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WO2006009435A2 - Procede de detection et de mesure d'un ligand - Google Patents

Procede de detection et de mesure d'un ligand Download PDF

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Publication number
WO2006009435A2
WO2006009435A2 PCT/NL2005/000517 NL2005000517W WO2006009435A2 WO 2006009435 A2 WO2006009435 A2 WO 2006009435A2 NL 2005000517 W NL2005000517 W NL 2005000517W WO 2006009435 A2 WO2006009435 A2 WO 2006009435A2
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WO
WIPO (PCT)
Prior art keywords
ligand
liquid
analog
extracting phase
binding
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PCT/NL2005/000517
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English (en)
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WO2006009435A3 (fr
WO2006009435A8 (fr
Inventor
Rentje Frederik Witkamp
Wouter Henricus Johannes Vaes
Micha Manuele MÜLLER
Original Assignee
Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
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Publication of WO2006009435A2 publication Critical patent/WO2006009435A2/fr
Publication of WO2006009435A3 publication Critical patent/WO2006009435A3/fr
Publication of WO2006009435A8 publication Critical patent/WO2006009435A8/fr

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Classifications

    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • 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
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances

Definitions

  • the invention relates to a method for the detection of binding between a ligand and its binding partner. More in particular, the present invention relates to an improved competitive binding assay for detecting and measuring a ligand. For studying binding processes between (bio)molecules, in the field of
  • CBAs competitive binding assays
  • the working principle of CBAs is based on the competition between an unlabeled ligand (usually the substance to be analyzed) and a labeled analog of that ligand (usually a reagent) for a limited number of binding sites on a biological macromolecule (the binding partner or carrier, usually an antibody, a receptor, a polynucleotide, or a transport protein).
  • the concentration of the unlabeled ligand is inversely proportional to the amount of labeled analog bound to the binding partner.
  • the binding partner with the labeled analog and the unlabeled ligand bound thereto is usually removed from the solution so that all bound ligands can be separated from the unbound material in the sample to be investigated. This separation of bound and dissolved phase may for instance take place by immobilization of the binding partner, for instance with the aid of an immobilized antibody, and removal of the reaction liquid.
  • the binding partner may, for instance, be coupled to the surface of a reaction vessel, e.g. to the surface of a well of a microtiter plate manufactured from a suitable plastic material, in order to thus facilitate the washing and removing of excess unbound labeled analog.
  • Such a CBA is referred to as a heterogeneous (solid phase) CBA.
  • An example of a heterogeneous CBA is the "radioreceptor assay" in which the binding site is located on a membrane or tissue receptor. By means of the assay, the concentration of a substance (usually a hormone) is determined, where a mixture of the sample to be investigated and a known amount of the substance to be tested which is radioactively labeled are exposed to a known amount of immobilized receptors for that substance to be tested.
  • the amount of the substance to be tested in the sample to be investigated is determined from the proportion of receptors which are occupied by radioactively labeled molecules of the substance to be tested, assuming that labeled and unlabeled molecules randomly bind to receptor binding sites.
  • Most enzyme-linked immunosorbent assays (ELISAs) are also forms of heterogeneous CBAs.
  • the pharmacological binding capacity of a receptor for a substance to be investigated is determined.
  • the separation of the ligands bound to the binding partner from the solution can be done physically; such as for instance with the aid of a filter, or chemically, for instance with the aid of active carbon. Then, an analysis carried out for the separated components should give a decisive answer whether the new substance has indeed displaced the known ligand.
  • heterogeneous CBAs are lengthy, laborious, have limited applicability and are largely manual. Despite the fact they are used very often, such assays are not very suitable for high-throughput screening (HTS) applications.
  • An example of a homogeneous CBA system is the enzyme-multiplied immunoassay technique (EMIT; Rubenstein et al. 1972, Biochem. Biophys. Res. Commun., 47:846), which is inter alia used for the analysis of urine for, for instance, cocaine. This analysis is based on the competition for binding sites on an antibody between the cocaine in the sample and the cocaine labeled with, for instance, the enzyme glucose -6-phosphate dehydrogenase (G6PDH).
  • G6PDH glucose -6-phosphate dehydrogenase
  • the cocaine concentration in the sample can be determined by spectrophotometric determination of the rate at which nicotinamide adenine dinucleotide (NAD) is reduced by G6PDH.
  • NAD nicotinamide adenine dinucleotide
  • HTS high-throughput screening
  • This increase in absorbed unbound ligand which can be determined in a conventional manner, thus indicates the presence of 0 substances which compete for binding sites on the receptor. Such substances are, for instance, potential medicines.
  • the absorption such as it plays a role in the present invention is based on migration of ligand from one physical phase to another physical phase. With the aid of the invention described herein, it is possible to detect ligands and optionally determine the 5 concentration thereof.
  • the invention in a first aspect, relates to a method for detecting and/or measuring a ligand in a liquid.
  • the method comprises the steps of: a) providing a binding partner of the ligand in the liquid; b) subjecting the liquid to conditions in which the ligand can bind to the binding partner for o providing an amount of bound ligand and an amount of free ligand in the liquid; c) subjecting the liquid to negligible depletion extraction in which a part of the amount of free ligand is extracted by an extracting phase, this extracting phase having the form of a surface coating on the wall of a liquid container or on particles mixable with the liquid and d) detecting and/or 5 measuring the extracted ligand.
  • the method preferably comprises the step of: e) determining the amount of bound ligand and/or free ligand present in solution.
  • a process of negligible depletion extraction takes place.
  • a negligibly small part for instance less than 20%, preferably less o than 10%, more preferably less than 2%, still more preferably less than 1%, still more preferably less than 0.5%, still more preferably less than 0.1%, still more preferably less than 0.01% of the free ligand is extracted by an extracting phase.
  • at least 0.1%, more preferably at least 0.01%, still more preferably at least 0.001% and still more preferably at least 0.0001% of the free ligands present in solution is extracted.
  • the invention relates to a method for detecting and/or measuring a ligand in a liquid, where (primarily) not the ligand itself, but a labeled analog of the ligand to be measured is detected. Therefore the present invention also relates to a method for detecting and/or measuring a ligand in a liquid, comprising the steps of: a) providing a binding partner of the ligand and a detectable analog of the ligand in the liquid; b) allowing the occurrence of competitive binding between the ligand and the analog to the binding partner in order to provide an amount of bound analog and an amount of free analog in the liquid; c) subjecting the liquid to negligible depletion extraction, in which a part of the amount of free analog is extracted by an extracting phase, the extracting phase having the form of a surface coating on the wall of a liquid container or on particles mixable with the liquid and d) detecting and/or measuring the extracted analog.
  • the method further comprises the step of: e) determining the amount of bound analog and/or free analog present in solution and still more preferably the method further comprises the step of: f) determining the amount of bound ligand and/or free ligand present in solution.
  • the binding partner is chosen from the group consisting of receptors, polynucleotides, binding proteins, cofactors and antibodies.
  • the ligand is a hydrophobic ligand.
  • the ligand is a pharmaceutical to be investigated, more preferably a steroid, benzodiazepine or antibiotic.
  • the analog is a radioactively labeled form of the ligand.
  • the extracting phase comprises lipophilic polymers.
  • the ligand and/or the analog is a polynucleotide
  • the extracting phase comprises a polynucleotide whose nucleotide order is at least partly complementary to the nucleotide order of at least a part of the ligand and/or the analog, and the polynucleotide which is in the extracting phase is able to extract the ligand and/or the analog from the liquid under conditions of negligible depletion.
  • At least 5-20, preferably at least 8-16, and more preferably at least 10-14 substantially consecutive nucleotides are complementary to substantially consecutive nucleotides of the ligand.
  • the extracting phase preferably comprises a solid phase.
  • the solid phase preferably comprises polysiloxanes, and more preferably the solid phase is polydimethylsiloxane.
  • the extracting phase used in a method according to the invention preferably has a partition coefficient of between 2 and 20.
  • the extracting phase comprises a scintillator for detection of radioisotopes.
  • the present invention further relates to a liquid container for detecting and/or measuring a ligand in a liquid, which container is provided with an extracting phase which has the capability to extract the ligand from the liquid phase, the extracting phase having the form of a surface coating on the wall of a liquid container or on particles mixable with the liquid, and which container is further provided with detection means, preferably a scintillator for detection of radioisotopes.
  • a liquid container provided with an extracting phase with the capability to extract substances from a liquid is inter alia known from WO 98/41855.
  • An advantage of the container according to the present invention over the one described in WO 98/41855 is that the present container comprises specific detection means.
  • the detection means are comprised in the extracting phase so that they can be brought in the immediate proximity of and, if necessary, into direct contact with the extracted ligand.
  • Detection means which are preferably used include detection means for detection of fluorescently labeled substances, such as for instance fluorescence quenchers. Fluorophores can also be used as detection means.
  • the use of the detection means in a container according to the invention of course depends on the ligand or analog to be detected and/or measured.
  • a fluorescence quencher is suitable as a detection means.
  • the ligand or analog may be labeled with a fluorescence quencher, in which case a fluorophore used as a detection means in a liquid container according to the invention is suitable.
  • a skilled person is able to use a suitable detection means depending on the ligand or analog to be detected.
  • Detection means which are preferably used are detection means for detection of radioactively labeled substances.
  • detection means are known to a skilled person and are, for instance, used in so-called scintillation proximity assays.
  • the detection means are scintillators.
  • 2,5-diphenyloxazole is used as a scintillator.
  • the extracting phase comprises a polynucleotide whose nucleotide order is partly complementary to the nucleotide order of at least a part of a polynucleotide ligand to be measured and which is able to extract the ligand from the liquid under conditions of negligible depletion.
  • the polynucleotide of the extracting phase may comprise 3-40 nucleotides.
  • the polynucleotide of the extracting phase in the polynucleotide of the extracting phase, at least 5-20, preferably at least 8-16, and more preferably at least 10-14 substantially consecutive nucleotides are complementary to substantially consecutive nucleotides of a polynucleotide ligand to be measured.
  • the polynucleotide of the extracting phase comprises 5-20, more preferably 8-16, more preferably 10-14 nucleotides.
  • oligonucleotides can also be referred to as oligonucleotides. Both DNA and RNA nucleotides can be used, as well as nucleotide mimetics such as PNA.
  • the complementarity of the polynucleotide of the extracting phase does not need to be 100% to still realize a useful negligible depletion extraction. It is, for instance, possible that the extracting polynucleotide is complementary to a polynucleotide ligand for only about 60%, about 70%, about 80%, about 90% or about 95%. This percentage is understood to mean that, in a plurality of directly adjacent nucleotides in a polynucleotide, one or more nucleotides may be present which are not complementary to the polynucleotide ligand and therefore do not participate in the hybridization reaction (with 95% complementarity, one in twenty nucleotides is not complementary).
  • the complementarity is 100%.
  • the extracting polynucleotide and/or the polynucleotide ligand can comprise more nucleotides than those that participate in the hybridization reaction which is the result of the at least partial complementarity between these molecules.
  • the above-mentioned complementarity percentage only relates to the nucleotides involved in the hybridization reaction.
  • the extracting phase comprises polysiloxanes and a preferred polysiloxane is poly dime thylsiloxane.
  • the extracting phase used in a liquid container according to the invention preferably has a partition coefficient of between 2 and 20.
  • the present invention further relates to the use of a liquid container according to the present invention in a method for detecting and/or measuring a ligand in a liquid.
  • a liquid container according to the invention is used in a method for detecting and/or measuring a ligand in a liquid according to the present invention.
  • a very suitable use of a method according to the invention resides in determining the binding capacity of pharmaceutical ligands. For instance, it can very simply be determined whether pharmaceutical substances have a high plasma-protein binding by use of a method according to the invention, preferably in combination with a liquid container according to the present invention.
  • the present invention further relates to a test kit for carrying out a method according to the invention, which test kit comprises a liquid container according to the invention.
  • detecting and/or measuring a ligand as stated in a method according to the present invention is in principle understood to mean all possible detection and measurement determinations. Possibilities for such determinations are, for instance, but not exclusively, determining the concentration of the ligand and/or determining the binding activity of the ligand.
  • detecting means finding or determining the presence of a ligand, both qualitatively and quantitatively, preferably qualitatively.
  • Measuring comprises, for instance, measuring the free concentration of a ligand, determining whether a substance is a ligand, testing the presence of binding partners of a ligand, evaluating (blood) plasma -protein binding of a ligand.
  • Detecting and/or measuring a ligand therefore also comprises the study of binding between a ligand and one or more binding partners.
  • Such an aspect of the invention, where binding between a ligand and binding partner is studied, may inter alia be carried out by use of different concentration ratios of ligand and analog of a ligand as set forth herein.
  • Methods according to the present invention are suitable to be used as high throughput screening method in the pharmaceutical industry. Further, the methods can be used quantitatively in analytical chemistry and for research of, for instance, pharmacokinetics.
  • the advantage of the methods according to the invention is that determinations are carried out such that only a small part of the free ligand (and, if applicable, of the unconjugated analog) present in a sample is subjected to the detection and/or measurement conditions so that measurement without appreciable difference of the system to be measured is possible. For instance, it is not necessary to add active carbon to the measuring system, or to remove the bound ligand (or, if applicable, the conjugated analog) by means of filtration.
  • a "chemical analysis” is defined as the determination of the composition of a sample or one of the chemical (e.g. binding) properties of a substance.
  • a “substance to be analyzed” is defined as the component of the sample of which the concentration, composition or the capacity to bind to a binding macromolecule is determined in a chemical analysis.
  • a "receptor” is defined as a molecule or surface in a cell which recognizes and binds a specific (messenger) molecule, which results in a biological response.
  • a "ligand” is defined as an ion, a molecule, or a molecular group which binds to another chemical entity, such as for instance an antibody, a receptor or a transport protein, for forming a larger complex or for bringing about a biological effect.
  • a "binding partner” is defined as a member of a specific binding pair, i.e. one of two different molecules having an area on the surface or a cavity which specifically binds to, and is therefore by definition complementary to, a particular spatial and polar organization of the other molecule.
  • the members of a specific binding pair may be a ligand and receptor; members of an immunological binding pair, such as an antigen - antibody binding pair; members of a non -immunological binding pair such as (strept)avidin and biotin; two complementary nucleic acids, both ribonucleic acids and deoxyribonucleic acids and hybrids between them; or any other binding molecule such as a lectin.
  • Complementary specific binding pairs bind to each other with great affinity, such as for instance a ligand and its complementary receptor.
  • One of the two complementary members of a specific binding pair can be referred to by the term "binding partner" for the other member.
  • a "reagent” is defined as a substance or mixture which is usable in chemical analysis or synthesis.
  • a “competitive binding assay” is defined as an assay based on the competition between a labeled and an unlabeled ligand in the reaction with a binding partner (for instance an antibody, a receptor, or a transport protein).
  • a binding partner for instance an antibody, a receptor, or a transport protein.
  • conjugation and conjugating are defined as the process of chemical binding and, herein, a “conjugate” is defined as the product of that binding.
  • Conjugation and (competitive) binding are, as used herein, essentially the same processes, in which a ligand or an analog thereof to be detected or to be measured binds to a binding partner.
  • Negligible depletion extraction is an assay process in which, generally with the aid of an extracting polymer, negligible amounts of free ligand present in solution are removed from the solution by extraction in or to an extracting (usually solid) phase, as a result of which a negligible equilibrium shift occurs between free ligand and bound ligand in a solution.
  • An example of a negligible depletion extraction assay is "negligible depletion solid-phase microextraction" (ND-SPME) (Vaes et al. 1997. Chem Res Toxicol 10:1067-72; Heringa et al. 2002. Anal Chem 74:5993-7).
  • solid phase In general, a solid phase is used for the extraction, but a polymeric liquid or liquid crystalline may be used as well.
  • the present invention is not limited to extracting solid phases which can remove ligands from a liquid. Therefore, in a general sense, this is referred to as an extracting phase herein.
  • This extracting phase is characterized in that the bound ligand is absorbed therein and is thereby removed from the liquid as a free ligand.
  • a characteristic of the negligible depletion extraction which is part of the aspects of the present invention is that only a small part of the total amount of free ligand or analog is extracted.
  • a "free" ligand or analog has the meaning of a ligand or analog freely present in solution, both in contrast with a ligand or analog bound to the binding partner and in contrast with a ligand or analog extracted by the extracting phase. So, herein, a free ligand or analog is unbound and not extracted.
  • SPME Solid phase microextraction
  • the relevant compound can be desorbed from the fiber and then be analyzed by means of techniques appropriate to that end such as for instance GC or HPLC.
  • One of the properties of SPME is that only the freely dissolved fraction of the compound is available for partitioning to the extraction tool.
  • Another property of SPME is that the method can be used in such a manner that only small amounts are extracted from the sample, which makes negligible depletion extraction possible. These two properties make SPME tools excellently suitable for measuring free concentrations.
  • the techniques described up to now which use negligible depletion extraction add a solid phase to the reaction mixture, such as the polymer-coated fiber of Heringa et al.
  • the present invention provides the solid phase as e.g.
  • the pharmaceutical and food industry are generally looking for compounds (competitors) which have a good affinity for a particular macromolecule, and can thereby displace the natural or biological ligand of this macromolecule. If a known amount of a known ligand and a macromolecule are brought together in solution, then the amount of ligand which is still freely present in solution will depend on the concentration and affinity of the competitors present (e.g. a potential pharmaceutical). Binding to macromolecules is generally an equilibrium process.
  • the equilibrium established between the amount of the known (natural) ligand which is free in solution and the amount of the known ligand which is bound to the macromolecule can be influenced by a substance which also binds to the macromolecule.
  • This latter substance, the competitor compound sought, can be found by using a method according to the invention in the following manner.
  • the concentration of the ligand in the extracting phase is a good reflection of the concentration of the ligand freely present in solution, and is thus not bound to the binding partner (e.g. the receptor).
  • the extraction then needs to have a negligible influence on the concentration of the ligand so that the equilibrium between the free and bound ligand is not disturbed.
  • the concentration of the free ligand present in the extracting phase can be determined with various methods, which methods will be described in more detail hereinbelow.
  • Methods according to the invention can be used to determine the presence of a wide range of substances with a specific binding property.
  • One condition is that one or more specific binding partners can be used for this.
  • the pair formed by the substance to be determined (herein also referred to as the ligand) and the binding partner may for instance be chosen from the following combinations, in which each individual member of the pair may be the ligand or the binding partner, on the condition that the ligand or analog thereof bound to the binding partner is substantially not extracted by the extracting phase:
  • Suitable ligands which can be measured by means of a method according to the present invention are low -molecular hydrophobic molecules.
  • low-molecular hydrophobic molecules such as steroids and benzodiazepines (such as valium), different hydrophobic antibiotics and substances such as vitamins, particularly vitamin D, are measured.
  • suitable ligands are Quercetin, estradiol, estriol, cannabinoids, ACE-inhibitors (angiotensin II antagonists) .
  • low-molecular is understood to mean a molecular weight of 100-2000 Da, preferably in a range from about 150 to about 1000 Da.
  • a macromolecular ligand is a polynucleotide.
  • a very suitable polynucleotide which can be measured by means of a method according to the invention comprises an oligonucleotide, for instance an oligonucleotide of 10-40 nucleotides.
  • a detectable analog of a ligand may, for instance, comprise a labeled form of the respective ligand, such as a fluorescently labeled ligand, or conversely a fluorescence quencher, and is preferably a radioactively labeled analog of the ligand whose binding to the binding partner or carrier is investigated.
  • binding partners for use in a method according to the invention, all individual members of the above-mentioned combinations of ligands and binding partners, such as receptors, polynucleotides, binding proteins, cofactors or antibodies, can be used.
  • Very suitable binding partners are binding partners which result in a bound ligand or analog which cannot be extracted by the extracting phase due to their molecular weight or due to their size, while ligand can be extracted by the extracting phase.
  • macromolecular structures with a Mw > 1000-2000 Da are too large to be extracted by the extracting phase. Therefore, preferably binding partners are used which cause the molecular weight of the ligand to increase to a value higher than 1000 Da, more preferably higher than 2000 Da.
  • receptors such as the vitamin D receptor, cannabinoid receptor, androgen receptors, recombinant proteins, plasma or plasma proteins, antibodies, estrogen receptors, BSA, angiotensin II antagonists, and SHBG are used as binding partners.
  • Other suitable binding partners are, for instance, cell systems, e.g. cells or tissues or homogenates thereof, which contain a particular binding macromolecule or homogenates of tissue or cells which contains a particular binding macromolecule, etc.
  • a binding partner is a macromolecule to which a known substance can bind and for which substance a competitor is sought.
  • the substance sought is an unknown substance to be tested (i.e. the ligand to be investigated) which may, for instance, be an agonist or antagonist.
  • the binding partner can be provided in any suitable concentration.
  • the binding partner is provided in a concentration in the range from about 1 attomol/L to about 10 mol/L, preferably from 1 attomol/L to about 10 nanomol/L, more preferably in the range from about 1 attomol/L to about 1 nanomol/L, and still more preferably from about 1 attomol/L to about 10 femtomol/L.
  • a binding partner is provided in a concentration in a range from about 10 attomol/L to about 10 femtomol/L.
  • the advantage of a method according to the present invention is that the binding partner can be applied in very small amounts.
  • the detectable analog of the ligand to be investigated can, if present, be provided in any suitable concentration.
  • the detectable analog of the ligand to be investigated can be provided in a concentration in the range from about 1 attomol/L to about 1 mol/L, preferably from about 1 picomol/L to about 1 micromol/L.
  • a solvent for providing a solution of a binding partner of the ligand in principle many types of solvents can be used. It is important that the solvent makes the binding between binding partner and one of the two forms of the ligand possible.
  • water is used as a solvent.
  • buffers are excellently suitable as an aqueous solvent.
  • a skilled person is able to realize a suitable reaction environment, in which binding occurs between the binding partner and one of the two forms of the ligand. With great preference, aqueous solutions in the form of a phosphate-buffered saline (PBS) are used as a reaction mixture for such a binding reaction.
  • PBS phosphate-buffered saline
  • TRIS/BSA buffers in which BSA is used for improving the solubility of the ligand or analog are very suitable as well.
  • the choice of the solvent or medium depends on the ligand to be detected.
  • a method for detecting and/or measuring a ligand will be carried out in a liquid.
  • a method according to the present invention is in principle not limited to the detection and/or the measurement of ligands in a liquid.
  • any sample is suitable to be subjected to a method according to the invention. If the sample is a solid or semi-solid sample, the ligands to be investigated therein can be subjected to a method according to the invention by including the ligands in a liquid, preferably an aqueous liquid, preferably a medium in which binding between a binding partner and a ligand and/or analog and also extraction of free ligand (and/or free analog) by the extracting phase can take place.
  • a method for detecting and/or measuring a ligand according to the present invention comprises the step of providing a binding partner of the ligand in the liquid.
  • a binding partner of the ligand is contacted with the ligand. This may, for instance, be done by separately adding the ligand and the binding partner to a reaction mixture in which binding can take place. It is also possible to add the binding partner to a (liquid) sample with the ligand and then form a reaction mixture thereof.
  • a method for detecting and/or measuring a ligand according to the present invention comprises the step of subjecting the liquid to conditions in which the ligand can bind to the binding partner in order to provide an amount of bound ligand and an amount of free ligand in the liquid.
  • the liquid is of course understood to mean the liquid containing the ligand, which liquid may have the form of a reaction mixture.
  • subjecting the ligand to conditions in which the ligand can bind is understood to means that the conditions for binding of the ligand to the binding partner not only need to be facilitated, but also need to be maintained for some time in order to enable binding of ligand to the binding partner and consequently an equilibrium between bound and free ligand.
  • the pH, the temperature or a different condition parameter is important, as well as the time during which the reaction components are exposed to the conditions for binding.
  • suitable periods are periods varying from a few seconds to a few hours.
  • such a binding reaction is carried out for a period of about 1 hour to about 24 hours.
  • the formation of free ligand and bound ligand is an equilibrium reaction in which the period in which the equilibrium is established can vary per ligand and per binding partner.
  • a skilled person will be familiar with the possibilities for optimization of both the reaction environment and the reaction condition on the basis of the description hereinbelow.
  • a method for detecting and/or measuring a ligand according to the present invention further comprises the step of subjecting the solution to a negligible depletion extraction in which a part of the amount of free ligand is extracted the extracting phase.
  • the liquid i.e. the reaction mixture with the free and bound ligands therein, needs to be contacted with an extracting phase.
  • an extracting phase may comprise an extracting surface.
  • the surface of the inside of a reaction vessel may comprise an extracting phase in which extraction can take place.
  • Such extracting phases can very suitably be applied in the form of a coating or cover layer to a surface which can be contacted with the reaction mixture so that that surface is provided with extracting properties.
  • Suitable coatings or cover layers preferably comprise lipophilic polymers such as for instance polysiloxanes.
  • Other polymers such as polyacrylates, divinylbenzene, carboxylated PVC, cellulose acetate, mixtures of polymers, "molecular imprinted polymers" (MIPs), protein receptors such as (strep t)avidin may also be used as an extracting phase.
  • polymers such as polydimethylsiloxane (PDMS) are used, more preferably with a Log Kd for the analog of between 1 and 8, preferably between 2 and 5.
  • a very suitable coating layer is formed by a substrate -immobilized polynucleotide which is complementary to (a part of) a polynucleotide ligand to be measured or to be detected, or to which a ligand can bind in a different manner.
  • the substrate-immobilized polynucleotide may, for instance, be immobilized on the inside of a reaction vessel (e.g. the container according to the invention) with the aid of a (strept)avidin coating applied to the substrate and a biotin label applied to the polynucleotide.
  • Such a binding layer may also be suitably applied to solid -phase particles present in the sample solution, e.g. beads from glass or plastic.
  • the reaction mixture is therefore subjected to negligible depletion extraction in which an extracting phase is used which can only bind a small part of the ligands freely present in solution in order to provide negligible depletion of the fraction of free ligands and/or analogs.
  • an extracting phase which can only bind a small part of the ligands freely present in solution in order to provide negligible depletion of the fraction of free ligands and/or analogs.
  • the amount of the extracting phase can be limited, for instance by limiting the dimensions of the thickness of an extracting coating layer or the surface an extracting coating layer or by, for instance, limiting the amount of an extracting coating layer applied to an inner wall of a reaction vessel.
  • a reaction mixture is contacted with an extracting phase which is located on the inside of a reaction vessel (liquid container) and which comprises an extracting polysiloxane in the form of a cover layer of which the amount is limited so that only a small part of the free ligands can be extracted thereby.
  • the limitation in the capacity (that is, inter alia the extraction efficiency) of the extracting phase to extract free ligands is in any case such that less than 20%, preferably less than 10%, more preferably less than 1% of the total amount of free ligands present can be extracted by the extracting phase.
  • more than 0.001% of the fraction of free ligands is extracted by the extracting phase.
  • the amount of the extracting phase in combination with the extracting capacity of the extracting phase is exactly known so that it is known what fraction of the total number of free ligands can potentially be extracted. This fraction can simply be determined in a control experiment.
  • the extraction of the free ligands (L so1 ) by the extracting phase can substantially take place by absorption, but adsorption, adhesion or binding is suitable as well.
  • the amount of extracting phase used to extract a predetermined fraction (for instance about 5%) or particular amount of the free ligands (L so1 ) thereof by means of the extracting phase depends on the partition coefficient (Kd) of the extracting phase used. With a partition coefficient of 10, the ratio of the amount (concentration) of ligand extracted by the extracting phase (L s P hase ) to the amount (concentration) of ligand freely present in solution (L so/ ) will be 10:1.
  • the extracting phase is substantially such that no bound (conjugated) ligand (L con J) is bound thereto and preferably no free binding partner either.
  • This can be provided by providing an extracting phase into which the bound ligands (the conjugates) (L cm J) and preferably also free binding partners cannot penetrate due to their size, but into which free ligands (L so1 ) can penetrate, for instance by means of diffusion, and can then absorb.
  • the analog is preferably a detectable form of the ligand, for instance a labeled form.
  • a method for detecting and/or measuring a ligand according to the present invention comprises the step of detecting and/or measuring the extracted ligand.
  • the determination thereof may, for instance, be done by separating the liquid in which free ligand is present from the extracting phase and determining the amount of ligand in the extracting phase. The determination may be done by use of, for instance, chromatographic, (mass) spectrometric, fluorometric and radiochemical detection techniques.
  • An alternative method for detecting and/or measuring a ligand according to the present invention uses a detectable analog, which method in principle comprises the same steps as those set forth hereinabove.
  • This alternative method comprises the step of a) providing a binding partner of the ligand and a detectable analog of the ligand in the liquid. This can be done in a similar manner as described hereinabove.
  • the alternative method further comprises the step of b) allowing the occurrence of competitive binding between the ligand and the analog of the binding partner in order to provide an amount of bound analog and an amount of free analog in the liquid.
  • This step is in principle the same as the step of subjecting the liquid to conditions in which the ligand can bind to the binding partner in order to provide an amount of bound ligand and an amount of free ligand, with this difference that, in the presence of analog, competition for binding sites will occur between ligand and analog.
  • the alternative method further comprises the step of c) subjecting the liquid to negligible depletion extraction whereby a part of the amount of free analog is extracted by an extracting phase, this extracting phase having the form of a surface coating on the wall of a liquid container or on particles mixable with the liquid.
  • this step it again holds true that it can largely be carried out as described hereinabove.
  • the alternative method comprises the step of d) detecting and/or measuring the extracted analog.
  • This step provides an advantage over the variant of the method mentioned first because a detectable analog can be used therein, which can be detected and/or measured in a much simpler manner than the ligand in the first method.
  • a method according to the invention can comprise the step of detecting the fraction of the analog of the ligand to be investigated extracted by the extracting phase (A s P hase ). The purpose of this step is to determine the fraction (concentration) of the free analog in solution (A so1 ) in order to be able to determine the fraction of the analog bound to the binding partner (A con ->) therefrom.
  • A A so1 + A con J + A s P hase , in which A is the analog, A so1 is the fraction of free analog in solution, A con J is the fraction of bound analog, Asphase j g the fraction of analog in extracting phase.
  • L may, for instance, be determined by making use of one calibration line, that is, by calibration of the assay with at least two known concentrations of ligand L, from which an unknown concentration L can be "read". In the present specification, in this case this is called a step of "measuring (the concentration of) a ligand". So, such a measurement may comprise a quantitative measurement.
  • An alternative method for determining L is connected with detecting
  • Detection of the fraction of the analog extracted by the extracting phase may for instance be done by separating the reaction mixture or the solution, with therein inter alia the ligands and analogs freely present in solution and the bound ligands and analogs, from the extracting phase, removing the detectable analog from the extracting phase, for instance by use of an organic solvent, and detecting the thus released analog, while the manner of detection will depend on the label used.
  • Detection may take place by use of, for instance, chromatographic, (mass) spectrometric, fluorometric and radiochemical detection techniques.
  • detection of the fraction of the ligand (L s P hase ) and/or analog (A s P hase ) extracted by the extracting phase may also be done by separating the reaction mixture or the solution from the extracting phase and detecting the ligand and/or analog without removing the ligand and/or analog from the solid phase.
  • a scintillation proximity assay SPA
  • SPA scintillation proximity assay
  • the scintillation proximity assay is based on the fact that 6 rays are emitted by compounds labeled with weak radioisotopes (such as 3 H, 125 1, 33 P and 35 S) can only cover a limited distance in an aqueous environment before their energy is lost completely. However, these emissions can be detected with high sensitivity if the compounds labeled with radioisotopes are brought in the immediate proximity of an extracting phase provided with a scintillator (such as for instance 2,5-diphenyloxazole) so that a specific emission is obtained. Radio-labeled compounds which are freely present in solution are not detected because they are too far removed from the scintillating extracting phase.
  • SPA technology is inter alia commercially available under the name FlashPlate® from NEN Life Science Products.
  • the present invention further relates to a liquid container for detecting and measuring a ligand in a liquid, which container is provided with an extracting phase having the capability to extract the ligand from the liquid phase, this extracting phase having the form of a surface coating on the wall of a liquid container or on particles mixable with the liquid, and which container is further provided with detection means.
  • a liquid container may, for instance, comprise a bottom, walls and an opening, the walls being at least partly provided with the extracting phase.
  • the extracting phase is provided with detection means, such as for instance scintillators for the detection of radioisotopes.
  • WO 02/101084 also discloses a container with a solid phase and scintillators; however, in this container (which not at all used for a method according to the present invention), the solid phase is not present as a coating of the wall of the container or on particles mixable with the liquid.
  • Suitable primary scintillators include organic compounds with phenyl, naphthyl and/or biphenyl groups, and oxazole and oxadiazole and related substances.
  • a (solid) scintillator preferably compounds like 2,5-diphenyloxazole (PPO), butyl phenyl biphenyloxadiazole (butyl-PBD), and p-terphenyl (TP) are used.
  • Suitable secondary scintillators include diphenyloxazolyl benzene (POPOP), l,4-bis(2-methylstyryl) benzene (bis- MSB), 2-(l-naphthyl)-5-phenyloxazole (NPO), dibiphenylyloxazole (BBO), diphenylhexatriene (DPH) or tetraphenylbutadiene (TPB).
  • POPOP diphenyloxazolyl benzene
  • bis- MSB 2-(l-naphthyl)-5-phenyloxazole
  • NPO 2-(l-naphthyl)-5-phenyloxazole
  • BBO dibiphenylyloxazole
  • DPH diphenylhexatriene
  • TPB tetraphenylbutadiene
  • the scintillator can be used in any known manner and may, for instance, very suitably be provided in a coating layer.
  • a method for providing a scintillator in a coating layer may, for instance, comprise mixing a suitable polymeric or solid phase with a suitable scintillator and applying a layer of this mixture to a support, such as the wall of the relevant container, optionally followed by curing of the cover layer thus applied.
  • a part of the inside of the container is coated with a lipophilic polymer such as for instance a polysiloxane or other lipophilic polymer as described hereinabove and a scintillator is used therein for detection of weak radioisotopes (such as 3 H, 125 I, 33 P and 35 S).
  • a lipophilic polymer such as for instance a polysiloxane or other lipophilic polymer as described hereinabove and a scintillator is used therein for detection of weak radioisotopes (such as 3 H, 125 I, 33 P and 35 S).
  • PDMS polydimethylpolysiloxane
  • the container need not be a conventional liquid container.
  • the liquid container may, for instance, also be a hollow fiber or a polystyrene or glass container in the form of a microtiter plate, such as a 96-well plate. This makes the present invention excellently suitable for high throughput screening assays.
  • ligands in a liquid can be detected qualitatively and quantitatively.
  • binding experiments can be carried out, for instance in screening reactions in which detecting radioactivity by use of a liquid container according to the invention is an indication of binding and/or competition.
  • new potential active substances can be identified which bind to particular receptors.
  • the method can be used as analytical chemical determination. Further, the method can be used in the determination of partition coefficients between a particular substance and water as it is important in pharmacokinetics for determination of plasma binding of pharmaceuticals.
  • the invention further relates to a test kit for detecting and/or measuring a ligand in a liquid, comprising a liquid container according to the invention.
  • the test kit may further comprise an instruction leaflet in which the application possibilities of the test kit and, for instance, a suitable method for detecting and/or measuring a ligand in a liquid are described.
  • the test kit comprises one or more suitable binding partners for a ligand to be detected and/or measured.
  • the test kit comprises one or more suitable analogs.
  • the different components can be comprised in combination in a test kit according to the invention.
  • the binding partners and analogs may be packaged in any suitable manner.
  • additional materials will be used in a method carried out with the aid of a liquid container according to the invention and may be present in a kit.
  • various reagents such as reaction buffers will be present in a test medium, as well as stabilizers for the test medium and the test components.
  • supplements will be comprised, for instance supplementary proteins, such as albumin; surfactants, particularly nonionic surfactants; binding promoters, for instance polyalkylene glycols and dextranes; preservatives; and antimicrobial agents, and the like.
  • the test kit can be used for diagnostics and research as described hereinabove.
  • Example 1 competitive binding assay of estradiol in competition with estriol in the presence of an E2-antiserum
  • estriol is used as a ligand and [ 3 H] 17 ⁇ - estradiol as an analog to determine the binding of estriol to E2-antiserum in a buffer.
  • a [ 3 H]17 ⁇ -estradiol dilution with a desired concentration of approx. 5 40 pg/10 ⁇ l (corresponding with approx. 25,000 DPM/10 ⁇ l) is used.
  • a standard series comprising 12 standard concentrations (0-1600 pg/ml of ligand) is composed by dissolving a known amount of ligand in a known amount of Tris/BSA working buffer (0.01 mol/1 Tris-HCl [pH 7.5]; 1 mg/ml BSA). Each standard concentration has such a concentration 0 that 200 ⁇ l thereof yields the desired concentration of the standard series.
  • a 5,00Ox diluted solution is used of rabbit serum generated against estradiol (rabbit anti-estradiol).
  • estradiol rabbit serum generated against estradiol
  • a skilled person will be familiar with the possibilities to make an anti-estradiol antiserum coming from rabbits.
  • 5 A glass (injection) vial (Packard, miniature vial 6 ml) was provided with a PDMS coating.
  • the PDMS coating has the function of extracting surface.
  • the coated vials are filled with 200 ⁇ l of the respective standard concentration, lO ⁇ l of the working tracer and 100 ⁇ l of the antiserum 0 working dilution (5,00Ox).
  • the vials are supplemented with Tris/BSA working buffer to a total volume of 1 ml per vial. Then, the vials are incubated (this may, for instance, be done overnight at approx. 4°C or 90 minutes at 37°C).
  • the above example describes a simple, quick and accurate method for testing, for instance, substances suspected of estrogenicity for estrogenicity.
  • Example 2 Analysis of the free oligonucleotide concentration in blood plasma with the aid of streptavidin-coated microtiter plates.
  • General principle A streptavidin-coated microtiter plate is coated with an oligonucleotide template as an extracting phase. Depending on the number of nucleotides, this oligonucleotide template has a particular oligonucleotide chain length and serves as a "capture" oligo of an oligonucleotide ligand to be investigated.
  • a biotin molecule is coupled with which the oligonucleotide template binds to the streptavidin in the well (the well of the microtiter plate). In view of the very high biological affinity between streptavidin and biotin, this binding will be very strong and will take place relatively rapidly (approx. 30 minutes).
  • the chain length of the respective oligonucleotide template is chosen.
  • the oligonucleotide template is diluted with sterilized demineralized water.
  • the plasma binding of an oligonucleotide ligand comprising 20 nucleotides is investigated.
  • the nucleotide sequence of this ligand was 5'-CTG*C*T*A*GCCTCTGGATTTGA-3' (in which * stands for a [ 35 S] label).
  • a biotinylated oligonucleotide template of 12 nucleotides is used as an extracting phase.
  • the nucleotide sequence of this 12-mer oligo was 5'-AGAGGCTAGCAG-'3 and is partly complementary to the oligonucleotide ligand.
  • biotinylated oligonucleotide template a stock solution of 1 mM was diluted further to a 10 ⁇ M working solution. Of this working solution, 100 ⁇ l was provided in each well of a 96 -well microtiter 5 plate in which streptavidin had already been immobilized and incubated for 30 minutes at 37°C by the manufacturer. After incubation, the wells were rinsed with 4 x 300 ⁇ l wash buffer (25 mM TRIS/HCl (pH: 7.2); 0.15 M NaCl; 0.1% Tween) with the aid of a multi-channel pipette.
  • wash buffer 25 mM TRIS/HCl (pH: 7.2); 0.15 M NaCl; 0.1% Tween
  • the plasma dilution series (in PBS) was: 99; 90; 75; 50; 25; 20; 15; 10; 5; 4; 3; 2,5; 2 and 1% plasma in PBS.
  • Each 200 ⁇ l of plasma dilution was spiked with 10 ul solution of the 20-mer oligonucleotide to be measured after which it was incubated for a total of 2 hours at 37°C.
  • the contents of the wells were rinsed with 5 ice-cold ( ⁇ 4°C) demineralized water.
  • the wells were filled with approx. 200 ⁇ l of scintillation liquid (Optiphase Hisafe II, Wallac) after which the microtiter plates were analyzed with the aid of a Liquid Scintillation Counter (LSC).
  • LSC Liquid Scintillation Counter
  • the measured counts come from free ligand which can bind to the o extracting phase, while the fraction of ligand bound by plasma has been removed during rinsing.
  • the measured counts present in the coated wells have been plotted in a graph versus the volume fraction of plasma so that a decrease curve of the free oligonucleotide concentration is obtained (see also Figure 1).
  • Figure 1 shows the decrease curve of the free oligonucleotide 5 concentration with an increasing plasma fraction.
  • the above example describes a simple, quick and accurate method for determining free concentrations of oligonucleotides in plasma. This may, for instance, be important when an oligonucleotide is used therapeutically, where it is important to know whether all active substance is bound by the o plasma or whether a free (active) fraction remains as well.
  • the extraction efficiency for this oligo of 12 nucleotides is about 5%, which can be considered negligible.
  • Extension of the complementary chain length increase of the complementarity between extracting phase and ligand) would result in a too high and therefore non-negligible depletion extraction.

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Abstract

L'invention se rapporte à un procédé de détection de la liaison entre un ligand et son partenaire de liaison et fournit donc un dosage de liaison compétitif amélioré en vue de la détection et de la mesure d'un ligand. Cette invention concerne également un contenant pour liquides permettant de détecter et de mesurer un ligand dans un liquide, lequel comprend, en plus d'une phase d'extraction capable d'extraire un ligand de la phase liquide, des moyens de détection afin de détecter des ligands ou des analogues de ceux-ci.
PCT/NL2005/000517 2004-07-19 2005-07-18 Procede de detection et de mesure d'un ligand WO2006009435A2 (fr)

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