibility with its intended use, such as the color desired for said label, fluorescent and/or excitation efficiency, energy transfer efficiency, or in the case of CD cat ⁇ alysts, specificity or affinity with the desired substrate to provide a suitable recognition site. Also, several guest molecules, either identical or different, can be included. Examples include fluorophores, scintil ⁇ lators, CL compounds or suitable hydroxylated compounds described herein.
  • Tne desired guest mol ⁇ ecule can be converted to a carbonyl chloride derivative by treatment cf an available carboxylic acid with thionyl chloride, which can subsequently be coupled through the appropriate spacer to the host CD.
  • suitable guest molecules are any suitable aliphatic, aromatic or hetero- cyclic inclusion compounds including; carboxylic fluorescein dyes such as 9- (carboxyethyl)-3-hydroxy-6-oxo-6H-xanthenes (eg.
  • chlor ⁇ ides dinitro-naphthalenes, toluidinylnaphthalene-sulfonates, 10-methyl- anthracene-9-carboxaldehydes, 10-chloro-9-anthracenemethanols, chloro- diphenylanthracenes, 9-fluorenylmethyl chlorcformate, fluorenone-4-carbonyl chloride, 1-fluorenecarboxy ⁇ c acid, dinitrofluorenes, pyrenebutyric acids, rhodamines, folic acids, among others.
  • the desired guest molecule can be coupled through an available amino group, such as with acridine yellow, acriflavines, aminoanthracenes, 9- (methy!aminomethyl)anthracenes, aminofluoranthenes, amincfluorenes. amino- fluorenones, aminopyrenes, and others, using appropriate bifunctional coupl ⁇ ing agents, or a carbodiimide method.
  • the desired guest molecule can alsc be coupled through an available sulfhydryl (eg.
  • sulfonates 8-anili-1-naphtha!ene sulfonates (ANS), 2-p-toluidinyl ⁇ naphthalene sulfonates (TNS), among others.
  • ANS 8-anili-1-naphtha!ene sulfonates
  • TMS 2-p-toluidinyl ⁇ naphthalene sulfonates
  • a variety of previously descr ⁇ ibed coupling agents can be used for coupling through an available carboxy!, amino or sulfhydryl on the host CD.
  • said carboxylic acid or amino group can be introduced onto the guest through suitable derivatiza ⁇ tion, as with europium chelates and ruthenium chelates (eg. dipyridines of Ege, supra) .
  • said host CD can be suitably capped if desired, to increase the affinity between the coupled guest and CD host.
  • a ino-CD's are prepared wherein a suitable amino group is preferably coupled to one end.
  • a suitable amino group is preferably coupled to one end.
  • CD hydroxyls are protected with benzoate esters, the primary end is selectively deprotected and amino derivatized (eg. Szejtli or Boyer, supra).
  • a suitable amino-derivatized solid support is prepared.
  • porous glass beads or predried silica gel is amino-derivatized with (3-aminopropyl)trimethoxysilane.
  • suitable materials such as those used in chromatography (eg. Smokova-Keulemansova, supra), can be used for a solid support.
  • Said solid support can be in the form of particles, beads, fibers, plates, and tubing walls, and composed of styrenes, acryla ides, silica gels, solid or porous glass, metals, dextran ⁇ , and celluloses, among others that are suitably derivatized as needed and com ⁇ patible with the reactions used.
  • a suitable intermediate compound such as DTBP, DSP, DTSSP, EADB, SPDP, and photoactive couplers like DTBPA, SADP, SAND, and SASD.
  • a cleavable disulfide coupling agent such as DTBP, DSP, DTSSP, EADB, SPDP, and photoactive couplers like DTBPA, SADP, SAND, and SASD.
  • Other suitable agents are periodate cleavable, such as DST and sulfo- DST, and hydroxylamine cleavable at the ethyl ester linkage, such as EGS and sulfo-EGS.
  • Said intermediate substance is then treated for additional coupling, with a suitable, noncleavable bifunctional coupling agent previously descr ⁇ ibed.
  • a suitable, noncleavable bifunctional coupling agent previously descr ⁇ ibed.
  • amino compounds such as proteins, polya ino acids,
  • One or more of said Amino-CD's is coupled to said intermediate on the support through the available CD amino group. Remaining hydroxyl groups or, the resulting immobilized CD's are deprotected as needed and coupled to a suitable spacer for subsequent coupling to the ⁇ esired inclusion compound or fluorophore.
  • said hydroxyls can be oxidized to dialdehydes, or treated with epichlorohydrin, and coupled to diaminohexan ⁇ . Or, treated witn acetic or succinic anhydride to give carboxylates that are converted to NHS esters through reaction with carbodiimides and N-hydroxysuccinimide, and then coupled to diaminohexane.
  • said immobilized CD's are prepared with one or more functional groups available (eg. aminos), attacned through suitable spacers, to the CD's.
  • Said immobilized CD's can now be coupled to a variety of inclusion compounds and fluorophores, including those described herein (or CL compounds), using any suitable coupling agent for coupling to amino or other groups.
  • suitable coupling agent for coupling to amino or other groups.
  • the following examples are illustrative. a. Amino Fluorophores.
  • Coupling to fluorophores with available amine groups such as acid blacks, acridine yellow, acriflavine, 2-aminoanthra- cene, 6-aminochrysene, 1-amino-4-chloro-naphthalene, 3-aminofluoranthene, 2- aminofluorene, 2-amino-9-fluorenone, 2-amino-3-chloro-7-nitro-9-fluorenone, 1-aminopyrene, bismark browns, N,N"-bis(3-aminophenyl)-3,4,9,10-perylene- teracarboxylic diimide, ethidium bromide, fluoresceinamine, reactive blues, phycoerythrins, phycocyanins, among others, is done by combining said immo ⁇ bilized CD with any suitable bifunctional, amino-coup!ing agent previously described, such as DMA, DMS, DSS, among others, and said fluorophor
  • Carboxylic Acid Fluorophores Coupling to fluorophores with avail ⁇ able carboxylic acid groups, such as carboxylic fluorescein dyes such as SF- 505, SF-512, SF-519, and SF-526, (see Prober, supra), 9-acridinecarboxylic acid, 1-fluorenecarboxylic acid, indoleacetic acid, IO-methylanthracene-9- carboxaldehydes, mordant oranges, mordant reds, mordant yellows, naphthyl- acetic acids, N-(4-nitrobenzoyl)-6-aminocaproic acid, phenolphthalein car- binol disulfate, protoporphyrins, 1-pyrenebutyric acid, quinolinecarooxylic acid, retinoic acid, rhodamine B's, among others, is done by converting an available carboxy!ate on said fluorophore to an NHS ester through
  • fluorophores with reducible sulfonates are 8-anilo- "1 - naphthalene sulfonate, 2-p-toluidinylnaphthalene- ⁇ -sulfonate, acid blacks, acid blues, acid greens, acid reds, acid violets, acid yellows, alizarins, direct blues, direct reds, texas reds, and others.
  • fluorophores with reducible sulfonates are 8-anilo- "1 - naphthalene sulfonate, 2-p-toluidinylnaphthalene- ⁇ -sulfonate, acid blacks, acid blues, acid greens, acid reds, acid violets, acid yellows, alizarins, direct blues, direct reds, texas reds, and others.
  • coupling to said immobilized CD is done by combining said immobilized CD with any suitable heterobifunctiona!
  • Coupling of CD's to fluorophores can also be done by combining in the dark, said immobilized CD with any suitable hetero- bifunctional, photoactive amino-coupling agent previously described, such as HSAB, NHS-ASA, among others, under suitable conditions for coupling. Then, said fluorophore is added and the photoactive group is activated with a suitable light source to initiate coupling.
  • any suitable hetero- bifunctional, photoactive amino-coupling agent previously described, such as HSAB, NHS-ASA, among others
  • tne cleavable disulfide is treated with a suitable reducing agent such as dithiothreitol , among others, providing the free label with a functional group for coupling to any suitable substance.
  • a suitable reducing agent such as dithiothreitol
  • CD Labels with Antenna Substances It has beer: dis ⁇ covered that a new CD label composition with potentially greater luminescent or catalytic efficiency can be synthesized by coupling certain light and/or energy collecting substances (herein called "antenna" substances), to sai CD l abel s .
  • Said antenna substances can be coupled to said label in various ways to promote the most efficient chemiluminescent, fluorescent, or catalytic activity.
  • said antennas can be suitably coupled to the CD molecule, to the fluorophore guest, or to an intermediate substance that is part of the CD label.
  • Certain photosynthetic antenna substances eg. chlorophylls, pigments
  • Suitable antenna substances are any aliphatic, aromatic or hetsrocyclic compounds that are capable of collecting light energy or photons. Examples include fo ⁇ c acids, carotenoids, retinols, retinals, rhodopsins, viologens, chlorophylls, bacteriochlorophylls, phycobiliproteins, phycoerythrins, phycocyanins, open chain tetrapyrroles (bilins), blue fluorescent proteins (eg. from luminescent bacteria), green fluorescent proteins (eg. from renilla, etc.), tryptophan and/or tyrosine-containing substances (eg. poly ⁇ peptides), fluorophores, scintillators, and various derivatives, analogs and precursors of said antenna substances.
  • n a multi ⁇ ple of 1 or more, the general structure is:
  • CD Catalyst Labels A CD catalyst lace! comprises a CD label with one or more catalytic groups coupled to it, and includes a suit ⁇ able functional group or coupling group for coupling. Said labe! can also be in the form of said multiple CD labe!, or duplex CD, and is prepared using any suitable methods described herein.
  • a suitable scheme for syn ⁇ thesizing CD catalyst labels of this invention is as follows;
  • an Amino-CD or group of CD's, is prepared wherein a suitable amino group is added and protected (eg. with halogenatec alkyl- phalimide), at one end.
  • a suitable amino group is added and protected (eg. with halogenatec alkyl- phalimide), at one end.
  • Remaining hydroxyl groups on the CD are deprotected as n ⁇ dec and derivatized (or coupled to a suitable spacer) and subsequently coupled to the desired catalytic groups and capture guests as desired.
  • Said protected amino group is suitably deprotected and then coupled to the substance to be labeled or any suitable coupling group (eg. bifunc ⁇ tional coupling agent), can be added, such as NHS, maleimido, or photoactive coupler by known methods.
  • any suitable coupling group eg. bifunc ⁇ tional coupling agent
  • a protected (eg. ester), carboxylate group can be added in place of said protected amino.
  • said carboxylic group is deprotection (eg. hydrolysis), and is derivatized to a coupling group (eg. NHS, or maleimide, etc.), using known methods.
  • a suitably protected amino CD, or group of CD's is coupled to any suitable support through a cleavable coupling agent, as described herein.
  • said CD is cleaved from the support, leaving a functional group (eg. sulfhydryl), that can be coupled directly to the desired substance, or derivatized to produce a coupling group for subsequent coupling, as described herein.
  • a functional group eg. sulfhydryl
  • any suitable intermediate substance can be included in said CD catalyst labels wherein a plurality of catalyst CD's are coupled to said intermediate which includes a suitable coupling group for subsequent coupling to any desired substance.
  • CD Activator Labels A suitable scheme fcr synthesizing CD activator labels is as follows;
  • any suitable CD label described herein is prepared wherein a suitable amino group has been added and protected (eg. with halogenated alkylphalimide), at one end.
  • Said amino group is suitably deprotected and coupled directly (or coupled through a suitable spacer), to a suitable activator substance through a suitable coupling agent, previously described.
  • any suitable CD label described herein car be coupled through a coupling group already on said label.
  • the N-succinimidyl coupling group on any NHS-CD label described herein will react with primary and secondary aliphatic amines.
  • Any suitable enzymes including glucose oxidases, peroxidases, or alkaline phosphatases, are labeled by mixing 50 icrograms of protein in 0.2 ml of 0.1 M phosphate buffer (PB), pH 8, with approximately 10 microliters of acetonitrile cont ⁇ aining 2.5 micrograms of dissolved NHS-CD label, in a glass vial for 15 minutes.
  • PB phosphate buffer
  • the mixture is applied to a column of G25 equilibrated with PBS composed of PB, pH 6.3, containing 0.15 M NaCl and 0.02% sodium azide.
  • the column is eluted with PBS and labeled fractions in the void volume are pooled and concentrated if necessary, by vacuum dialysis.
  • the reaction conditions may be varied appropriately, such as including spacers, to obtain an enzyme that is suitably coupled to a plurality cf NHS- CD labels and sufficiently retains the desired enzymatic activity.
  • Such conditions that may be appropriately varied are, amounts of reagents, times, temperature and the use of other compatible buffers, solvents and additives.
  • said NHS-CD label can be substituted for a label with a different coupling group or functional group.
  • conditions can be appropriately modified by one skilled in the art.
  • CD's are capped with triamine compounds (eg. basic fuchsins, etc., through spacers), one cf the amino' ⁇ can be left available for subsequent coupling to a bifunctional agent to introduce any desired coupling group. Also, when CD's are capped with reagents that produce ketones, the available ketone is subsequently reacted with an amino-carboxylic acid or diamino com ⁇ pound to produce the corresponding oxime coupling.
  • triamine compounds eg. basic fuchsins, etc., through spacers
  • one cf the amino' ⁇ can be left available for subsequent coupling to a bifunctional agent to introduce any desired coupling group.
  • the available ketone is subsequently reacted with an amino-carboxylic acid or diamino com ⁇ pound to produce the corresponding oxime coupling.
  • tc introduce a carboxylic acid group that can be coupled through a mixed anhydride reaction (eg. Erlanger, supra), or derivatized to give an NHS coupling group.
  • a mixed anhydride reaction eg. Erlanger, supra
  • a multiple CD labe is defined herein as a CD label comprising a plurality of any CD's.
  • Said [CD]n labels can include fluorophore CD labels, chemiluminescent CD labels, catalyst CD labels, activator CD labels, as well as labels with captured guests, and various derivatives and/or capping substances, and antenna substances, as described previously.
  • Said [CD]n labels overcome the problem of labeling a substance with a plurality of CD molecules that are greater than the number of coupling sites available. These labels allow the covalent coupling of a plurality of CD molecules to proteins such as antibody, avidin or streptavidin, or ligands, antigens, or nucleic acids, and other substances, such as magnetic particles, through the appropriate functional groups.
  • One composition comprises two or more CD labels coupled to an apprcpriate intermediate compound so that at least one coupling group is left available for subsequent coupling to the substance to be labeled.
  • Said coupling group is coupled directly to the substance to be labeled or, an appropriate func ⁇ tional group on the- label is first derivatized with any suitable coupling agent such as succinimidyl, maleimidyl, imidoester, aldehyde, or photoactive agents including nitrenes, among others previously described.
  • Said intermediate compound can b ⁇ any of the intermediate coupling s o- stances previously described including said hydroxylated compounds, carbohy ⁇ drates, sulfur containing and amino containing compounds.
  • compositions comprises a grouping of CD molecules coupled tc eacr. other through various known m ⁇ ans to form a dimer, trimer or polymer cf CD molecules.
  • Said grouping of CD's also includes suitable functional groups ' for derivatizing and/or coupling to (labeling), the desired substance.
  • said CD grouping can be coupled to an intermediate substance that includes a suitable functional group or coupling agent to facilitate said labeling.
  • said functional group or agent is appro ⁇ priately protected, if necessary, during coupling of said CD grouping to said intermediate compound. Said functional group is then de-protectec for coupling and/or derivatization.
  • A. A Multiple CD* Label Coupled Through Hydroxylated Compounds.
  • Hydroxylated compounds such as oligosacharrides, celluloses, dextrans, polysaccharides, amino sugars, glu- cosa ines, galactosamines, polysorbates, hyaluronic acids, heparins, polyene antibiotics, polyvinyl alcohols (eg. Szejtli, supra), oligonucleotides, proteins, polypeptides, and polyaminoacids, among others, are suitable wherein a carboxylic acid group is available.
  • carboxylic acid group can be added by known methods. Hydroxylated carboxylic acids such as cholic acid, gallic acid, digallic acid, citrazinic acid, fluores- cin, or polytyrosine, including their various derivatives, can also be used.
  • the desired carboxylic acid group of said hydroxylated com ⁇ pound is suitably protected by known methods, leaving one or more hydroxyls available for coupling to CD's.
  • one or more carboxylic acid groups can be reacted with a suitable alcohol to form a protective ester.
  • a suitable alcohol for example, a benzyl alcohol is used to produce a hydroxylated benzyl ester.
  • Cyclodextrin which can be suitably complexed, derivatized and/or capped before coupling, is combined with said hydroxylated protected ester and a suitable coupling agent.
  • coupling is done with epichlorohydrin, or an epoxy compound previously described, in suitable solvent, and mixed under suitable conditions until coupling is completed between a plurality of said CD and the available hydroxyls on the protected ester.
  • the product a plurality of CD molecules coupled to said hydroxylated ester (ester-[CD]n), is appropriately treated if necessary to protect any remaining uncoupled hydroxyl groups on the CD molecules.
  • Said ester-[CD]n is suitably hydrolyzed (eg. hydrogen bromide in acetic acid), to cleave the benzyl ester and produce multiple CD's coupled to a carboxylic acid (Acid-[CD]n).
  • Said Acid-[CD]n is combined with N-hydroxy ⁇ succinimide in anhydrous DMF, and mixed with N,N'-dicylohexylcarbodiimide for 2 hours to overnight at RT. Dicylohexyl urea is removed by precipita ⁇ tion after adding a few drops of glacial acetic acid.
  • the product, a plura ⁇ lity of CD molecules coupled to an N-hydroxysuccinimidyl derivative (NHS- [CD]n) is collected by evaporation.
  • hydroxylated compounds may be used, including any appropriate polyamino adds and their derivatives and analogs with available hydroxyls, including polytyrosines, polytyrosine-lysines, polytyrosine-lysine-cysteins, polytryp- tophans; and various hydroxylated polymers and their combinations, derivat ⁇ ives and analogs.
  • Aromatic Hydroxylated Compounds also, aromatic hydroxylated com ⁇ pounds can be used. If necessary, a carboxylic acid group can be added and protected as needed, by known methods. Examples are; various flavone deriv- atives and analogs including dihydroxyflavones (chrysins), trihydroxyflav- ones (apigenins), pentahydroxyflavones ( orins), hexahydroxyflavones ( yri- cetins), flavyliums, quercetins, fisetins; various antibiotics including teramycins, tetracyclines, chlorotetracyclines, clomocyclines, guamecy- clines, a photericins, filipins, fungichro ins; various cevine derivatives and analogs including verines, desatrines, veratridine; various sulfur and mercapto derivatives and analogs including dihydroxy-2-mercaptopyrimidine
  • CD labels Immobilized on a Solid Support. It has been dis ⁇ covered that a [CD]n label can be synthesized with a more predictable number of CD molecules per label, giving new advantages of uniform structure and chemical properties. These CD labels also are suitable for coupling a plur ⁇ ality of CD molecules to a substance in a single step. The synthesis approach is to immobilize the initial CD molecule to a solid support, and then attach additional CD molecules to the first in a controlled, step-wise manner. After sufficient CD's have been linked together, the entire group is cleaved from the solid support for use as a [CD]n label.
  • the CD molecules used in this procedure can include captured guests and antenna substances, and be suitably derivatized and/or capped before coupl ⁇ ing to incorporate other desirable features.
  • each CD molecule (or dimer, or trimer), that is coupled has a well defined structure to facilitate the production of CD labels with uniform and con ⁇ sistent properties.
  • Said solid support can be in the form of particles, beads, fibers, plates, and tubing walls, and composed of styrenes, acrylamides, silica gels, solid or porous glass, metals, dextrans, and celluloses, among others that are suit ⁇ ably derivatized as needed and compatible with the reactions used.
  • the coupling agent used to couple the initial CD to the support is preferably one that is readily cleaved when desired, and the coupling agent used to couple subsequent CD's is preferably noncleavable.
  • the initial coupling agent is also a bifunctional reagent such as those with a cleavable disulfide group, including DTBP, DSP, DTSSP, EADB, SPDP, and pho ⁇ toactive couplers like DTBPA, SADP, SAND, and SASD.
  • Other suitable agents are periodate cleavable, such as DST and sulfo-DST, and hydroxylamine cleav ⁇ able at the ethyl ester linkage, such as EGS and sulfo-EGS.
  • the label is cleaved from the support after synthesis, leaving a suitable functional group for subsequent reactions.
  • the remaining functional group is derivatized to an NHS ester coupling group (to give an NHS-[CD]n) by various known means.
  • the func ⁇ tional group is derivatized to a photoactive coupling group (to give Photo- [CD]n).
  • the derivatizing coupling agents used can be a variety reagents previously described.
  • D. Multiple CD Label Containing a CD Grouping In this example, a highly porous support, (eg.
  • porous glass beads or predried silica gel 5-6 gm
  • a suitable spacer is coupled to said amino-derivatized support through a suitable cleavable disulfide coupling agent such as DSP.
  • a suitable cleavable disulfide coupling agent such as DSP.
  • a dia ⁇ mino substance such as 1,12-diaminododecane is used.
  • the solvents used can be anhydrous such as methanol, methylene chloride, or pyridine or they can be suitable aqueous buffer solutions, as conditions require.
  • Said 1,12-diaminododecane-coupled support is then combined with excess DSS to allow coupling of the DSS to the coupled 1,12-diaminododecane.
  • the excess DSS is removed, giving DSS-activated 1,12-diaminododecane coupled to the support.
  • a CD or CD group ⁇ ing eg. dimer, or polymerized CD
  • amino derivatized CD is a mono-amino preparation with a suitable spacer, wherein all of the CD molecules have the amino group on the same (eg. primary), side. Said CD molecules are allowed to couple and the excess is removed, leaving CD's coupled to the 1,2-diaminododecane through the DSS coupling agent.
  • Said coupled CD is then suitably coupled to additional CD's to form a grouping of CD's.
  • said CD is suitably oxidized (eg. sodium periodate or oxidase enzyme), to produce dialdehydes on the coupled CD (Dial-CD).
  • Dial-CD is then added excess, amino-derivatized CD so that coupling occurs between the dialdehyde of the previously coupled CD and the amino group of said amino-derivatized CD's added, and excess CD is removed.
  • said grouping of CD's can be synthesized by coupling addi ⁇ tional CD's using known crosslinking agents, including epichlorohydrin, or an epoxy compound previously described.
  • the dialdehyde or other coupling procedure of this step is repeated until the desired number of CD's have been added to form a grouping of CD's coupled to said spacer.
  • the [CD]n composition is recovered from the support by cleaving the initial coupling agent used.
  • the cleavable disulfide is treated with a suitable reducing agent such as dithiothreitol , among others.
  • the Schiff bases are suitably reduced (eg. NaBH 4 ).
  • the released CD label will have a sulfhydryl group available for subsequent coupling to any suitable substance.
  • said sulfhydryl is derivatized to an NHS ester, such as by coupling it to a heterobifunctional reagent (eg. MBS, SMCC or SMPB), with a maleimide and an NHS group at opposite ends.
  • a heterobifunctional reagent eg. MBS, SMCC or SMPB
  • said sulfhydryl is deriv ⁇ atized in the dark with APB or APTP to produce a photoreactive coupling group (eg. phenyl azide).
  • n a multiple of 1 or more
  • the general structure is: lCDJn-SPACER-NHS or JCD!n-SPACER-(phenyl azide)
  • any CD's, or CD labels are coupled to the same intermediate com ⁇ pound.
  • the CD's used in this procedure can include captured guests and antenna substances, and be suitably derivatized and/or capped before coupl ⁇ ing to incorporate other desirable features.
  • suitable derivatization a wide variety of substances are suitable as intermediates, such as thio- lated hydroxyl compounds including carbohydrates.
  • CHAPS is reduced with dithiothreitol to convert the sul- fonate group to a sulfhydryl (eg. Carlsson, supra), to produce 3-[(3-chol- amidopropyl)-dimethylammonio]-1-mercaptopropane (hereinafter CHAMP).
  • a suitable amino-derivatized support is prepared as described above. Said amino groups are then suitably thiolated to provide sulfhydryls (or 2- pyridyl disulphides using SPDP). Said CHAMP is coupled through its sulfhy ⁇ dryl to the available thiol or 2-pyridyl disulphide to form a cleavable dis ⁇ ulfide linkage to the support, and produce a CHAMP coupled support.
  • Said CHAMP coupled support is treated with a suitable noncleavable, hydroxyl crosslinking agent such as epichlorohydrin, among others, to give an epoxy-activated CHAMP, and exposed to an excess of CD molecules, that have been suitably derivatized as needed for coupling.
  • a suitable noncleavable, hydroxyl crosslinking agent such as epichlorohydrin, among others.
  • the product is 1, 2, or 3 CD molecules coupled to said CHAMP through the hydroxyl groups.
  • Said product is suitably cleaved and recovered from the support by treating with a suitable reducing agent such as dithiothreitol, among others.
  • a suitable reducing agent such as dithiothreitol, among others.
  • the released CD label will have a sulfhydryl group available for subsequent coupling to any suitable substance.
  • said sulfhydryl is converted to an NHS ester by coupling it to a heterobifunctional reagent with a maleimide and an NHS group at opposite ends.
  • the CD label can be further modified wherein specific derivatization and/or capping reactions are per ⁇ formed while the disulfide group is protected.
  • the desired guest mol ⁇ ecules and antenna substances can be included before or after coupling of the CD's.
  • cleavable groups eg. Ji, supra
  • other useful functional groups can be incorporated into the label that remain protected until cleaved. For instance, initial coupling through a cleavable ester will produce a carboxylic acid or hydroxyl group on the CD label after cleavage.
  • a variety of protecting and deprotecting schemes can be adapted to serve as temporary coupling sites on a solid support for synthesis of said CD labels. The major requirement is that subsequent reactions for coupling CD's do not cleave the label before synthesis is completed.
  • a suitable amino-derivatized support is prepared as descr ⁇ ibed above. Said amino groups are then suitably coupled to a dianhyride such as 3,4,9,10-perylenetetracarboxylic dianhydride to form an imide. Then, a suitably protected Amino-CD is coupled to the other end of the immo ⁇ bilized dianhydride. After appropriate synthesis of a multiple CD as before, the label is cleaved by treatment with hydrazine, leaving an amino group on the label. Suitably, this procedure can also be done on a hydroxy ⁇ lated support material.
  • PREPARATION XIV Multiple CD Molecules Coupled to Carbohydrates
  • These label compositions allow labeling a substance with a plurality of CD's that are greater than the number of coupling sites available.
  • the use of a car ⁇ bohydrate provides new properties for coupling a plurality of CD's to a variety of other substances such as lectins, cell receptors, nucleic acids, proteins such as antibody, avidin or streptavidin, and other substances.
  • a useful label is produced by using one or more carbohydrates or sac ⁇ charides as part of the label.
  • Said carbohydrate is any carbohydrate cont- aining substance, including oligosaccharides, mono-, di- and polysacchar- ides, amino sugars, sulfo-sugars, deoxysugars, glycosides, lectin-binding carbohydrates, aldoses, ketoses, pentoses, arabinoses, riboses, xyloses, hexoses, glucoses, fructoses, galactoses, mannoses, sorboses, glucosamines, sucroses, lactoses, maltoses, raffinoses, soluble starches, amylopectins, pectins, agars, agaroses, dextrans, celluloses, hyaluronic acids, heparins, nucleosides, nucleotides, glycoproteins, and any suitable polymers, deriva
  • One approach is to couple two or more CD molecules to said carbohydrate either directly or through an intermediate coupling agent.
  • the resulting label can then be bound noncovalently to the appropriate lectin or receptor that binds the carbohydrate on the label.
  • the approach is to couple two or more CD molecules to said carbohydrate either directly or through an intermediate so that at least one functional group is left available on the label.
  • Said functional group is then coupled to the substance to be labeled or, derivatized with any suitable coupling agent such as succinimidyl, maleimidyl, imidoester, aldehyde, or photoactive agents including nitrenes, among others previously described.
  • any suitable coupling agent such as succinimidyl, maleimidyl, imidoester, aldehyde, or photoactive agents including nitrenes, among others previously described.
  • said functional group is appro ⁇ priately protected, if necessary, during coupling.
  • Said functional group is then de-protected for coupling and/or derivatization.
  • Another method for introducing said functional groups into said carbohy ⁇ drate is to derivatized a small number of the existing hydroxyl groups with a bifunctional coupling agent that will not react significantly during coupling of one or more CD's. A suitable number of hydroxyl groups are left for coupling said carbohydrate to said CD.
  • various amino groups that may be present on said carbohydrate can be derivatized using known methods to produce an appropriate protecting group such as a benzyl ester.
  • said carbohydrate can have one or more functional groups preferentially derivatized.
  • pairs of hydroxyls are reacted with appropriate aldehydes or ketones to pro ⁇ prise protective derivatives such as acetals (eg. isopropy!1denes), which can be subsequently hydrolyzed.
  • pairs of vicinal hydroxyls are oxidized to produce aldehyde groups. Said oxidation suitably is done with sodium periodate or with oxidizing enzymes using known methods, so that sufficient hydroxyls are left un-oxidized for coupling with CD molecules.
  • NHS-TCDln Labeled Protein the N- succinimidyl coupling group on NHS-[CD]n, previously described, reacts with primary and secondary aliphatic amines on the protein.
  • Any suitable pro ⁇ tein, including antibody, antigen, or avidin (or streptavidin) is labeled by mixing 50 micrograms of protein in 0.2 ml of 0.1 M phosphate buffer (PB), pH 8, with approximately 10 microliters of acetonitrile containing 2.5 micro- grams of dissolved NHS-[CD]n, in a vial for 15 minutes.
  • PB phosphate buffer
  • the mixture is applied to a column of G25 equilibrated with PBS composed of PB, pH 6.3, containing 0.15 M NaCl and 0.02% sodium azide.
  • the column is eluted with PBS and labeled fractions in the void volume are pooled and concentrated if necessary, by vacuum dialysis.
  • the reaction conditions may be varied appropriately, such as the inclu ⁇ sion of an intermediate coupling substance, to obtain a labeled protein or nucleic acid that is suitably coupled to a plurality of NHS-[CD]n and suffi ⁇ ciently retains the binding properties needed for use as a tracer.
  • Such conditions that may be appropriately varied are, amounts of reagents, times, temperature and the use of other compatible buffers, solvents and additives.
  • said NHS-[CD]n is substituted for one of said labels from previous preparations wherein said label contains an N-hydroxysuccinimidy! coupling group.
  • said NHS-[CD]n can be substituted for one of said labels from previous preparations wherein said label contains a coupling group or functional group that is not an N-hydroxysuccinimidyl coupling group.
  • n a multiple of 1 or more, the general structure is:
  • maleimido-CD Labeled Protein In this example, aleimido-CD (Mal-CD), is used as a label by covalently coupling it to a protein such as antibody, avidin or streptavidin.
  • the protein to be labeled is modified with the thiolating agent S-acetylmercaptosuccinic anhydride (SAMSA) to provide accessible sulfhydryl functional groups for coupling with maleimide residues on said Mal-CD.
  • SAMSA S-acetylmercaptosuccinic anhydride
  • the mixture is reacted long enough to incorporate an average of one or more thiol groups per protein molecule without excessive inactivation, suitably 40 minutes at room temper ⁇ ature.
  • the mixture is applied to a 1 X 25 cm column of Sephadex G25 and eluted with PB at 4 * C.
  • the derivatized protein and Mal-CD are combined in PB and allowed to couple for one to several hours, and CD labeled protein is purified using Sephadex as above.
  • any desired CD label described previously can be noncovalently coupled to a biotinylated protein such as antibody.
  • a biotinylated protein such as antibody.
  • any CD label that is first coupled to avidin or streptavidin can be noncovalently coupled to the biotinylated protein by allowing the coupled avidin or strep ⁇ tavidin to bind to the biotin on the protein.
  • Another suitable scheme for noncovalent coupling is to produce a "second" antibody that is specific for a "first" (antigenic), ant ⁇ ibody, and will bind to it. Then, a CD label that is suitably coupled to said second antibody, can be noncovalently coupled to the appropriate first antibody when the antibodies are allowed to bind. This way, any CD label that is coupled to an antibody specific for another antibody, can be non ⁇ covalently coupled to an antibody of a different specificity.
  • antibody binding proteins such as protein A or protein G, among others, can be suitably coupled to any CD label. Said CD labeled proteins can then be readily coupled to the appropriate antibodies that have recep ⁇ tors for said proteins.
  • anti-CD antibodies that are specific for CD, or a CD derivative, or a CD label.
  • said antibody is pro ⁇ substituted by immunizing any animal (eg. a mouse), with a polymerized form of CD molecules with adjuvant as needed.
  • Said anti-CD antibodies are combined with the appropriate CD label (eg. fluorophore, catalytic, etc.), to form soluble immune complexes herein called "CD anti-CD", or "CAC”, wherein a plurality of CD labels are bound in a single complex.
  • CD label eg. fluorophore, catalytic, etc.
  • a second antibody is produced in a different animal (eg. rabbit), that is specific for the antibodies used to make CAC.
  • said second antibody is anti-mouse IgG.
  • a third antibody specific for the analyte, is raised in a mouse (eg. monoclonal), which is allowed to bind to said analyte that is immobilized (eg. in a tissue sample or sandwich type assay).
  • said CAC can function as a noncovalent labeling complex, wherein said second antibody forms a binding bridge between said third antibody and said CAC.
  • different colored antibody tracers can be prepared by labeling with different colored CD fluorophore labels, previously described.
  • any of the CD labels or multi ⁇ ple CD labels, described herein, can be coupled to the desired ligand.
  • ligands with available amino groups are coupled to NHS-CD labels, or Dial-CD's, and those with available sulfhydryls are coupled to maleimido- CD labels or SH-CD's using known methods.
  • others can be readily der ⁇ ivatized as needed.
  • any steroids, or suitable drugs or other organic compounds of interest can be converted (eg. via acylation), to their corresponding succinate or oxime derivatives. Said derivatives are then readily coupled to Amino-CD's using a well known, mixed anhydride reac ⁇ tion with carbodiimide.
  • any suitable cyclic or heterocyclic compound eg. ligand
  • certain fluorophores, drugs, antibiotics, dyes, and nucleic acids can be suitably halogenated using appropriate protecting and deprotecting methods as needed.
  • Said halogenated compound is further derivatized for subsequent coupling to any CD derivative, CD label, or multiple CD label of this invention as follows:
  • THP tetrahydropyranyl acetal
  • Said coupling is done by combining said halogenated compound (2-3 gm), with said THP protected hydroxyalkyne (500-700 g), in 160 ml of deoxy- genated triethylamine, with addition of appropriate amounts (50-70 mg) of bis(triphenylphosphine)palladium chloride, herein called (Ph3P)2PdCl2, and Cul.
  • the mixture is stirred at 55 "C under N2 for 4 Hr or until coupling is completed.
  • a glass lined pressure bomb in a heated oil bath is used.
  • the product a THP-hydroxyalkyne compound
  • the product is collected as a yellow oil by evaporation, dissolved in chloroform, and washed with 5% disodium EDTA/H2O.
  • the product is recrystallized by redissolving in chloro ⁇ form, precipitating with methanol, collected by filtration and drying.
  • the THP protective group is removed to expose (deprotect), the alkyne hydroxyl for coupling to the labels of this invention.
  • Said THP on said THP-hydroxyalkyne compound (2-3 gm) is removed by acid catalysis in a solution of 30 ml of CH ⁇ Cl ⁇ /methanol/CFsCO ⁇ H (15:10:5), at 25 ⁇ C for 0.5-1 Hr, and recrystallized as above.
  • Said compound with added alkyne hydroxyl is suitably coupled directly to an Amino-CD of this invention through a suitable amino group provided as described herein.
  • the hydroxyl can be converted to a hemisuccinate using succinic anhydride as described by Steiner, A.L., et al, Proc. Nat. Acad. Sci. U.S.A. 64, 367-373 (1969), among others.
  • succinic anhydride as described by Steiner, A.L., et al, Proc. Nat. Acad. Sci. U.S.A. 64, 367-373 (1969), among others.
  • sebacoyl dichlorides can be used as disclosed by Bailey, J.M., et al, IN: "The Reticuloendothelial System and Atheros ⁇ clerosis", Diluzio, N.R.
  • a variety of coupling agents that will crosslink said hydroxyl with an Amino-CD group such as epoxys (eg. 1,4-butanediol diglycidyl ether, Vretblab, below), epi- chlorohydrin, sulfonyls, carbonyls, chlorocarbonates, anhydrides, or cyano ⁇ gen bromide, among others, can be used with appropriate modification to ensure that the labeled compounds are still functional.
  • epoxys eg. 1,4-butanediol diglycidyl ether, Vretblab, below
  • epi- chlorohydrin eg. 1,4-butanediol diglycidyl ether, Vretblab, below
  • epi- chlorohydrin eg. 1,4-butanediol diglycidyl ether, Vretblab, below
  • epi- chlorohydrin eg. 1,4-butanediol diglycidyl ether
  • said deprotected alkyne hydroxyl is suitably sulfonylated by reacting with an appropriate sul ⁇ fonylating reagent such as tosyl chloride.
  • an appropriate sul ⁇ fonylating reagent such as tosyl chloride.
  • 200-300 mg of said compound with deprotected alkyne hydroxyl is combined with 200-300 mg of p- toluenesulfonyl chloride in 20 ml of pyridine and stirred at 25 * C for 18 Hr, evaporated and recrystallized.
  • the product, tosylated-hydroxyalkyne compound can then be coupled to a maleimido-CD label of this invention.
  • Said tosylated- hydroxyalkyne compound can be further derivatized through well known methods to replace the tosylated hydroxyl with an amino group or a sulfhydryl group, among others, and subsequently coupled to a suitably derivatized CD or CD label of this invention.
  • said amino-derivatized compound is readily coupled to the N-hydroxysuccinimide derivatized labels described herein.
  • said sulfhydryl-derivatized compound is readily coupled to the maleimido-derivatized or sulfhydryl-derivatized labels described herein.
  • Said coupling can also be done through many well known coupling agents prev ⁇ iously described.
  • ligands and other compounds that already have available hydroxyls can be coupled without derivatization, or by using suitable protection and deprotection methods as needed.
  • n a multiple of 1 or more, the general structure is:
  • An antibody captor-activator (AbCA) is comprised of an antibody captor and an activator coupled in close proximity so that when a CD tracer is bound in the immediate vicinity, it can be activated to produce light.
  • Said antibody captor is any suitable antibody, including monoclonal antibodies, or anti ⁇ body fragments or derivatives, that binds specifically to the analyte of interest.
  • Said captor is preferably in "excess" so that all of the analyte in a sample can be bound to the captor.
  • Said captor and activator are insolubil- ized or immobilized by being coupled directly, or through an intermediate coupling substance, to each other, or in close proximity to each other on the same support.
  • said captor and activator are coupled to a suitable support or container such as a membrane, test tube, bag, icrotiter well, polymeric tubing, flow cell or cuvette.
  • Said support or container is composed of any suitable material such as celluloses, nitro- celluloses, nylon, polyvinylidene difluoride (PVDF), polystyrenes, polyeth- ylenes, polypropylenes, glass, plastics, resins, and other polymers, among others.
  • PVDF polyvinylidene difluoride
  • Said captor and activator can be coupled to the same support using many well known methods in the art of immobilizing antibodies and enzymes and taking the necessary precautions to ensure sufficient antibody and enz ⁇ yme activity.
  • Another useful composition comprises variations of said AbCA wherein said captor antibody of each AbCA has specificity for a different analyte or a different form of said analyte.
  • two or more said varieties of AbCA are associated with the same support or carrier.
  • said different AbCA's are suitably mixed or are located in separate zones on said support.
  • said captor and activator are coupled to the same colloidal substance.
  • Said colloidal substance is compo ⁇ sed of proteins, nucleic acids, celluloses, resins, latexes, carbohydrates, aminostyrenes, or any suitable polymeric substance.
  • said colloidal substance can be in the form of particles, beads, vesicles, liposomes, artificial cells, flakes, threads or filaments, including magnetic particles described herein.
  • said antibody captor is coupled to an activator (eg. glu ⁇ cose oxidase), directly, or through a suitable intermediate coupler such as protein.
  • Said coupled captor and activator can then be coupled to a col ⁇ loidal particle of dextran, acrylamide or latex, using known coupling meth ⁇ ods.
  • said captor and activator can be coupled directly to said colloidal particle so that they are in close proximity.
  • the test device comprises one or more varieties of an immo ⁇ bilized antibody captor-activator (AbCA, above), combined with an appro ⁇ priate carrier material as the support, and a supporting member.
  • Said sup ⁇ porting member will determine the actual configuration or shape of the test device and can be any suitable material that is compatible with the assay components.
  • Said supporting member can be rounded, flattened, tubular or hollow or any desired shape including the shape of a bead, ball, tape, stick, rod, sheet, wafer, disk, block, plate, tube or well.
  • said supporting member can be associated or combined with a photo- multiplier tube, the window end of a fiber optic device or any photodiode, biosensor or solid state, charge-coupled device capable of detecting elec ⁇ tromagnetic radiation signals such as luminescence, fluorescence, color changes and spectrophotometric signals.
  • said device can be inserted into, sandwiched between or otherwise closely positioned with a reading instrument composed of one, or a plurality of, photodiodes that sur ⁇ round the device.
  • the carrier can take a variety of useful forms such as porous fibers, filaments, sponges, foams, gels, fabrics, papers, meshes, matrices and the like for holding or entrapping said AbCA.
  • Said carrier can be comprised of various appropriate materials such as polymeric teflons, including polytetrafluoroethylene (PTFE), acrylamides, celluloses, polystyrenes, nylons and the like.
  • PTFE polytetrafluoroethylene
  • said carrier can be an appropriate adhesive or glue that binds said AbCA to said support member.
  • said device is configured like a "dip stick” or "test strip”.
  • said captor-activator is preferably in excess so that all of the analyte in a sample will be bound to the captor.
  • Said activator is an enzyme that will generate one or more substances needed to cause a lumin escent reaction when exposed to appropriate coenzymes and/or substrates.
  • the activator is glucose oxidase enzyme, which generates H2O2 in the presence of oxygen and glucose.
  • Said test device is prepared by combining said AbCA (one or more), with said carrier and supporting member, so that when the test device is exposed to a sample solution, the desired analytes and CD tracers in the sample will penetrate the carrier and selectively bind to the AbCA.
  • said CD tracer is antibody specific for said analyte, labeled with a plurality of a suitable fluorophore CD, described previously.
  • the carrier and supporting member must be of compatible materials so that no excessive interference occurs with the test.
  • the test device can be packaged and stored for later use either in appropriate stabilizing solution, or preferably, suit ⁇ ably dried before storage.
  • Appropriate stabilizers and other additives can be included to improve the storage life and performance of the reagents in the device.
  • Said stabilizers and other additives can include various proteins, BSA, polyvinyl alcohol, surfactants, buffers, thioredoxins, pre ⁇ servatives, and the like.
  • said dip stick device can be used for fluorescent detection using incident light for activation.
  • PREPARATION XXI Synthesis of an Immobilized Luminescer Test Device Said test device is prepared by combining a suitable captor such as captor antibody, with a plurality of CD label previously described, with an appro ⁇ priate carrier material and a supporting member.
  • a fluorophore CD label is coupled to said captor antibody or to an intermediate coupling substance, or directly to said carrier, in close proximity to the captor antibody.
  • Said supporting member will determine the actual configuration or shape of the test device and can be any suitable material that is compatible with the assay components.
  • Said supporting member can be rounded, flattened, tubular or hollow or any desired shape including the shape of a bead, ball, tape, stick, rod, sheet, wafer, disk, block, plate, tube or well.
  • said supporting member can be associated or combined with a photo- multiplier tube, the window end of a fiber optic device or any photodiode, biosensor or solid state, charge-coupled device capable of detecting elec ⁇ tromagnetic radiation signals such as luminescence, fluorescence, color changes and spectrophotometric signals.
  • said device can be inserted into, sandwiched between or otherwise closely positioned with a reading instrument composed of one, or a plurality of, photodiodes that sur ⁇ round the device.
  • said device is configured like a "dip stick” or "test strip”. Said device is exposed to a suitable solution containing analyte and to a solution with the appropriate activator labeled tracer (eg. glucose oxidase coupled to antibody specific for the analyte). Under suitable conditions, said analyte will bind to said captor antibody of the device. Said acti ⁇ vator coupled tracer will bind to captured analyte and then activate the fluorophore CD label of said device when appropriate substrate and peroxy- oxalate is available.
  • analyte eg. glucose oxidase coupled to antibody specific for the analyte
  • Said acti ⁇ vator coupled tracer will bind to captured analyte and then activate the fluorophore CD label of said device when appropriate substrate and peroxy- oxalate is available.
  • PREPARATION XXII Mercantile Kits Any of the preparations disclosed are readily incorporated into a mercantile test kit. Said test kit can con ⁇ tain one or more of the said preparations as needed, and other appropriate reagents and/or solutions for performing the intended assays, with suitable containers and instructions for storage and use. Said preparations and reagents can be packaged in various forms, including frozen and/or lyophil- ized.
  • a heterogeneous ligand binding assay is one that requires separation of the bound and free fractions before measurement.
  • a homogeneous assay has the advantage of not requiring a separation step before measuring the results.
  • buffer solutions and incubation conditions can be used to provide conditions conducive to specific binding between ligands, ligators, captors, and CD tracers.
  • Suitable buffer solutions eg. pH 6.5 - 8
  • CD derivatives, labels and tracers of this invention can be used in many unexpected ways, including substitution into any known tracer method. Therefore, the invention is not limited to the examples described herein, which are illustrative.
  • METHOD A Competitive Heterogeneous Assay Using a Fluorophore CD Label
  • a CD tracer prepared from a ligand (eg. antigen), that is identical to, or homologous to the analyte, with one or more CD fluorophore labels coupled to it.
  • the CD tracer will then compete with the analyte for specific binding by a predetermined amount of specific ligator (eg. antibody, receptor, etc.). Any bindable analyte can be determined by this method, including ones of small molecular weight that have only one binding site.
  • specific antibody is the ligator, which can be used in soluble form to bind the analyte, or 1t can be suitably insolubilized.
  • said antibody is chemically coupled or absorbed to an appropriate mobile or immobile support material (immunosorbent) such as a filter, polymeric bead, liposome, magnetic particle or the inside of a transparent test tube or a section of nylon or other polymeric tubing, as described pre ⁇ viously.
  • the immunosorbent is prepared with a predetermined concentration of antibody that binds a known, constant quantity (total count) of CD tracer.
  • the analyte is brought into contact with said ligator (eg. immunosorbent), and with a suitable, predet ⁇ ermined amount of tracer to allow competitive or displacement binding between said ligator, analyte, and tracer.
  • said tracer may be added simultaneously with the analyte, or added a suit ⁇ able time after the analyte.
  • the binding reaction is done from 5 minutes to several hours at 5 to 35 * C, in an aqueous solution of .01 M phosphate, pH 7.5, with other additives as needed.
  • unbound material is separated from the bound, suitably by washing said immu ⁇ nosorbent with appropriate buffer.
  • Other known separation methods are used depending on the binding reaction and/or immunosorbent, such as precipita ⁇ tion and/or centrifugation, chromatography, dialysis, filtration, or mag ⁇ netic manipulation.
  • precipita ⁇ tion and/or centrifugation chromatography
  • dialysis filtration
  • mag ⁇ netic manipulation e.g., a second antibody (soluble or antigen complexed or insoluble), to bind to the lig ⁇ ator.
  • suitable separation materials include latex beads, polymeric gels, salts, etc. Separation can also be done using electrophoresis or high pressure liquid chromatography, (HPLC).
  • the amount of tracer in the bound and/or unbound fractions is selectively determined through CL activation with the appropriate reagents, as described previously, and in a dark environment, light emission is measured and/or recorded.
  • a sufficient amount of peroxyoxalate and peroxide such as H2O2, among others, is added in a suitable buffer solution.
  • H2O2 may be supplied by oxidizing enzymes such as glucose oxidases in the presence of appropriate substrate.
  • any suitable activation and measurement procedure can include the steps of bringing the appropriate reagents in contact with said tracer to be measured, in a continuous, or stopped flow detection system using appropriate light detection instruments.
  • CD labels can be used with appropriate modification of the method.
  • labels such as multiple NHS-CD labels, chemi- luminescent CD labels, activator CD labels, as well as labels with captured guests, and various derivatives and/or capping substances, and antenna sub ⁇ stances, as described previously, can be used in place of said fluorophore CD labels.
  • hydrazide eg. luminol
  • the activating reagents would consist of H2O2 and appropriate catalyst as descr ⁇ ibed previously.
  • the amount of detectable CD tracer remaining is indirectly proportional to the amount of analyte that was pres ⁇ ent in the sample.
  • METHOD B Sandwich Assay Using a Fluorophore CD Label
  • This method is readily adaptable for use with said AbCA test device described herein, and in 96 well microtiter plates and other supports used in immunoassays. Analytes determined by this method are generally those of larger molecular weight that have more than one binding site.
  • a captor is prepared comprising ligator specific for said analyte, suf ⁇ ficient to bind all of said analyte, coupled to a suitable mobile or immo ⁇ bile support substance or carrier, as previously described.
  • a CD tracer is prepared comprising a ligator specific for said analyte coupled to one or more CD labels.
  • the analyte is brought into con ⁇ tact with said captor and with a suitable amount of CD tracer to allow spec ⁇ ific, "sandwich" binding between said captor, analyte, and tracer.
  • antibody captor sufficient to bind all of said analyte, is coupled to a suitable mobile or immobile substance to produce an immuno ⁇ sorbent, as previously described.
  • said antibody is coupled to the inside of a polymeric test tube.
  • the analyte is brought into contact with said immunosorbent to allow specific binding of said analyte with said immu ⁇ nosorbent.
  • said CD tracer brought into contact with said ana ⁇ lyte is antibody specific for said analyte, labeled with a plurality of a suitable fluorophore CD.
  • the specific binding reactions are carried out in appropriate buffer sol ⁇ ution to allow sufficient binding of analyte with immunosorbent, and tracer with analyte.
  • the binding reaction is done from 5 minutes to several hours at 5 to 35 "C, in an aqueous solution of .01 M phosphate, pH 7.5, with other additives as needed.
  • the unbound tracer fraction is separated from the bound, such as by washing with appropriate buffer solution.
  • Other known separation methods are used depending on the binding reaction and/or immunosorbent, such as precipita ⁇ tion and/or centrifugation, chromatography, dialysis, filtration, or mag ⁇ netic manipulation.
  • the amount of tracer in the bound and/or unbound fractions is selectively determined through CL activation with the appropriate reagents, as described previously, and in a dark environment, light emission is measured and/or recorded.
  • CL activation is done with addition of peroxyoxalate and peroxide such as H2O2, in a suitable buffer solution.
  • H2O2 may be supplied by oxidizing enzymes such as glucose oxidases in the presence of appropriate substrate.
  • said captor, analyte, and tracer may be brought into contact simultaneously or in steps.
  • the antibody of said CD tracer and said fluorophore CD label can be coupled noncovalently by various means before use or during the assay.
  • a CD labeled second antibody can be used, or in this case, a biotinylated antibody is used.
  • Avidin or streptavidin, coupled as previously described with a plurality of fluorophore CD labels, is added in a suitable buffer solution.
  • any suitable activation and measurement procedure can include the steps of bringing the appropriate reagents in con ⁇ tact with said tracer to be measured, in a continuous, or stopped flow detection system using appropriate light detection instruments.
  • CD labels can be used with appropriate modification of the method.
  • labels such as multiple NHS-CD labels, chemi ⁇ luminescent CD labels, activator CD labels, as well as labels with captured guests, and various derivatives and/or capping substances, and antenna sub ⁇ stances, as described previously, can be used in place of said fluorophore CD labels.
  • a captor activator is prepared composed of ligator (captor) specific for the analyte, sufficient to bind all of said analyte, coupled to a suitable mobile or immobile support substance or carrier, as previously described. Also coupled directly with, or in close proximity to, the ligator is a suf ⁇ ficient amount of CL activator, as described previously, that will generate one or more substances needed to cause a luminescent reaction when exposed to appropriate coenzymes and/or substrates.
  • the activator is coupled directly, or through an intermediate coupling substance to the captor, or coupled in close proximity to, the captor.
  • a CD tracer is prepared comprising a ligator specific for said analyte coupled to one or more CD labels.
  • the analyte is brought into contact with said captor activator, and with a suitable amount of tracer to allow “sandwich" binding between said captor, analyte, and tracer.
  • said captor activator is an antibody captor activator (AbCA, above), wherein sufficient antibody to bind all of said analyte is coupled to a suitable mobile or immobile substance as previously described.
  • said AbCA is coupled to the inside of a polymeric test tube and the activator is glucose oxidase enzyme (or apoenzyme), which potentially generates H2O2 in the presence of oxygen, glucose and coenzyme as needed.
  • the analyte is brought into contact with said AbCA to allow specific binding of said analyte with said AbCA.
  • said tracer brought into contact with said analyte is antibody specific for said analyte, labeled with a plurality of a suitable fluorophore CD, described previously.
  • the specific binding reactions are carried out in appropriate buffer sol ⁇ ution to allow sufficient binding of analyte with AbCA, and tracer with ana ⁇ lyte, under conditions that do not cause significant irreversible inactiva ⁇ tion of said activator.
  • the binding reaction is done from 5 minutes to several hours at 5 to 35 * C, in an aqueous solution of .01 M phosphate, pH 7.5, with other additives as needed.
  • the amount of bound tracer is selectively determined wherein a sufficient amount of peroxyoxalate, oxygen and substrate for the activator enzyme is added in a suitable buffer solution.
  • suitable amounts of scavengers eg. catalase enzyme
  • suitable amounts of scavengers that slow down or inhibit any significant CL reaction in the bulk solution but do not inhibit CL in the immediate vicinity of the analyte, can be included.
  • said captor-activator, analyte, tracer, scavenger and buffer solution containing peroxyoxalate, oxygen and glucose may be brought into contact simultaneously or in steps.
  • the antibody and fluorophore CD label can be coupled noncovalently by various means before use or during the assay.
  • a CD labeled second antibody can be used, or in this case, a biotinylated antibody is used.
  • Avidin or streptavidin, coupled as previ ⁇ ously described with a plurality of fluorophore CD labels, is added in a suitable buffer solution that contains sufficient amounts of peroxyoxal te, oxygen and substrate for activation.
  • suitable amounts of scavengers can also be included.
  • any suitable activation and measurement procedure can include the steps of bringing the appropriate reagents in con ⁇ tact with said tracer to be measured, in a continuous, or stopped flow detection system using appropriate light detection instruments.
  • the peroxyoxalate in the bulk solution with unbound tracer is not substantially activated because little or none of the activating sub ⁇ stances are available in the bulk solution. Also, with very little or no analyte in the sample, no significant binding occurs with the CD tracer.
  • other CD labels can be used with appropriate modification of the method. For example, labels such as multiple NHS-CD labels, chemi ⁇ luminescent CD labels, activator CD labels, as well as labels with captured guests, and various derivatives and/or capping substances, and antenna sub ⁇ stances, previously, can be used in place of said fluorophore CD labels.
  • CL labels can be substituted, such as multiple acridinium ester labels to produce new methods.
  • isoluminols, ABEN, or suitable hydrazide CL label preparations previously described can also be substituted wherein the activating buffer solution would be at pH 7- 8, and contain glucose, oxygen and appropriate catalysts such as perox- idases, hemins or hematins as needed.
  • catalysts or cooxidants that are possible are various transition metal salts such as cobalt(II), cop- per(II) and ferricyanide.
  • a new captor-luminescer method provides for more versatility through the reversal of certain components.
  • a plurality of luminescer eg. fluorophore CD label
  • said tracer is replaced with an activator labeled tracer composed of activator (eg. glucose oxid ⁇ ase), covalently coupled to said ligator (eg. antibody).
  • noncovalent binding can be used wherein a biotinylated ligator (eg. antibody) is used and the tracer used is composed of activator coupled to avidin or streptavidin in place of the fluorophore CD.
  • a biotinylated ligator eg. antibody
  • the tracer used is composed of activator coupled to avidin or streptavidin in place of the fluorophore CD.
  • the dif ⁇ ferent colored CD tracers used in the methods described below consist of ligands or ligators coupled to a CD label, or [CD]n label, complexed with a different colored guest fluorophore or a different guest CL compound as des ⁇ cribed previously.
  • Said labels can also include multiple NHS-CD labels, chemiluminescent CD labels, activator CD labels, captured guests, and vari- ous derivatives and/or capping substances, and antenna substances, as descr ⁇ ibed previously.
  • said CD tracers are prepared so that they meet the require ⁇ ments of (1) the fluorophore guests are suitably efficient emitters, (2) the emission spectra or colors, are easily distinguishable, and (3) the labels do not significantly impair specific binding reactions.
  • Said fluorophore CD labeled tracers are activated by any suitable CL activation, or energy transfer reaction described previously, such as com ⁇ bining them with a suitable peroxyoxalate and peroxide.
  • TCPO is added (eg. 600 mg/1 in 10 ⁇ 3 M phosphate buffer, pH 6), and in a dark environment, the activation is started with the addition of H2O2 (eg. 10 -5 - 10 -6 M).
  • H2O2 eg. 10 -5 - 10 -6 M
  • Other substances are suitably included in the buffer such as solv ⁇ ents, ethyl acetate, stabilizers and surfactants, as needed.
  • peroxides eg. H2O2
  • peroxidizing enzymes such as glucose oxidases in the presence of appropriate substrate.
  • Said reagents can be combined with said CD tracers to activate, or, tracers are suitably separated from the other assay components (eg. by wash ⁇ ing, precipitation, centrifugation or column chromatography), and are detected in suitable containers or a continuous, or stopped flow detection system.
  • Detection is done in a dark environment with a photometer or other light detector that can discriminate between the emission wavelengths, or colors, of the CD labels used.
  • said light detector employs photomulti- pliers, or charge coupled devices (CCD), (eg. video camera), or photodiodes with appropriate filters and/or grids as needed.
  • CCD charge coupled devices
  • a scanning system eg. using fiber optics
  • CD tracers can be specifically bound to discrete zones on a suit ⁇ able support material and thereby discriminated one from the other based on their position and/or different color.
  • said support material is an immunosorbent with different ligands or ligators coupled in discrete zones. Then through specific binding, directly or in sandwich fashion, each CD tracer used with different specificity, will bind to its corresponding ligand or ligator.
  • the light emission data is collected in a computerized, auto ⁇ mated system. Using known statistical methods, it can then be determined which labeled substances (by color, signal intensity, position, etc.), are present.
  • METHOD E Competitive Heterogeneous Assay Using Different Colored CD Labels This is an improvement over the competitive heterogeneous method described above. With each additional type of analyte tested for, a cor ⁇ responding number of different colored CD tracer types and ligator types are used.
  • each tracer type or variety comprises a ligand (eg. antigen), that is identical to, or homologous to one the of the ana ⁇ lytes being tested for, and is labeled with a different colored fluorophore CD label.
  • each type of ligator used eg. immunosorbent antibody
  • the analytes are brought into contact with said ligators (eg. immunosorbent), and with a suitable, predetermined amount of appropriate tracers to allow competitive or dis ⁇ placement binding between said ligator, analyte, and tracer.
  • said tracers may be added simultaneously with the ana ⁇ lytes, or added a suitable time after the analytes.
  • CD tracer in the bound and/or unbound fractions is selectively determined through CL activation with the appropriate reagents, as described previously, and detected and discriminated by the color of light emitted by the CD label.
  • each tracer type or variety comprises a ligator (eg. antibody), with correspond ing specificity for binding to one of the analytes being tested for, that is labeled with a different colored fluorophore CD label.
  • a corresponding type of captor is used (eg. immunosorbent anti ⁇ body), with corresponding specificity for one of the analytes being tested for.
  • the specific binding reactions are carried out in appropriate buffer sol ⁇ ution to allow sufficient binding of said analytes with said corresponding captors, and tracers with corresponding analytes. After sufficient time and appropriate conditions for sufficient binding, the unbound tracer fractions are separated from the bound as previously described.
  • captors, analytes, and tracers may be brought into contact simultaneously or in steps.
  • the amount of each tracer is selectively determined wherein said bound and/or unbound fractions of different colored CD tracers are CL activated with the appropriate reagents, (eg. energy transfer reaction), and are detected and discriminated by the color of light generated through activa ⁇ tion of the CD label, as described previously.
  • the appropriate reagents eg. energy transfer reaction
  • each tracer type or variety comprises a ligator (eg. antibody), with correspond ⁇ ing specificity for binding to one of the analytes being tested for, that is labeled with a different colored fluorophore CD label.
  • a corresponding type of captor eg. AbCA antibody
  • the specific binding reactions are carried out in appropriate buffer sol ⁇ ution to allow sufficient binding of said analytes with said corresponding AbCA's, and tracers with corresponding analytes.
  • said captor, analyte, and tracer may be brought into contact simultaneously or in steps.
  • the amount of each bound tracer is selectively determined wherein said different colored CD tracers are CL activated with the appropriate reagents, and are detected and discriminated by the color of light generated through activation of the CD label, as described previously.
  • Catalyst CD Labels With the versatility of the CD tracers of this invention, the unexpected discovery has been made for using catalyst CD labels in specific binding assays.
  • Said catalyst CD tracers used in the methods described below consist of ligands or ligators coupled to a CD label, or [CD]n label, suitably derivatized to provide certain cata ⁇ lytic activity to said CD label as described previously.
  • Said labels can also include multiple NHS-CD labels, and various derivat- ives and/or capping substances, activator and antenna substances, as descr ⁇ ibed before.
  • said catalyst CD tracers are prepared that func ⁇ tion in specific binding reactions and are capable of generating a detec ⁇ table product.
  • CD catalyst tracers of this inven ⁇ tion With the surprising versatility of the CD catalyst tracers of this inven ⁇ tion, it has been discovered that more than one type of analyte can be tested for simultaneously.
  • the principal requires the use of different CD catalyst tracers that produce catalytic products distinguishable by their colored or fluorescent or CL products.
  • Said different CD catalyst tracers consist of ligands or ligators coupled to a CD catalyst label, or [CD]n cat ⁇ alyst label, derivatized with one or more different catalytic groups, that is specific for a different substrate, previously described.
  • Said labels can also include multiple NHS-CD labels, activator CD labels, and various derivatives and/or capping substances, and antenna substances, as described previously.
  • each tracer type or variety comprises a ligator (eg. antibody), with correspond ⁇ ing specificity for binding to one of the analytes being tested for, that is labeled with a different CD catalyst label.
  • a corresponding type of captor eg. immunosorbent antibody
  • said CD catalyst tracers to be detected are exposed to the appropriate substrates for an appropriate period (eg. 1 minute to several hours), and are detected by measurement of the catalytic products.
  • An improvement on the method comprises the use of a solution containing said substrates that includes free CD (or CD derivatives), to improve solvency and/or detectability of the catalytic products (eg. Beaty, et al, U.S. Pat. No. 4,511,651, 1985, and Kaufman, U.S. Pat. No. 4,451,563, 1984).
  • Detection is done using a spectrophotometer or, in a dark environment with a photometer or other light detector that can discriminate between the wavelengths, or colors, of absorption, or fluorescent or CL emission of each catalytic product.
  • said light detector employs photomultipliers, or charge coupled devices (CCD), (eg. video camera), or photodiodes with appropriate filters and/or grids as needed.
  • CCD charge coupled devices
  • a scanning type of system eg. using fiber optics
  • different CD catalyst tracers can be specifically bound to discrete zones on a suitable support material and thereby discriminated one from the other based on their position and/or different color.
  • said sup ⁇ port material is an immunosorbent with different ligands or ligators coupled in discrete zones. Then through specific binding, directly or in sandwich fashion, each CD catalyst tracer used with different specificity, will bind to its corresponding ligand or ligator.
  • the absorption or light emission data is collected in a com ⁇ puterized, automated system. Using known statistical methods, it can then be determined which labeled substances (by color, signal intensity, posi ⁇ tion, etc.), are present.
  • reaction conditions can be modified appropriately (eg. pH, temperature, buffers, etc.), to perform said reac ⁇ tions, including suitable modification of substrates as needed.
  • suitable modification of substrates e.g. pH, temperature, buffers, etc.
  • certain esterified or phosphorylated substrates may require more (or less), carbons between the species to be cleaved, and/or certain derivatives may be more effective substrates than others due to hydrophobicity and/or steric requirements.
  • a CD catalyst label with a suitable catalyst group (eg. imidazole), is used to hydrolyze nitrophenyl acetate to produce a colored product detec ⁇ table by an increase in spectrophotometric absorbance.
  • a CD catalyst label with a suitable catalyst group can also be used to hydrolyze o-nitrophenol- galactopyranoside or chlorophenol red galactopyranoside.
  • naphthyl acetate can be cleaved to produce naphthol which is detectable as a colored diazoniu precipitate.
  • a CD catalyst label with a suitable catalyst group is used to hydrolyze a 7-hydroxycoumarin ester of 5-(2,4-dinitrophenyl)- aminopentanoic acid to produce 7-hydroxycoumarin, detectable by an increase in fluorescence.
  • a suitable catalyst group eg. imidazole
  • a CD catalyst label with a suitable dephosphorylating catalyst group is used to cleave phosphate from a phosphorylated substrate normally used with alkaline phosphatase, to produce a detectable product. For instance, nitrophenyl phosphate or naphthyl phosphate is cleaved to produce a colored product. Or, a chemiluminescent product is produced through cleavage of a phosphorylated dioxetane, including adamantyl 1,2-dioxetane phosphates, among others.
  • METHOD I Competitive Assay Using Catalyst CD Labels This is a compe ⁇ titive heterogeneous ligand binding assay, requiring a CD tracer prepared from a ligand (eg. antigen), that is identical to, or homologous to the ana ⁇ lyte, with one or more CD catalyst labels coupled to it. The CD tracer will then compete with the analyte for specific binding by a predetermined amount of specific ligator (eg. antibody, receptor, etc.). Any bindable analyte can be determined by this method, particularly those of smaller molecular weight that have only one binding site.
  • ligand eg. antigen
  • specific antibody is the ligator, which can be us ⁇ d in soluble form to bind the analyte, or it can be suitably insolubilized.
  • said antibody is chemically coupled or absorbed to an appropriate mobile or immobile support material (immunosorbent) such as a filter, polymeric bead, liposo e, magnetic particle or the inside of a transparent test tube or a section of nylon or other polymeric tubing, as described pre ⁇ viously.
  • the immunosorbent is prepared with a predetermined concentration of antibody that binds a known, constant quantity (total count) of CD tracer.
  • the analyte is brought into contact with said ligator (eg. immunosorbent), and with a suitable, predet ⁇ ermined amount of tracer to allow competitive or displacement binding between said ligator, analyte, and tracer.
  • said tracer may be added simultaneously with the analyte, or added a suit ⁇ able time after the analyte.
  • a CD catalyst tracer composed of ligand (eg. antigen), suitably coupled with a suitable CD catalyst label (eg. histamine derivat ⁇ ized), is suitably incubated and bound to a ligator such as immobilized ant ⁇ ibody. After sufficient time and appropriate conditions for sufficient binding, unbound material is separated from the bound as described above in said competitive heterogeneous assay. The amount of tracer in the bound and/or unbound fractions is selectively exposed to the appropriate substr ⁇ ate, and is detected by measurement of the catalytic product.
  • ligand eg. antigen
  • a suitable CD catalyst label eg. histamine derivat ⁇ ized
  • said histamine derivatized CD catalyst label will hydrolyze p-nitrophenyl acetate in suitable solvent, to p-nitrophenol , detectable by an increase in spectrophotometric absorbance at 400 nm.
  • a CD catalyst tracer is prepared comprising a ligator specific for said analyte coupled to one or more CD catalyst labels.
  • the analyte is brought into contact with said captor and with a suitable amount of CD tracer to allow specific, "sandwich" binding between said captor, analyte, and tracer.
  • antibody captor sufficient to bind all of said analyte, is coupled to a suitable mobile or immobile substance to produce an immuno ⁇ sorbent, as previously described.
  • said antibody is coupled to the inside of a polymeric test tube.
  • the analyte is brought into contact with said immunosorbent to allow specific binding of said analyte with said immu ⁇ nosorbent.
  • said CD tracer brought into contact with said ana ⁇ lyte is antibody specific for said analyte, labeled with a plurality of a suitable CD catalyst label (eg. histamine derivatized), described previ ⁇ ously.
  • a suitable CD catalyst label eg. histamine derivatized
  • the specific binding reactions are carried out in appropriate buffer sol ⁇ ution to allow sufficient binding of analyte with immunosorbent, and tracer with analyte.
  • the binding reaction is done from 5 minutes to several hours at 5 to 35 "C, in an aqueous solution of .01 M phosphate, pH 7.5, with other additives as needed.
  • the unbound tracer fraction is separated from bound such as by washing.
  • Other known separation methods are used depending on the binding reaction and/or immunosorbent, such as precipitation and/or centrifugation, chromato ⁇ graphy, dialysis, filtration, or magnetic manipulation.
  • the amount of tracer in the bound and/or unbound fractions is selectively determined by exposure to the appropriate substrate, and detecting the catalytic product.
  • said histamine derivatized CD catalyst label will hydrolyze p-nitrophenyl acetate in suitable solvent, to p-nitrophenol, detectable by an increase in spectrophotometric absorbance at 400 nm.
  • said captor, analyte, and tracer may be brought into contact simultaneously or in steps.
  • the antibody of said CD tracer and said CD catalyst label can be coupled noncovalently by various means before use or during the assay.
  • a CD labeled second antibody can be used, or in this case, a biotinylated antibody is used.
  • Avidin or streptavidin, coupled as previously described with a plurality of CD catalyst labels, is added in a suitable buffer solution.
  • any suitable activation and measurement procedure can include the steps of bringing the appropriate reagents in contact with said tracer to be measured, in a continuous, or stopped flow detection system using appropriate detection instruments.
  • a captor activator is prepared composed of ligator (captor) specific for the analyte, sufficient to bind all of said analyte, coupled to a suitable mobile or immobile support substance or carrier, as previously described. Also coupled directly with, or in close proximity to, the ligator is a suf ⁇ ficient amount of "CD catalyst activator".
  • Said CD catalyst activator is a substance, such as an enzyme, that will generate one or more substances needed to cause a CD catalyst reaction when exposed to appropriate coenzymes and/or substrates.
  • said CD catalyst activator can be an enz ⁇ yme such as oxidases, peroxidases, phosphatases, transferases, etc.
  • alkaline phosphatase can be a catalyst activator that removes one or more phosphates from a phosphorylated substance (eg. phosphorylated nitrophenyl acetate), to pro ⁇ prise a substrate (eg. nitrophenyl acetate), that is subsequently hydrolyzed (or oxidized), by a CD catalyst label.
  • glutamyltransferase can be a catalyst activator that removes a glutamyl group from a substance that is subsequently catalyzed by a CD catalyst label.
  • the activator is coupled directly, or through an intermediate coupling substance to the captor, or coupled in close proximity to, the captor.
  • a CD tracer is prepared comprising a ligator specific for said analyte coupled to one or more CD catalyst labels.
  • the analyte is brought into contact with said captor activator, and with a suitable amount of tracer to allow "sandwich" binding between said captor, analyte, and tracer.
  • said captor activator is an antibody captor activator (AbCA, above), wherein sufficient antibody to bind all of said analyte is coupled to a suitable mobile or immobile substance as previously described.
  • said AbCA is coupled to the inside of a polymeric test tube and the activator is alkaline phosphatase enzyme.
  • the analyte is brought into contact with said AbCA to allow specific binding of said analyte with said AbCA.
  • said tracer brought into contact with analyte is antibody specific for said analyte, labeled with a plurality of a suitable catalyst CD, (eg. imidazole catalyst), descr ⁇ ibed previously.
  • a suitable catalyst CD eg. imidazole catalyst
  • the specific binding reactions are carried out in appropriate buffer sol ⁇ ution to allow sufficient binding of analyte with AbCA, and tracer with ana ⁇ lyte, under conditions that do not cause significant irreversible inactiva ⁇ tion of said activator.
  • the binding reaction is done from 5 minutes to several hours at 5 to 35 ⁇ C, in an aqueous solution of .01 M phosphate, pH 7.5, with other additives as needed.
  • the amount of bound tracer is selectively determined wherein a sufficient amount of substrate (eg. phosphorylated hydroxycoumarin ester of 5-(2,4- dinitrophenyD-aminopentanoic acid), for the activator enzyme is added in a suitable buffer solution.
  • substrate eg. phosphorylated hydroxycoumarin ester of 5-(2,4- dinitrophenyD-aminopentanoic acid
  • the activator will dephosphorylate this ester, converting it to a suitable substrate for the catalyst CD label.
  • scavengers that slow down or inhibit any significant dephosphorylation reaction in the bulk solution but do not inhibit it in the immediate vicinity of the ana ⁇ lyte, can be included.
  • said captor-activator, analyte, tracer, scavenger and buffer solution containing substrate may be brought into contact simultaneously or in steps.
  • the antibody and catalyst CD label can be coupled noncovalently by various means before use or during the assay.
  • a CD labeled second antibody can be used, or in this case, a biotinylated antibody is used.
  • Avidin or streptavidin, coupled as previ ⁇ ously described with a plurality of catalyst CD labels, is added in a suit ⁇ able buffer solution that contains sufficient amounts of substrate for activation.
  • suitable amounts of scavengers can also be included.
  • any suitable activation and measurement procedure can include the steps of bringing the appropriate reagents in contact with said tracer to be measured, in a continuous, or stopped flow detection system using appropriate detection instruments.

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Abstract

De nouvelles compositions et de nouvelles méthodes permettent de procéder à des essais immunitiares avec des marqueurs en cyclodextrine (CD). Les marqueurs comprennent une molécule ''hôte'' de CD qui contient une molécule ''invitée'' fluorescente ou chimioluminescente, avec un ''groupe de couplage'' agencé sur la CD. Les marqueurs sont utiles pour marquer diverses substances, telles que ligands, antigènes et anticorps, qui servent d'indicateurs lors d'essais immunitaires et d'autres essais par liaison avec des ligands. On décrit des procédés d'essais de type compétitif et non compétitif en ''sandwich'', ainsi que des essais hétérogènes et homogènes. Les propriétés et les avantages de l'invention sont: 1) indicateurs sûrs non radioactifs; 2) des signaux d'une efficacité potentiellement supérieure et une plus longue durée de conservation; 3) des groupes de couplage pour des marquages en une seule étape; 4) une détection chimioluminescente plus sensible; 5) des marqueurs multiples en CD qui assurent un marquage efficace; 6) similarité chimique/physique, qui facilite la synthèse et l'utilisation; 7) des marqueurs de couleurs différentes avec des propriétés similaires; 8) des procédés faciles à automatiser, pour des applications à des volumes importants. L'invention concerne également des procédés et des marqueurs en CD par catalyse.
PCT/US1990/005759 1989-10-10 1990-10-09 Marqueurs en cyclodextrine pour essais immunitaires et analyses biochimiques WO1991005605A1 (fr)

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US6048736A (en) * 1998-04-29 2000-04-11 Kosak; Kenneth M. Cyclodextrin polymers for carrying and releasing drugs
US6120987A (en) * 1997-12-12 2000-09-19 Cis Bio International Non-aggregated fluorescent conjugates and the process for their preparation
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EP1418430A2 (fr) 1991-05-22 2004-05-12 Dade Behring Marburg GmbH Essai homogène pour déterminer un analyte
EP1088547B1 (fr) * 1999-09-02 2007-04-18 Wacker Chemie AG Compositions cosmetiques contenant des adduits moléculaires de biotine et/ou de ses esters avec des cyclodextrines
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WO2015021418A1 (fr) * 2013-08-09 2015-02-12 Bio-Rad Laboratories, Inc. Détection de protéine au moyen de cyclodextrines modifiées
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CN111936861A (zh) * 2018-03-30 2020-11-13 希森美康株式会社 对脂蛋白质的吸收能力进行测定的方法及试剂
US10890848B2 (en) 2017-12-20 2021-01-12 Samsung Electronics Co., Ltd. Material for detecting photoresist and method of fabricating semiconductor device using the same
CN112229991A (zh) * 2020-10-16 2021-01-15 厦门圣科环保科技股份有限公司 一种纳米捕获器件及其制备方法和用途
CN116183901A (zh) * 2023-01-04 2023-05-30 烟台大学 一种基于环糊精-金刚烷化铱络合物主客体组装标记的蛋白标记物荧光检测方法
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KR102515008B1 (ko) * 2018-10-12 2023-03-29 기초과학연구원 인공결합쌍을 이용한 단백질 검출 방법 및 검출용 키트
CN112229991A (zh) * 2020-10-16 2021-01-15 厦门圣科环保科技股份有限公司 一种纳米捕获器件及其制备方法和用途
CN112229991B (zh) * 2020-10-16 2022-08-16 厦门圣科环保科技股份有限公司 一种纳米捕获器件及其制备方法和用途
CN116183901A (zh) * 2023-01-04 2023-05-30 烟台大学 一种基于环糊精-金刚烷化铱络合物主客体组装标记的蛋白标记物荧光检测方法
CN116183901B (zh) * 2023-01-04 2025-04-18 烟台大学 一种基于环糊精-金刚烷化铱络合物主客体组装标记的蛋白标记物荧光检测方法

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