+

WO1996039518A1 - Polypeptides fixant le plomb et nucleotides codants afferent - Google Patents

Polypeptides fixant le plomb et nucleotides codants afferent Download PDF

Info

Publication number
WO1996039518A1
WO1996039518A1 PCT/US1996/009258 US9609258W WO9639518A1 WO 1996039518 A1 WO1996039518 A1 WO 1996039518A1 US 9609258 W US9609258 W US 9609258W WO 9639518 A1 WO9639518 A1 WO 9639518A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
monoclonal antibody
ser
acid sequence
nucleic acid
Prior art date
Application number
PCT/US1996/009258
Other languages
English (en)
Inventor
Dwane E. Wylie
Osvaldo Lopez
Peter Joseph Murray
Original Assignee
Bionebraska, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bionebraska, Inc. filed Critical Bionebraska, Inc.
Priority to AU60946/96A priority Critical patent/AU6094696A/en
Priority to US08/767,128 priority patent/US6111079A/en
Publication of WO1996039518A1 publication Critical patent/WO1996039518A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Heavy metal can exhibit dual roles.
  • lead is used in glass making and in chemical manufacturing operations. Yet when ingested by mammals, such as from drinking water, lead may be highly toxic in very small amounts. Hence, detection and quantification of minute concentrations of a heavy metal, such as lead, in drinking water and other media would serve exploratory, safety and regulatory goals.
  • Monoclonal antibodies of course, come to mind as especially suited agents for practice of this technique. Since Kohler and Milstein published their article on the use of somatic cell hybridization to produce monoclonal antibodies (Nature 256:495 (1974)), immunologists have developed many monoclonal antibodies which strongly and specifically immunoreact with antigens.
  • Disadvantages of antigen measurement methods based on such antibodies include: the complicated reagents involved in detection, lack of simple tests that discriminate among antigens, cross-reactivity with chelates of other antigens and cross-reactivity with the chelating agent itself.
  • Other instances of monoclonal antibody combinations with metals involve metal tags. The metals or metal chelates are bound to the antibody at a site remote from the antigen binding site or sites. The metal tag is not the antigen. Instead, the metal tag is used to indicate the presence of the monoclonal antibody when it reacts with its specific antigen. See for example, V.P. Torchilian et al., Hybridoma, 6., 229 (1987); and C.F.
  • the present invention provides a metal binding polypeptide which selectively binds a heavy metal, such as a lead cation.
  • the metal binding polypeptide includes an amino acid sequence for a variable region from a monoclonal antibody, wherein the monoclonal antibody immunoreacts with a lead cation.
  • the metal binding polypeptide may include an amino acid sequence for a heavy chain Fd fragment (consisting of the heavy-chain variable region and heavy-chain constant region 1 domains) from the monoclonal antibody.
  • the metal binding polypeptide may further include a heavy chain Fc fragment fused to the heavy chain Fd fragment or a phage coat protein or portion thereof fused to the heavy chain Fd fragment.
  • the metal binding polypeptide may include an amino acid sequence for a light chain from the monoclonal antibody.
  • Another embodiment of the invention provides a recombinantly produced Fab fragment that immunoreacts with a lead cation.
  • the recombinantly produced Fab fragment includes an amino acid sequence for a variable region from a monoclonal antibody which immunoreacts with the lead cation.
  • the Fab fragment includes a heavy chain Fd fragment or a light chain from the monoclonal antibody.
  • the present invention also provides a purified antibody which includes a Fab fragment.
  • the Fab fragment immunoreacts with a lead cation and includes an amino acid sequence selected from a group of sequences for a variable region of certain specified monoclonal antibodies.
  • the Fab fragment heavy chain preferably includes an amino acid sequence selected from a group of the sequences for the heavy chain variable region of the specified monoclonal antibodies.
  • the Fab fragment light chain includes an amino acid sequence selected from a group of the sequences for the light chain variable region of the specified monoclonal antibodies.
  • the monoclonal antibody may be a recombinantly produced monoclonal antibody.
  • the invention is also directed to a heavy chain of the monoclonal antibody.
  • the heavy chain preferably includes a sequence selected from a group of the sequences for the heavy chain variable region of certain specified monoclonal antibodies.
  • the invention is also directed to a light chain of the monoclonal antibody. As with the heavy chain, the light chain preferably includes a sequence selected from a group of the sequences for the light chain variable region of certain specified monoclonal antibodies.
  • Yet another embodiment of the invention is directed to an isolated nucleic acid sequence coding for a variable region of a monoclonal antibody, e.g., the heavy chain variable region or the light chain variable region of the monoclonal antibody.
  • the monoclonal antibody immunoreacts with a lead cation.
  • the isolated nucleic acid sequence may code for the heavy chain Fd fragment, the entire heavy chain or the entire light chain of the monoclonal antibody.
  • the present invention is also directed to an expression cassette.
  • the expression cassette includes a nucleic acid sequence coding for a variable region of the monoclonal antibody which immunoreacts with a lead cation.
  • the nucleic acid sequence coding for the variable region is operably linked to a promoter functional in a vector.
  • the expression cassette may include the promoter operably linked to a nucleic acid sequence coding for a heavy chain Fd fragment of the monoclonal antibody.
  • the expression cassette may include the promoter operably linked to a nucleic acid sequence coding for a light chain of the monoclonal antibody.
  • the expression cassette may also include a leader sequence located between the promoter and the nucleic acid sequence coding for the monoclonal antibody chain.
  • the leader sequence may function to direct the heavy or light chain to a membrane in a host cell or to cause the antibody chain to be secreted by the host cell.
  • the present invention provides a fusion protein which includes a phage coat protein or portion thereof fused to an amino acid sequence for a heavy chain variable region from the monoclonal antibody.
  • the fusion protein preferably includes the heavy chain Fd fragment of the monoclonal antibody.
  • the fusion protein may be present as part of the coat of a phage and, preferably, the coat of a filamentous phage.
  • Another embodiment of the present invention is directed to an expression cassette coding for a fusion protein.
  • This expression cassette includes a first nucleic acid sequence coding for a heavy chain Fd fragment of a monoclonal antibody which reacts with a lead cation.
  • the first nucleic acid sequence is linked for co-expression to a second nucleic acid sequence, such as a nucleic acid sequence coding for a phage coat protein or a portion thereof, to form a nucleic acid sequence encoding the fusion protein.
  • the fusion protein includes the heavy chain Fd fragment fused to the phage coat protein or portion thereof.
  • the expression cassette coding for the fusion protein also includes a promoter that is functional in a vector.
  • the promoter is operably linked to the first and second DNA sequences and provides for expression of the fusion protein.
  • the expression cassette may also include a leader sequence which directs expression of the fusion protein to a membrane of a host cell. The leader sequence is located between the promoter and the nucleic acid sequence coding for the fusion protein.
  • the expression cassette may include a third nucleic acid sequence coding for a peptide linker. The third nucleic acid sequence is typically located between the first and second nucleic acid sequences.
  • the expression cassette may optionally include a fourth nucleic acid sequence coding for a light chain of a monoclonal antibody.
  • the light chain is a light chain of a monoclonal antibody that immunoreacts with a lead cation.
  • the present invention also provides a phagemid vector which includes one of the expression cassettes described above.
  • the invention is also directed to methods for detecting, removing, adding, or neutralizing the heavy metals in biological and inanimate systems through the use of the metal binding polypeptides, heavy and light chains, fusion proteins, recombinantly produced Fab fragments and monoclonal antibodies described above.
  • the advantages of the invention include among others : the lack of complication by additional reagents, a high discrimination against similar metallic cations, lack of cross-reactivity with similar metallic cations, and lack of cross-reactivity with test reagents.
  • the metal binding polypeptide of the invention binds with a heavy metal per se, and preferably with a lead cation per se.
  • the metal binding polypeptide binds with a heavy metal cation which is at least partially exposed, i.e., a heavy metal cation which is not complexed or enveloped.
  • the heavy metal cation may be associated with a spacer arm which includes a sulfur atom as a Lewis base group.
  • the state of the heavy metal during the immunoreaction is preferably one of non-coordination with any other substance; in other words, bare or exposed.
  • the metal binding polypeptide exhibits a substantially high degree of specific immunoreactivity toward the heavy metal.
  • the metal binding polypeptide includes a portion of a recombinantly produced Fab fragment (e.g., the light chain or the heavy chain Fd fragment of the Fab fragment) and has an association constant for a heavy metal such as a lead cation that is about 10,000 fold greater than the association constant for the immunogen compound without the heavy metal.
  • a heavy metal such as a lead cation
  • metal binding polypeptide is immunospecific for a particular member of a group of very similar heavy metals.
  • the monoclonal antibody will typically exhibit a relative association constant for such a particular heavy metal that is about 10,000 fold greater than that for the other heavy metals of such a group.
  • the hybridoma of the invention which produces the monoclonal antibody, is formed from immune cells that are specific for the heavy metal.
  • the formation may be accomplished by fusion of an immortal mammal cell line and mammalian immune cells from a second mammal previously immunized with an immunogen compound which contains the heavy metal.
  • a lead cation monoclonal antibody produced by this first type of hybridoma is hereinafter referred to as a "Group 1 lead cation monoclonal antibody.”
  • the hybridoma may be formed by fusion of an immortal mammal cell line and mammalian immune cells from a second mammal previously immunized with a second monoclonal antibody capable of immunoreacting with the heavy metal.
  • a lead cation monoclonal antibody produced by this latter type of hybridoma is hereinafter referred to as a "Group 3 lead cation monoclonal antibody.” Selection of the appropriate hybridoma may be carried out by cross- screening the secreted monoclonal antibody against the heavy metal and against controls.
  • the immunogen compound of the invention is typically composed of a biopolymer carrier, a spacer arm covalently bonded to the carrier and the heavy metal coordinated to the spacer arm.
  • the spacer arm is preferably semi-rigid and has at least one heavy metal coordination site. This arrangement maintains the heavy metal in at least a partially exposed state and prevents substantially complete inclusion or chelation of the heavy metal by spacer arm and/or carrier.
  • the biopolymer carrier may be a polysaccharide, a synthetic polyamide or preferably a protein. Preferred classes include blood or tissue sera proteins.
  • the spacer arm is generally no more than about 25 atoms in length.
  • the spacer arm is composed of one of three classes: an oligopeptide, an aliphatic compound or an aliphatic fragment. More preferably, the spacer arm is an oligopeptide.
  • the first two classes are generally each substituted with no more than about 2 pendent Lewis acid or base groups, and a coupling group for forming a covalent bond with the protein carrier.
  • the aliphatic fragment is substituted by a coupling group for forming a covalent bond with the protein carrier, and a carboxylic acid, hydroxyl, mercapto, amine or other group adapted for interacting with the heavy metal.
  • the coupling group is an amine, carboxylic acid, aldehyde, hydroxyl or mercapto group.
  • a preferred spacer arm for metallic cations is an oligopeptide or aliphatic compound having no more than about 2 pendent Lewis base groups wherein the deformation of the electron shell of the Lewis base group is approximately of the same character as the deformation of the electron shell of the metallic cation.
  • Especially preferred Lewis base groups for transition elements and the heavy metals are those containing sulfur, such as a sulfonate or thiol group.
  • spacer arms which include glutathione and cysteine, mercapto ethanol amine, dithiothreitol, amines and peptides containing sulfur, and the like.
  • Other preferred spacer arms include a carboxylic acid group or a phosphorus-containing Lewis base group such as a phosphonate anion.
  • the metallic cations are derived from metals such as period four transition metals, and period five, six and seven metals, transition elements and inner transition elements.
  • the metallic cations of special mention as the heavy metal include those derived from zinc, lead, cadmium, bismuth, cobalt, arsenic, chromium, copper, nickel, strontium and mercury.
  • the metallic cations are lead cations, e.g., lead(II) cations.
  • the present invention also provides a method of forming a hybridoma which produces an antibody capable of immunoreacting with a lead cation and preferably with an exposed form of the lead cation.
  • the method includes inoculating a mammal with a solution containing second antibodies which are capable of immunoreacting with the lead cation.
  • Immune cells are isolated from the mammal and fused with an immortal mammal cell line from a second mammal.
  • the resulting fused cell lines are selected for hybridomas which produce monoclonal antibodies capable of immunoreacting with a lead cation (Group 3 lead cation monoclonal antibodies; "Group 3 Pb mAbs”) .
  • Methods according to the invention utilize the metal binding polypeptide for detection, removal, neutralization or addition of the heavy metal respectively in, from, within or to a solid, liquid or gaseous medium. These methods utilize features such as metal binding polypeptide immobilization, heavy metal immobilization, competitive binding, and means employing an oscillating probe, a micromagnetic probe and other physiochemical methods typically used to monitor antigen-antibody interactions. Methods for detection that are based upon heavy metal immobilization may indicate the presence of the heavy metal-metal binding polypeptide conjugate (e.g., a lead cation-Fab fragment conjugate) by known immunologic assay techniques. In a first step, the heavy metal may be coordinated with an immobilized spacer arm.
  • the heavy metal may be coordinated with an immobilized spacer arm.
  • the spacer arm can be any of the foregoing that will hold the heavy metal in at least a partially exposed state. It need not be the same spacer arm of the immunogen compound used to develop the metal binding polypeptide. Non-immobilized materials are then removed from the mixture holding the immobilized spacer arm-heavy metal. Addition of the metal binding polypeptide (e.g., Fab fragment) , removal of uncomplexed metal binding polypeptide and immunoassay complete the steps for this detection method.
  • the metal binding polypeptide e.g., Fab fragment
  • Methods for detection that are based upon an immobilized metal binding polypeptide may utilize a radioactive version of the heavy metal or a similar tagged form thereof.
  • tags include fluorescent, colorimetric and other spectrally active groups that can be coordinated or bonded to the heavy metal like the spacer arm.
  • a preferred tag is a spacer arm containing a spectrally active group.
  • Methods for detection that are based upon an oscillating probe utilize either an immobilized spacer arm for the heavy metal or preferably immobilized metal binding polypeptide.
  • This method measures the change in frequency of an oscillating surface as a function of the change in weight of that surface due to the binding of the non-immobilized heavy metal or metal binding polypeptides are immobilized on the surface of a high frequency oscillating probe.
  • the probe is placed into a medium containing an unknown quantity of heavy metal. Binding of the heavy metal to the immobilized metal binding polypeptide will change the oscillation frequency of the probe. Hence, the degree of change will indicate the level of heavy metal present.
  • an especially preferred method for detection utilizes an oligopeptide having reactive group(s) capable of coordinating with the metal cation.
  • the oligopeptide and the metal binding polypeptide specific for the metal cation unknown are added to the aqueous medium.
  • the medium then is assayed for the presence of metal binding polypeptide cation conjugate.
  • the interaction of the metal binding polypeptide with the metal cation is independent of the order of addition of the reactants and is independent of the identity of the oligopeptide.
  • a fixed support is utilized. Here, either the oligopeptide or the metal binding polypeptide is immobilized on the fixed support.
  • the method is then conducted as related above.
  • the invention contemplates methods for heavy metal removal from, heavy metal neutralization within or heavy metal addition to biological or inanimate systems.
  • an effective amount of the metal binding polypeptide is combined in some fashion with at least part of the system.
  • metal binding polypeptide-heavy metal conjugate is removed by separation means such as immunoprecipitation, immobilization, chromatography, filtration and the like.
  • Pursuant to the neutralization method the metal binding polypeptide-heavy metal conjugate remains in the system until it is removed by non-specific means.
  • the metal binding polypeptide-heavy metal conjugate also remains in the system and the heavy metal is actively incorporated or otherwise used therein.
  • the metal binding polypeptide may be combined with a pharmaceutically acceptable carrier.
  • the metal binding polypeptide will not of itself cause an undesirable immune response of the biological system.
  • the biological systems contemplated according to the invention include unicellular organisms, multicellular simple organisms, cellular component systems, tissue cultures, plants and animals, including mammals.
  • the present invention also contemplates methods for removing heavy metallic cations or radioactive compounds from human fluids such as blood, serum or lymph by utilization of immobilized metal binding polypeptides.
  • An extracorporeal shunt placed in the patient permits removal of the body fluid and its reintroduction. Passing the body fluid extracorporeally through a bed of immobilized metal binding polypeptide accomplishes the desired removal.
  • the present invention also contemplates a kit for assaying the presence and quantity of heavy metal in a biological or inanimate system.
  • the kit includes aliquots of metal binding polypeptides in the appropriate buffer, as well as a fixed support for absorption of the heavy metal, washing solutions, reagents such as enzyme substrates, and metal binding polypeptide specific antisera conjugated to a detectable substrate.
  • Figures 1A-1B depict the nucleotide and deduced amino acid sequences for amino acids 1 through 113 of the heavy chain variable regions of monoclonal antibodies which immunoreact with a lead cation.
  • the numbering scheme is according to Kabat et al. , Sequences of Proteins of Immunological Interest, vol. II. 5th edition, U.S. Department of Health and Human Services (1991) (hereinafter "Kabat”) .
  • Dashes indicate sequence identity with the 6B11 sequence; periods indicate gaps compared to the 1254 sequence.
  • Figure 2 depicts the nucleotide and deduced amino acid sequences for amino acids 5 through 108 of the light chain variable regions of monoclonal antibodies which immunoreact with a lead cation.
  • the numbering scheme is according to Kabat. Dashes indicate sequence identity with the 6B11 sequence.
  • Figures 3A-3B depict the nucleotide and deduced amino acid sequences for amino acids 1 through 113 of the heavy chain variable regions of monoclonal antibodies which immunoreact with a lead cation.
  • the numbering scheme is according to Kabat. Periods indicate gaps.
  • Figure 4 depicts the nucleotide and deduced amino acid sequences for amino acids 1 through 107 of the light chain variable regions of monoclonal antibodies which immunoreact with a lead cation.
  • the numbering scheme is according to Kabat.
  • Figure 5 depicts the nucleotide and deduced amino acid sequences for amino acids 1 through 113 of the heavy chain variable regions of monoclonal antibody 2B4 which immunoreacts with a lead cation.
  • the numbering scheme is according to Kabat.
  • Figure 6 depicts the nucleotide and deduced amino acid sequences for amino acids 1 through 113 of the heavy chain variable regions of monoclonal antibody 2E7 which immunoreacts with a lead cation.
  • the numbering scheme is according to Kabat.
  • Figure 7 shows the sensitivity of a set of Group 1 monoclonal antibodies (standardized to 50 ⁇ g/mL in PBS) to lead (as Pb(N0 3 ) 2 ).
  • the indicated amounts of lead were added to a microtiter plate coated with an amino acid polymer formed from glutamic acid, lysine, and tyrosine (hereinafter "EKY polymer”) and analyzed by ELISA.
  • EKY polymer amino acid polymer formed from glutamic acid, lysine, and tyrosine
  • Figure 8 shows the sensitivity of a second set of Group 1 monoclonal antibodies (standardized to 50 ⁇ g/mL in PBS) to lead (as Pb(N0 3 ) 2 ) •
  • the indicated amounts of lead were added to a microtiter plate coated with EKY polymer and analyzed by ELISA. The measured absorbances were corrected for the absorbance of an average of four control wells.
  • Figure 9 shows the sensitivity of a set Group 3 monoclonal antibodies (standardized to 50 ⁇ g/mL in PBS) to lead (as Pb(N0 3 ) 2 ) •
  • the indicated amounts of lead were added to a microtiter plate coated with EKY polymer and analyzed by ELISA. The measured absorbances were corrected for the absorbance of an average of four control wells.
  • Figure 10 shows the sensitivity of a second set of Group 3 monoclonal antibodies (standardized to 50 ⁇ g/mL in PBS) to lead (as Pb(N0 3 ) 2 ) .
  • the indicated amounts of lead were added to a microtiter plate coated with EKY polymer and analyzed by ELISA. The measured absorbances were corrected for the absorbance of an average of four control wells.
  • Figure 11 shows the sensitivity of a third set of Group 3 lead cation monoclonal antibodies (standardized to 50 ⁇ g/mL in PBS) to lead (as Pb(N0 3 ) 2 ) .
  • Monoclonal antibody 3A6 which is specific for the PR8 influenza virus, was included as a control.
  • the indicated amounts of lead were added to a microtiter plate coated with EKY polymer and analyzed by ELISA. The measured absorbances were corrected for the absorbance of an average of four control wells.
  • Metal binding polypeptides of the present invention are key to the development of methods for detecting, adding, neutralizing or removing minute quantities of heavy metals.
  • the invention contemplates metal binding polypeptides which selectively bind with a heavy metal, e.g. monoclonal antibodies to heavy metals.
  • a heavy metal e.g. monoclonal antibodies to heavy metals.
  • Hybridomas for the monoclonal antibodies and immunogen compounds for carrying the heavy metals and inducing immunogenicity are also included in the present invention.
  • the metal binding polypeptides may include a monoclonal antibody, a recombinantly produced Fab fragment or a fusion protein.
  • the fusion protein includes the heavy chain variable region of a monoclonal antibody which is capable of immunoreacting with a heavy metal, such as a lead cation.
  • the invention also provides methods for the detection, addition, neutralization or removal of heavy metals using the metal binding polypeptides.
  • the monoclonal antibodies of the invention are mammalian immunoglobulin proteins which have strong affinity constants for a specific heavy metal.
  • the monoclonal antibodies are from the IgG, IgA, IgM and IgE classes of antibodies, and more preferably, from the IgA, and IgM classes of antibodies.
  • the heavy metal monoclonal antibodies are typically characterized by selective immunoreactivity with a particular heavy metal and a substantially lower immunoreactivity with other similarly structured heavy metals.
  • the monoclonal antibodies have an association constant for the selected heavy metal that is at least about 10,000 fold greater than the association constant for any similarly structured heavy metal.
  • the especially preferred IgM and IgA classes of monoclonal antibodies of the present invention exhibit discriminatory dissociation constants of about 10 ⁇ 6 M to about 10 ⁇ 12 M.
  • Examples include monoclonal antibodies of the IgM class which are produced by hybridomas 6B11, 1254, 7D10, 4E8, 8E7, 14F11 (ATCC NO. HB 11330), 10G5 and 14G11 and have a dissociation constant for lead cation of less than about 10 -9M but do not bind cadmium, copper, zinc, nickel and cobalt cations to any appreciable extent.
  • Examples include monoclonal antibodies of the IgM and IgA classes which are produced by hybridomas derived from the spleen cells of a mammal injected with a solution of antibodies capable of immunoreacting with a lead cation.
  • Suitable examples include monoclonal antibodies of the IgM class which are produced by hybridomas 13D10, 2E7, and 6F5.
  • Monoclonal antibodies produced by hybridoma 11D11, 2B6, 13E8 and 8E7 are examples of antibodies of the IgA class which are capable immunoreacting with a lead cation.
  • the immunogen compounds for generation of the specific immunogenicity of the monoclonal antibodies are based upon the hapten-carrier concept.
  • the present invention broadens this concept so that the hapten is coordinated at the end of a spacer arm covalently bonded to the carrier.
  • the spacer arm is adapted so as to be semi-rigid and to hold the heavy metal in an exposed position relative to the carrier.
  • This arrangement is also adapted to maintain the heavy metal in a substantially exposed and preferably, essentially completely exposed state.
  • the spacer arm may be an oligopeptide, an aliphatic compound, or an aliphatic fragment.
  • the aliphatic compound or fragment may be covalently bonded to the carrier by means of a Schiff base reaction with an aldehyde group, an amide formation reaction with an amine or carboxylic acid group using a peptide activator such as carbodiimide, acid chloride and the like, an ester formation reaction with a hydroxyl or carboxylic acid group using a Schotten Bauman reaction, or azide or acid catalysis reaction, a sulfide reaction using a sulfide coupling agent, or other known coupling reactions for joining organic molecules to proteins.
  • the oligopeptide, aliphatic compound or fragment will contain backbone groups which provide semi-rigidity to the spacer arm.
  • Preferred groups for developing this semi-rigidity include peptide bonds, olefin bonds, olefinic conjugated systems, ester groups and enone groups.
  • one or more aromatic rings can be incorporated into the spacer arm to stimulate the development of an immune response.
  • the oligopeptide spacer arm has the following formula: - X - (R) - Y wherein X is a coupling group that will bond to the carrier, R is one or more amino acid residues and Y is the Lewis Acid or Base group(s) for heavy metal coordination.
  • the aliphatic compound or fragment spacer arm has the following formula:
  • X is a coupling group that will bond to the carrier
  • Q is a semirigid aliphatic moiety containing ester, amide, keto, olefin or aromatic groups and the like
  • Z is a Lewis acid or Base group(s) for heavy metal coordination.
  • the oligopeptide or aliphatic compound is used as the spacer arm to coordinate a metal cation.
  • the pendent Lewis base groups will preferably be positioned at the spacer arm end remote from the carrier. These Lewis base groups function as the coordination site or sites for the metal cation. It is preferable that the deformability of the electron shells of the Lewis base groups and the metal cations be approximately similar. Accordingly, sulfur groups can serve as the Lewis base groups when the metal cations are transition metals or inner transition elements.
  • the carrier of the immunogen compound is a large biopolymer that is known to participate in the development of hapten antigenicity.
  • Blood serum proteins, amylopectins, polysaccharides, fetal serum components, biologically acceptable natural and synthetic proteins and polyamides such as polyglycine can serve as the carriers.
  • Preferred carriers include serum and tissue proteins. Examples are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) . Other examples include ovalbumin and chicken gamma globulin. These carriers have sites for coordinate bonding of the spacer arm. Such sites are preferably populated by amine groups, carboxylic acid groups, aldehyde groups and/or alcohol groups.
  • hybridomas The production of hybridomas according to the invention generally follows the Kohler, Milstein technique cited supra. Many heavy metals, however, toxify the mammalian system being used as a source of immune cells. This effect makes it important to determine the highest allowable dose of heavy metal and/or immunogen compound that can be used over a substantially long period of time without killing the host.
  • Pursuant to the Kohler, Milstein technique immunization of the mammalian host may be accomplished within this dose parameter by subcutaneous or intraperitoneal injection of the immunogen compound in adjuvant. Administration is repeated periodically and preferably for at least four injections. Three days before the spleen is removed, a priming injection of immunogen compound is again administered.
  • the spleen cells are fused with immortal mammal cells such as mouse myeloma cells using the techniques outlined by Kohler and Milstein. Polyethylene glycol (PEG) or electrical stimulation will initiate the fusions.
  • immortal mammal cells such as mouse myeloma cells using the techniques outlined by Kohler and Milstein.
  • PEG Polyethylene glycol
  • electrical stimulation will initiate the fusions.
  • the fused cells are then cultured in cell wells according to culture techniques known in the art.
  • the induction of antibodies to heavy metals in a mammalian host may also be accomplished by subcutaneous or intraperitoneal injection of a lead cation monoclonal antibody solution in an adjuvant. These antibodies are typically derived from another animal of the same species.
  • the spleen cells are then isolated and fused with immortal mammalian cells as described above. As before, the fused cells are cultured and the cellular secretions are examined for the presence of the desired antibodies ("Group 3 monoclonal antibodies”) .
  • the latter technique based on the induction of antibodies using a second lead cation antibody may be more efficient than the method based on the injection of a Pb(II) /glutathione/carrier immunogen compound into a mouse.
  • Table 1 herein shows the relative success rate for induction of lead cation antibodies using the two techniques. The results shown in Table 1 demonstrate that the rate of generating lead cation monoclonal antibody producing hybridomas formation derived from injection of the 1254 mAb was greater than twenty fold more efficient than the method based on injection of the OVA/glutathione/Pb(II) immunogen compound.
  • a single set of fusions of spleen cells from animals injected with the 1254 mAb produced more lead cation antibodies than the total number of lead cation antibodies produced by five sets of fusions of spleen cells induced with the immunogen compound.
  • the selection technique for identifying the appropriate monoclonal antibody is an important aspect for determining the immunospecificity desired according to the invention.
  • the selection techniques according to the invention call for determining the binding affinity of the hybridoma cellular products against the heavy metal and against cross-reactive controls.
  • hybridoma culture fluid is tested in screening assays against the heavy metal, the carrier, the carrier-spacer arm product and the immunogen compound as well as optionally against the spacer arm- heavy metal coordinate. Screening assays can be performed by immunoenzymatic-assay, immunofluorescence, radioimmunoassay, immunoprecipitative assay or inhibition of biological activity.
  • the hybridoma cultures selected will exhibit strong binding characteristics to the heavy metal (and immunogen compound) and will not bind with the spacer arm-carrier product or with the carrier itself.
  • subcloning to refine the selected culture can be performed. These techniques are known to those skilled in the art. See for example Goding, James Goding, Monoclonal Antibodies: Principles and Practice. 2nd Edition, Academic Press, San Diego, CA 1986, the disclosure of which is incorporated herein by reference. Briefly, the appropriately selected cell culture is separated into one cell units which are then recultured. The subclone cultures are then again tested for specific immunoreactivity, lack of cross-reactivity and the amount of monoclonal antibody secreted. Those subcultures exhibiting the highest amounts of secreted monoclonal antibody are chosen for subsequent pilot development.
  • mAb 3A6 a monoclonal antibody specific for the PR8 influenza virus
  • Pb(II) the sensitivity of mAb 3A6 (a monoclonal antibody specific for the PR8 influenza virus)
  • Monoclonal antibody 4A10 was derived from the spleen cells of a mouse injected with a mercury cation.
  • the monoclonal antibodies produced by hybridoma 4A10 cross react with Pb(II) cations as well as immunoreacting with a mercury cation.
  • the set of Group 3 lead cation monoclonal antibodies exhibited lead sensitivity comparable to the "parent" Group 1 antibody, mAb 1254, and to other Group 1 lead cation monoclonal antibodies generated by injection of the OVA/glutathione/lead(II) immunogen compound.
  • the immunogenic host for these hybridomas was the BALB/c mouse and the fusion partner was chosen from the mouse myeloma cell lines P3X63-Ag8.653 or SP2/0.
  • Immunizations to induce Group 1 lead cation antibodies were accomplished with the immunogen compound formed from ovalbumin, glutathione and lead(II) cation functioning as the heavy metal in complete Freund's adjuvant.
  • Immunizations to induce Group 3 lead cation antibodies were accomplished with the lead cation monoclonal antibody designated 1254 ("mAb 1254”) in an adjuvant. All of the cell lines mentioned above are maintained in culture medium and in frozen medium at liquid nitrogen temperature.
  • PCR Amplification PCR amplification of Fd and K regions from the spleen messenger RNA (mRNA) of a mouse immunized with BSA-glutathione-lead cation may be performed as described by Sastry et al . , Proc. Natl. Acad. Sci U.S.A. , 86, 5728 (1989) .
  • the PCR amplification is performed with cDNA obtained by the reverse transcription of the mRNA with a primer specific for amplification of heavy chain sequences or light chain sequences.
  • the PCR amplification of mRNA isolated from spleen cells or hybridomas with oligonucleotides that incorporate restriction sites into the ends of the amplified product may be used to clone and express heavy chain sequences (e.g., the amplification of the Fd fragment) and . light chain sequences from mouse spleen cells.
  • the oligonucleotide primers which are analogous to those that have been successfully used for amplification of V H sequences (see Sastry et al . , Proc. Natl. Acad. Sci U.S.A., 86, 5728 (1989)) , may be used for these amplifications. Restriction endonuclease recognition sequences are typically incorporated into these primers to allow for the cloning of the amplified fragment into a ⁇ phage vector in a predetermined reading frame for expression.
  • Phage assembly proceeds via an extrusion-like process through the bacterial membrane.
  • Filamentous phage M13 has a 406-residue minor phage coat protein (cpIII) which is expressed before extrusion and which accumulates on the inner membrane facing into the periplasm of E. coli.
  • cpIII 406-residue minor phage coat protein
  • the two functional properties of cpIII, infectivity and normal (nonpolyphage) morphogenesis, have been assigned to roughly the first and second half of the gene.
  • the N-terminal domain of cpIII binds to the F' pili, allowing for infection of E. coli, whereas the membrane-bound C-terminal domain, P198-S406, serves the morphogenic role of capping the trailing end of the filament according to the vectorial polymerization model.
  • a phagemid vector may be constructed to fuse the antibody Fd chain with the C-terminal domain of cpIII (see Barbas et al. , Proc. Natl. Acad. Sci. USA. 88. 7978 (1991)) .
  • a flexible five-amino acid tether (GGGGS) which lacks an ordered secondary structure, may be juxtaposed between the expressed Fab and cpIII domains to minimize interaction.
  • the phagemid vector may also be constructed to include a nucleotide coding for the light chain of a Fab fragment.
  • the cpIII/Fd fragment fusion protein and the light chain protein may be placed under control of separate lac promoter/operator sequences and directed to the periplasmic space by pelB leader sequences for functional assembly on the membrane.
  • Inclusion of the phage FI intergenic region in the vector allows for packaging of single-stranded phagemid with the aid of helper phage.
  • the use of helper phage superinfection may result in expression of two forms of cpIII. Consequently, normal phage morphogenesis may be perturbed by competition between the cpIII/Fd fragment fusion protein and the native cpIII of the helper phage for incorporation into the virion.
  • the resulting packaged phagemid may carry native cpIII, which is necessary for infection, and the fusion protein including the Fab fragment, which may be displayed for interaction with an antigen and used for selection. Fusion at the C-terminal domain of cpIII is necessitated by the phagemid approach because fusion with the infective N-terminal domain would render the host cell resistant to infection.
  • the result is a phage displaying antibody combining sites ("Phabs”) .
  • the antibody combining sites, such as Fab fragments, are displayed on the phage coat.
  • This technique may be used to produce Phabs which display recombinantly produced Fab fragments (e.g., recombinantly produced Fab fragments that immunoreact with a lead cation) on the phage coat of a filamentous phage such as M13.
  • recombinantly produced Fab fragments e.g., recombinantly produced Fab fragments that immunoreact with a lead cation
  • phagemid vector which allows the display of antibody Fab fragments on the surface of filamentous phage, has been described (see Barbas et al., Proc. Natl. Acad. Sci. USA. 88. 7978 (1991)) .
  • Xhol and Spel sites for cloning PCR-amplified heavy-chain Fd sequences are included in pComb 3.
  • Sad and Xbal sites are also provided for cloning PCR-amplified antibody light chains. These cloning sites are compatible with known mouse and human PCR primers (see, e.g., Huse et al., Science, 246, 1275-1281 (1989)) .
  • the nucleotide sequences of the pelB leader sequences are recruited from the ⁇ HC2 and ⁇ LC2 constructs described in Huse et al, ibid, with reading frames maintained. Digestion of pComb 3 encoding a selected Fab with Spel and N el permits the removal of the gene III fragment. Because Spel and Nhel produce compatible cohesive ends, the digested vector may also be religated to yield a phagemid that produces soluble Fab. Phabs may be produced by overnight infection of phagemid containing cells (e.g., infected E. coli XL-1 Blue) yielding typical titers of 10 11 cfu/mL.
  • phagemid containing cells e.g., infected E. coli XL-1 Blue
  • the metal binding polypeptide can be used to advantage for detection, neutralization, addition or removal of heavy metals from biological or inanimate systems.
  • the presence of minute quantities of heavy metals in certain instances may be desirable.
  • the presence of inorganic moieties in such mixtures as doping materials for semi-conductors and integrated circuits contributes to the properties of the product. Quality control of the presence and concentration of these heavy metals is essential for the functioning of the product.
  • the detection methods of the invention enable ready and early measurement of the presence of such heavy metals and avoid later production or regulatory difficulties.
  • Heavy metals in biological or inanimate systems can also be removed by methods according to the invention. In the main, immobilization of the metal binding polypeptides on a solid support followed by its mixture with the materials of the biological or inanimate system will remove the heavy metals.
  • the immobilization of the monoclonal antibodies can be accomplished by techniques known to those of skill in the art. See, for example, Affinity Chromatography. CR. Fowe & P.D.G. Sean, John Wiley & Sons, London 1974, the disclosure of which is incorporated herein by reference. Removal is accomplished by passing a fluid mixture of the system ingredients suspected of having the heavy metals over the immobilized metal binding polypeptides. Of course, the metal binding polypeptides are designed to be specific for the heavy metal sought to be removed. An advantage of this method is the removal of undesirable heavy metals in the presence of similarly structured desirable metal species.
  • whole blood from a patient suffering from lead poisoning can be removed from the patient, optionally filtered to return the cellular blood components to the patient, and the serum or blood passed over immobilized metal binding polypeptides specific for the lead.
  • the purified serum or blood can then be returned to the patient.
  • the lead will be removed but other blood serum components such as zinc, calcium and the like will not.
  • a particular application of the present invention contemplates a method for the production of monoclonal antibodies specific for the lead cation or another toxic, heavy metal cation.
  • the heavy metal cation in question is combined into an immunogen compound as described above and suspended in an aqueous medium.
  • a preferred protein carrier for the immunogen compound in this instance is an albumin, e.g., ovalbumin. keyhole limpet hemocyanin is also a preferred carrier.
  • the preferred spacer arm in this instance is an oligopeptide which has sulfonate or carboxylate groups capable of coordinating with the heavy metal cation.
  • the suspension of immunogen compound is used to immunize a host mammal such as a mouse following the techniques outlined above.
  • the laboratory strain of mouse designated BALB/c is particularly preferred.
  • Spleen cells of the immunized host are collected and converted into a suspension. These spleen cells are fused with immortal cells as described above.
  • myeloma cells of the same animal species as the immunized host are used as the fusion partner.
  • a cell fusion promoter such as polyethylene glycol is employed to cause formation of the hybridoma cells.
  • the hybridoma cells are diluted and cultured in a medium which does not allow for the growth of unfused cells.
  • the monoclonal antibodies produced and secreted by the hybridomas are thereafter assayed for the ability to bind immunologically with the heavy metal cations used for immunization.
  • the antibodies are further selected for lack of cross-reactivity with carrier, with carrier- spacer arm and with similar metallic cations.
  • the preferred assay method in this context is an enzyme-linked immunosorbent assay.
  • the resulting monoclonal antibodies are specific for heavy metal cations and exhibit strong immunoreaction to the heavy metal cations in the presence of spacer arm or the spacer arm-carrier composition.
  • Preferred monoclonal antibodies are selectively immunoreactive with lead cations.
  • the present invention also provides methods and kits for detecting the presence of a toxic heavy metal cation such as a lead cation.
  • the methods and kits include a metal binding polypeptide which selectively binds the heavy metal.
  • an immobilized coordinating compound is combined with the unknown mixture containing the toxic heavy metal cation.
  • the heavy metal cation complexes with the coordinating compound and is immobilized thereto. Removal of the non-immobilized components leaves the immobilized toxic heavy metal cation.
  • Addition of the metal binding polypeptide specific for the targeted heavy metal cation forms an immobilized cation-metal binding polypeptide conjugate. Its presence and concentration can be assayed by an ELISA technique or other tagging or visualization technique known to those of skill in the art. In this process, of course, non- immobilized metal binding polypeptide is removed before the assay is conducted.
  • a kit for quantitatively measuring the presence of a heavy metal cation is a further aspect of the invention.
  • the kit includes a metal binding polypeptide specific for the toxic metal cation in question.
  • the metal binding polypeptide is preferably metered into several aliquots of varying, known concentration.
  • the kit may also include the immobilized coordination compound, preferably, attached to a solid support such as the well of a microtiter plate or a chromatographic material.
  • the kit also typically includes a visualization or tagging assay material for determination of the presence of the metal binding polypeptide-heavy metal cation conjugate. If desired, a meter or other device for detecting and signaling the level of visual or other reading from the assay may also be included.
  • Hybridoma antibodies were produced from the spleen cells of BALB/c mouse that had received multiple injections of lead(II) cations reacted with glutathione to produce a lead cation coordinate covalent compound, which was covalently bound to ovalbumin ("OVA/glutathione/Pb(II) antigen") .
  • Glutathione is a three amino acid residue peptide having one reactive sulfhydryl group which forms a coordinate bond with lead cations.
  • the OVA/glutathione/ Pb(II) antigen in complete Freund's adjuvant was utilized to assist in the elicitation of an immune response in the host animal.
  • hybridomas isolated some were determined to be producing monoclonal antibody specific for glutathione.
  • other hybridomas (6B11, 1254, 8E7, 10G5 and 14F11) were producing monoclonal antibodies that were strongly positive against glutathione-lead cation but negative against glutathione without lead cation. These antibodies were subcloned by the process of limiting dilution for further characterization.
  • Microtiter plates (EIA/RIA grade) were treated with BSA-glutathione, blocked with 1% polyvinyl alcohol in phosphate-buffered saline (“PBS”) and used for the
  • the monoclonal antibodies may be assayed with various other metals for inhibition of binding of the monoclonal antibodies to lead cations.
  • the cationic metals assayed may include the ions of zinc, copper, cadmium, nickel, and arsenic.
  • the inhibition of the binding of a monoclonal antibody which immunoreacts with immobilized glutathione-lead cations by various concentrations of divalent cations may be examined.
  • Metal ions at the indicated concentrations are incubated with culture fluid from the antibody in an ELISA plate. The absorbance at 405 nm may be determined for each sample, and the percent inhibition of each metal ion concentration determined by the formula given above.
  • the monoclonal antibodies can establish if the antibodies are specific for the lead cations per se and that glutathione is not needed for the monoclonal antibodies to react with and bind to the lead cations.
  • the monoclonal antibodies may also be assayed against BSA-glutathione, BSA-glutathione-lead cations, and BSA-lead cations. Comparison with a negative control consisting of a monoclonal antibody specific for an unrelated antigen permits a determination of whether the monoclonal antibodies bind to lead cation in the absence of glutathione.
  • PBS containing metal ions at the indicated concentrations may be added to microtiter wells to which BSA-glutathione has been absorbed. After incubation at room temperature for 30 minutes, the plates are washed to remove unbound metals, and the plates are used for a standard ELISA to detect lead cations.
  • the pellet was dissolved in 200 mL of 40% dimethylformamide pH 4.8, containing 200 mg of l-ethyl-3- (3- dimethylaminopropyl) -carbodiimide, and 1 g of either bovine serum albumin or OVA was added to the solution.
  • the reaction mixture was stirred at room temperature overnight.
  • the mixture was then centrifuged as above, resuspended in PBS, and dialyzed overnight at 4°C against 4 liters of PBS.
  • mice Multiple injections of the antigen prepared with 10 ug of protein per injection were made into BALB/c mice. Lead cation-glutathione-OVA emulsified in Freund's adjuvant was employed as the antigen. Complete adjuvant was used for the first two injections, while incomplete adjuvant was used for all subsequent injections. After the fourth injection, a drop of blood from the tail of each mouse was collected separately in 0.5 mL of PBS, and each sample was assayed by ELISA for the presence of antigen-specific antibody. The mice used for hybridoma production received an intraperitoneal injection consisting of 10 ug of antigen in PBS 3-4 days before cell fusion. 3. Hybridoma Production
  • the spleen was removed aseptically from a mouse, and the cells were isolated by placing the spleen in 5 mL of sterile PBS and teasing it with two sterile, 18-gauge hypodermic needles.
  • the cell suspension was added to an empty sterile, conical, 15-mL centrifuge tube and tissue fragments were allowed to settle for 1-2 minutes.
  • the cells still in suspension were placed in a tube similar to that above and centrifuged at 300 g for 10 minutes at room temperature. The cells were then washed 3 times by centrifugation in serum-free DMEM (Dulbecco's modified Eagle's medium).
  • Spleen cells were co-pelleted with P3X63-Ag8.653 myeloma cells at a ratio of 4 spleen cells to 1 myeloma cell.
  • the supernatant fluid was removed, and the pellet was suspended in 1 mL of 35% polyethylene glycol for 1 minute.
  • the polyethylene glycol was gradually diluted by addition of increasing amounts of serum-free DMEM over a period of 15 minutes.
  • the cells are then suspended in HAT medium (Monoclonal Antibodies. Kennett, McKean, Backitt, eds.
  • Polyvinyl chloride microtiter assay plates may be coated with antigen by addition of 50 ⁇ l of lead cation-glutathione-BSA or glutathione-BSA at a concentration of 5 ug/mL in PBS to each well of the plate. The plates are allowed to incubate at room temperature overnight to allow the antigen to dry on the plate. Next day the plates are blocked by addition of 200 ⁇ L of 1% polyvinyl alcohol (“PVA”) in PBS to each well; the addition of the PVA blocks the remaining protein-binding sites. The plates are incubated for 30 min at room temperature, then washed 3 times with ELISA wash (PBS with 0.1% of Triton X-100) , 3 times with milliQ water.
  • PVA polyvinyl alcohol
  • a 100 ppm solution of lead in lOOmM Hepes at pH 7 is then added to the wells (100 ⁇ L/well and allowed to incubate for 30-60 minutes at room temperature.
  • Fifty microliters of culture fluid to be assayed for the presence of antigen-specific antibody may be added to the appropriate well, and the plates are incubated at room temperature for 2 hours. The plates are again washed 3 times with ELISA wash, and 50 ⁇ L of goat anti-mouse serum (Cooper Biomedical) diluted 1:1000 in 2% BSA in PBS are added to each well.
  • each well at 405 nm may be measured with a UV/Vis spectrometer.
  • the use of other enzymes as sensors is also possible provided that such enzymes can be linked to an appropriate antibody, and catalyze a reaction which produces a color change.
  • beta galactosidase, urease, or horseradish peroxidase could be utilized in this context.
  • One hundred microliters of BSA-glutathione at a concentration of 5 ug/mL were added to the wells of a microtiter plate and allowed to dry overnight. The plates were then blocked with PVA as above.
  • One hundred microliters of PBS containing a known concentration of lead cations were added to triplicate wells on the plate, which were then incubated at room temperature for 30 minutes. After this incubation period the plates are washed with ELISA wash to remove unbound metal ions and then used in the standard ELISA to measure reactivity with a lead cation-specific antibody.
  • the primer used for cDNA synthesis of the variable region of a particular antibody polypeptide chain was also used for PCR amplification of that variable region, in conjunction with an appropriate V-region primer as described in Huse et al., Science, 246, 1275 (1989).
  • the PCR was performed as described in Sastry et al., Proc. Natl. Acad. Sci. USA, 8_6, 5728 (1989) .
  • Figures 1A-1B depict the nucleotide and deduced amino acid sequences for the heavy chain variable regions of a number of monoclonal antibodies that immunoreact with a lead cation. The following sequences are shown: the heavy chain variable region nucleotide acid sequence and deduced amino acid sequence for monoclonal antibody 6B11 (SEQ ID NO:l); the heavy chain variable region nucleotide sequence and deduced amiro acid sequence for monoclonal antibody 1254 (SEQ ID NO:3); the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 8E7 (SEQ ID NO:5); the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 10G5 (SEQ ID NO:7); and the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 14F11 (SEQ ID NO: 9) .
  • Figure 2 depicts the nucleotide and deduced amino acid sequences for the light chain variable regions of a number of monoclonal antibodies which immunoreact with a lead cation. The following sequences are shown: the light chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 6B11 (SEQ ID NO: 11); the light chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 1254 (SEQ ID NO:13); and the light chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 14F11 (SEQ ID NO:15).
  • Figures 3A-3B depict the nucleotide and deduced amino acid sequences for the heavy chain variable
  • RECTIFIED SHEET regions of a number of monoclonal antibodies that immunoreact with a lead cation.
  • the following sequences are shown: the heavy chain variable region nucleotide acid sequence and deduced amino acid sequence for monoclonal antibody 13D10 (SEQ ID N0:17); the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 11D11 (SEQ ID NO:19); the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 14G11 (SEQ ID NO:21); the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 6F5 (SEQ ID NO:23); the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 7D10 (SEQ ID NO:25); and the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 4E8 (SEQ ID NO:27
  • Figure 4 depicts the nucleotide and deduced amino acid sequences for the light chain variable regions of a number of monoclonal antibodies which immunoreact with a lead cation. The following sequences are shown: the light chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 10G5 (SEQ ID NO:29); the light chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 2E7 (SEQ ID NO:31); and the light chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 7D10 (SEQ ID NO: 33) .
  • Figure 5 depicts the nucleotide and deduced amino acid sequences for the heavy chain variable regions of
  • RECTIFIED SHEET (RULE 91) the monoclonal antibody 2B4 that immunoreacts with a lead cation.
  • the following sequences are shown: the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 2B4 (SEQ ID NO:35).
  • Figure 6 depicts the nucleotide and deduced amino acid sequences for the heavy chadn variable regions of the monoclonal antibody 2E7 that immunoreacts with a lead cation. The following sequences are shown: the heavy chain variable region nucleotide sequence and deduced amino acid sequence for monoclonal antibody 2E7 (SEQ ID NO: 37) .
  • most of the light chains of the lead-reactive monoclonal antibodies are members of the V k 32 light chain family, having a motif of four carboxylic acid-containing residues, e.g., glutamic acid and/or aspartic acid residues, in a stretch of five amino acids in the CDR1.
  • Results of molecular modeling indicate that one or more of the carboxyl groups of these residues are positioned at the appropriate distance to form coordinate interractions with lead.
  • the majority of the resulting lead-reactive monoclonal antibodies contained light chains of the V k 32 family.
  • the pelB leader sequences and cloning sites for the heavy-chain fragment and light chain may be derived from phagemids excised from ⁇ Hc2 and ⁇ Lc2 vectors as
  • the sequences may be modified to remove a redundant SacI site from Hc2 phagemid and a Spel site from the Lc2 phagemid.
  • the combinatorial phagemid vector pComb is constructed from these two modified phagemids by restricting each with Seal and EcoRI and combining them in a ligation reaction. Recombinan s are screened for the presence of two NotI sites yielding the combinatorial vector pComb.
  • the tether sequence GGGGS and gill fragment (gene coding for coat protein III of filamentous phage M13 (see Barbas, et al., Proc. Natl. Acad. Sci. USA, _88_, 7978 (1991)) from Spel to Nhel are the product of PCR of M13mpl8 (Yanisch-Perron, et al.. Gene, 33, 103-119 (1985) ) using the oligonucleotides 5'-GAGACGACTAGTGGTGGCGGTGGCTCTCCATTCGTTTGTGAATATCAA-3' (SEQ ID NO: 43) and
  • 5'-TTACTAGCTAGCATAATAACGGAATACCCAAAAGAACTGG-3' (SEQ ID NO:44) as reported in Barbas, et al., Proc. Natl. Acad. Sci. USA, 8_8, 7978 (1991) .
  • the lacZ promoter, operator, and Cap-binding site controlling light chain expression are the product of PCR with M13mpl8 using oligonucleotides 5'-TATGCTAGCTAGTAACACGACAGGTTTCCCGACTGG-3' (SEQ ID NO:45) and 5'-AGCTTTGAATTCGTGAAATTGTTATCCGCT-3' (SEQ ID NO:46) as reported in Barbas et al., ibid.
  • PCR fragments encoding the gill fragment and lacZ promoter are spliced by PCR overlap extension (see Horton et al., Gene, 77, 61-68 (1989)).
  • the resulting product is digested with Spel and EcoRI and ligated into the corresponding sites of pComb to yield pComb 3'.
  • pComb 3' is digested with Xhol and Spel and ligated with the corresponding 51-base-pair (bp) stuffer from pBluescript (see Short, et al., Nucleic Acids Res., 16, pp. 7583-7600 (1988)) (Stratagene) to yield pComb 3, an ampicillin-resistant phagemid.
  • bp 51-base-pair
  • a pComb 3 phagemid including a recombinantly produced Fab fragment that immunoreacts with a lead cation may be transformed into E. coli XLl-Blue cells.
  • the transformed E. coli XLl-Blue cells may be grown in super broth medium (SB; 30 g of tryptone, 20 g of yeast extract, 10 g of Mops per liter, pH 7) at 37°C supplemented with tetracycline at 10 ⁇ g/mi and carbenicillin at 50 ⁇ g/mL or chloramphenicol at 30 ⁇ g/mL. Cultures are grown to an OD 600 of 0.4 and infected with VCSM13 helper phage (phage to cell ratio, 20:1) and grown an additional hour. After one hour kanamycin is added (70 ⁇ g/mL) , and the culture is incubated overnight at 30°C.
  • SB super broth medium
  • kanamycin kanamycin
  • Phage may be isolated from liquid culture by polyethylene glycol 8000 and NaCl precipitation as described in Cwirla, et al. , Proc. Natl. Acad. Sci. USA. 87. pp. 6378-6382 (1990) . Phage pellets may be resuspended in phosphate-buffered saline (10 mM phosphate, pH 7.2, 150 mM NaCl) and stored at -20° C. 3. Single-Pass Enrichment Experiments
  • Phage expressing lead cation binding Fab fragments on their surface may be enriched by a modification of the panning procedure described by ParmLey, et al. , Gene, 73, pp. 305-318 (1988) .
  • a single well of a microtiter plate (Costar 3690) is coated overnight at 4°C with 25 ⁇ L of BSA-glutathione-lead cation at 2 mg/mL in 0.1 M bicarbonate, pH 8.5.
  • the well is washed once with water and blocked by filling the well with Blotto (1% (wt/vol) PVA in phosphate-buffered saline) and incubating the plate at 37°C for one hour.
  • Blocking solution is shaken out, and 50 ⁇ l of clonally mixed phage (typically 10 11 colony-forming units (cfu) ) is added, and the plate is incubated for an additional 2 hr at 37°C. Phage are removed, and the well is washed once with distilled water. The well is washed 10 times with TBS/Tween solution (50mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) over a period of one hour at room temperature.
  • TBS/Tween solution 50mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20
  • the well is washed once more with distilled water, and adherent phage are eluted by adding 50 ⁇ L of elution buffer (0.1 M HCl, adjusted to pH 2.2 with glycine) , and incubation at room temperature for 10 min.
  • the eluate is removed and neutralized with 3 ⁇ L of 2 M Tris base.
  • the initial phage input ratio may be determined by titering on selected plates.
  • the final phage output ratio may be determined by infecting 1 mL of logarithmic phase XLl-Blue cells with the neutralized eluate for 15 min at room temperature and plating equal aliquots on selective carbenicillin and chloramphenicol plates.
  • Phabs obtained from cultures of E. coli XLl-Blue cell may be transformed with a phagemid including a recombinantly produced Fab fragment that immunoreacts with a lead cation incubated at 37°C for two hours on BSA-Glutathione ELISA plates with or without lead nitrate.
  • a rabbit anti-M13 antiserum is typically used as a second antibody followed by affinity-purified goat-antirabbit serum conjugated with peroxidase.
  • 2,2' -Azino-Di- [3- ethylbenzthiazoline sulfonate] (ABTS) may be used as peroxidase substrate. The results are typically expressed as absorbance at 405 nanometers.
  • mice 1254 (mAb 8.15) .
  • the mice were injected biweekly with
  • mice 50 ⁇ g of antibody emulsified in Freunds complete adjuvant and bled seven days after each injection.
  • the mice were bled by nicking the tail and collecting 50 ⁇ l of blood, which was diluted to 500 ⁇ l in phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • the serum was tested using the same ELISA procedure as for screening the fusions (described below) .
  • the spleen cells of the mice were then used for production of hybridoma monoclonal antibodies (mAb's) as described below. Fusion Procedure for Hybridoma Production
  • a mouse was sacrificed by cervical dislocation, and its spleen was removed aseptically.
  • a single cell suspension was prepared from the spleen, and the cells were washed three times in serum-free Dulbecco's modified Eagle's medium (DMEM) by centrifugation at 400x g for 10 minutes, with 10 ml of DMEM per wash.
  • the number of viable spleen cells was determined by trypan blue exclusion with a hemacytometer.
  • the spleen cells were then mixed with SP2/0 myeloma cells at a ration of 4 spleen cells:1 myeloma cell.
  • the SP2/0 cells had previously been washed three time with serum-free DMEM as described above for the spleen cells.
  • the cell mixture was centrifuged as above, and the cell pellet was resuspended in 500 ⁇ l of 50% polyethylene glycol (PEG 1500, Sigma Chemical Co., St. Louis, MO) , which was added over a one-minute period.
  • the cell suspension was stirred for an addition minute, followed by addition of 10 ml of serum-free DMEM over the next two minutes. After stirring for an additional minute, the cells were centrifuged at 400x g for 10 minutes, and the pellet was resuspended in HAT medium to a final concentration of 1 x 10 7 spleen cells/ml.
  • HAT media Add the following to 500 milliliters of DMEM:
  • EKY polymer amino acid polymer consisting of glutamic acid, lysine, and tyrosine at a ratio of 6:3:1 respectively
  • EKY polymer amino acid polymer consisting of glutamic acid, lysine, and tyrosine at a ratio of 6:3:1 respectively
  • PBS amino acid polymer
  • One hundred microliters of the EKY polymer solution was added to each well of a high- binding polystyrene plate (Corning Costar, Cambridge, MA) . The plate was incubated at room temperature for thirty minutes, then rinsed three times with distilled water.
  • One hundred microliters of 1% polyvinyl alcohol (w/v) in PBS was added to each well, and the plate was incubated at room temperature for sixty minutes, after which it was rinsed as above.
  • SRM Standard Reference Material
  • One hundred microliters of hybridoma culture fluid to be assayed for the presence of lead cation monoclonal antibodies was added to the well of an antigen-coated microliter plate.
  • the assay plate was incubated for 30 minutes at room temperature.
  • the plate was then rinsed three times with ELISA wash (0.1% Triton X 100 in PBS) and three times with distilled water.
  • One hundred microliters of a 1:1000 dilution of goat anti-mouse IgA, IgG, IgM (Kirkegaard and Perry Laboratories, Gaithersburg, MD) conjugated to horseradish peroxidase was added to each well.
  • the plate was incubated at room temperature for thirty minutes, then rinsed as described above.
  • Positive hybridomas were subcloned by limiting dilution in 96-well microtiter plates in medium consisting of 50% HT medium and 50% complete DMEM conditioned by buffalo rat liver cells. Buffalo rat liver cells are known to secrete several somatomedin growth factors into the medium during growth. The somatomedin growth factors enhance the growth of cells in cloning situations.
  • the pellet was resuspended in and dialyzed against PBS at 4°C. The volume of the dialysate was measured, and two volumes of 60 millimolar sodium acetate, pH 4.0, was added to bring the final pH to 4.8.
  • Caprylic acid 60 millimolar sodium acetate, pH 4.0
  • Triton X-100 for 24 hours then against PBS for 24 hours.
  • ELISA was carried out essentially as described in subsection 2 above except that the lead nitrate solution was diluted to final concentrations of 100, 50, 10, 5, or 1 ⁇ g/ml using 0.1 M HEPES, pH 8.0. One hundred microliters of each of these solutions was then added to the plate coated with the polyamino acid mixture. Caprylic acid-precipitated mAb was adjusted to a concentration of 50 ⁇ g/ml, and 100 ⁇ l was added to the appropriate wells of the microliter plate. The plate was incubated for 30 minutes at room temperature, and the ELISA was continued as described above.
  • GGC ATG ATT CAT CCT TCC GAT AGT GAA ACT AGG TTA AAT CAG AAG TTC 192 Gly Met lie His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gin Lys Phe 50 55 60
  • GCA AGA AGG GGT AAC TCC GCC TGG TTT GCT TAC TGG GGC CAA GGG ACT 336 Ala Arg Arg Gly Asn Ser Ala Trp Phe Ala Tyr Trp Gly Gin Gly Thr 100 105 110
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • Gly Met lie His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gin Lys Phe
  • GCA AGA CAC GAA GGG TAT GGT AAC TAC GTG GCC TGG TTT GCT TAC TGG 336 Ala Arg His Glu Gly Tyr Gly Asn Tyr Val Ala Trp Phe Ala Tyr Trp 100 105 110
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • GGA AGG ATT GAT CCT TCC GAT AGT GAA ACT CAC TAC AAT CAA AAG TTC 192 Gly Arg lie Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gin Lys Phe 50 55 60
  • ATC CAA CTC AGC AGC CTG ACA TCT GAG GAC TCT GCG GTC TAT TAC TGT 288 lie Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • Gly Arg lie Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gin Lys Phe
  • Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 lie Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
  • GGC ATG ATT CAT CCT TCC GAT AGT GAA ACT AGG TTA AAT CAG AAG TTC 192 Gly Met lie His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gin Lys Phe 50 55 60
  • GCA AGA AGG AGG GAT TAC GAC CCG TTT GCT TAC TGG GGC CAA GGG ACT 336 Ala Arg Arg Arg Asp Tyr Asp Pro Phe Ala Tyr Trp Gly Gin Gly Thr 100 105 110
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • Gly Met lie His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gin Lys Phe
  • GCA AGA AGC GGC TAC GGC CAC TGG TAC TTC GAT GTC TGG GGC GCA GGG 336 Ala Arg Ser Gly Tyr Gly His Trp Tyr Phe Asp Val Trp Gly Ala Gly 100 105 110
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • ATC AGA TGC ATA ACC AGC ACT GAT ATT GAT GAT GAT ATG AAC TGG TAC 96 lie Arg Cys lie Thr Ser Thr Asp lie Asp Asp Asp Met Asn Trp Tyr 20 25 30
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • ATC AGA TGC ATA ACC AGC ACT GAT ATT GAT GAT GAT ATG AAC TGG TAC 96 lie Arg Cys lie Thr Ser Thr Asp He Asp Asp Asp Met Asn Trp Tyr 20 25 30
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • GCA AGG ATC TAC TAT GGT CAC TTG TGG TTT GCT TAC TGG GGC CAA GGG 336 Ala Arg He Tyr Tyr Gly His Leu Trp Phe Ala Tyr Trp Gly Gin Gly 100 105 110
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • Trp He Glu Trp Val Lys Gin Arg Pro Gly His Gly Leu Glu Trp He
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des polypeptides fixant un métal, lesquels comprennent une séquence d'acides aminés codant pour une région variable d'un anticorps monoclonal immunoréagissant avec un cation de plomb, et des nucléotides comprenant une séquence d'acide nucléique codant pour la région variable. L'invention concerne également des protéines de fusion comprenant la région variable à chaîne lourde de l'anticorps monoclonal ainsi qu'une protéine d'enveloppe de phage ou une partie de celle-ci. L'invention concerne également des bactériophages comprenant la protéine de fusion dans leur enveloppe. De plus, l'invention concerne des procédés de détection, d'extraction, d'addition ou de neutralisation de cations de plomb dans des systèmes biologiques ou inanimés à l'aide des polypeptides de fixation du plomb. En outre, l'invention concerne un procédé de formation d'un hybridome produisant des anticorps monoclonaux capables d'avoir une immunoréaction avec un cation métallique.
PCT/US1996/009258 1995-06-05 1996-06-05 Polypeptides fixant le plomb et nucleotides codants afferent WO1996039518A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU60946/96A AU6094696A (en) 1995-06-05 1996-06-05 Lead binding polypeptides and nucleotides coding therefor
US08/767,128 US6111079A (en) 1995-06-05 1996-12-04 Lead binding polypeptides and nucleotides coding therefore

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US46279895A 1995-06-05 1995-06-05
US08/462,798 1995-06-05
US54137395A 1995-10-10 1995-10-10
US08/541,373 1995-10-10

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US54137395A Continuation-In-Part 1995-06-05 1995-10-10

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US08/767,128 Continuation-In-Part US6111079A (en) 1995-06-05 1996-12-04 Lead binding polypeptides and nucleotides coding therefore

Publications (1)

Publication Number Publication Date
WO1996039518A1 true WO1996039518A1 (fr) 1996-12-12

Family

ID=27040456

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/009258 WO1996039518A1 (fr) 1995-06-05 1996-06-05 Polypeptides fixant le plomb et nucleotides codants afferent

Country Status (2)

Country Link
AU (1) AU6094696A (fr)
WO (1) WO1996039518A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023082A1 (fr) * 1998-10-19 2000-04-27 Yeda Research And Development Co. Ltd. Traitement du lupus erythemateux systemique par regulation negative de la reponse auto-immune a des autoantigenes
WO2002034788A1 (fr) * 2000-10-20 2002-05-02 Inhibitex, Inc. Anticorps monoclonaux diriges contre la proteine map et procede d'utilisation de ceux-ci dans le traitement ou la prevention des infections
EP1302477A1 (fr) * 2000-07-14 2003-04-16 Matsushita Electric Industrial Co., Ltd. Fructose peptidique et complexe de ce dernier associe a une proteine de liaison
WO2003050143A1 (fr) * 2001-12-11 2003-06-19 Expressive Constructs, Inc. Detection et elimination du plomb au moyen de proteines de liaison du plomb
WO2003059260A3 (fr) * 2001-12-21 2004-01-15 Biosynexus Inc Anticorps monoclonaux et chimeriques opsoniques specifiques de l'acide lipoteichoique de bacteries gram-positif
US6939543B2 (en) 1997-06-16 2005-09-06 Henry M. Jackson Foundation For The Advancement Of Military Medicine Opsonic and protective monoclonal and chimeric antibodies specific for lipoteichoic acid of gram positive bacteria
WO2005102387A3 (fr) * 2004-03-29 2006-03-09 Protein Design Labs Inc Utilisation therapeutique des anticorps anti-cs1
US7250494B2 (en) 1998-06-15 2007-07-31 Biosynexus Incorporated Opsonic monoclonal and chimeric antibodies specific for lipoteichoic acid of Gram positive bacteria
US8008450B2 (en) 2003-05-08 2011-08-30 Abbott Biotherapeutics Corp. Therapeutic use of anti-CS1 antibodies
US9309305B2 (en) 2011-06-10 2016-04-12 National Research Council Of Canada Anti-ricin antibodies and uses thereof
JP2016520533A (ja) * 2013-03-19 2016-07-14 ベイジン シェノゲン ファーマ グループ リミテッド エストロゲン受容体関連疾患を処置するための抗体及び方法
US9403913B2 (en) * 2014-09-16 2016-08-02 Ovascience, Inc. Anti-VASA antibodies, and methods of production and use thereof
US9862764B2 (en) 2014-12-19 2018-01-09 Novartis Ag Compositions and methods for antibodies targeting BMP6
US11434269B2 (en) 2016-06-15 2022-09-06 Novartis Ag Methods for treating disease using inhibitors of bone morphogenetic protein 6 (BMP6)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990010709A2 (fr) * 1989-03-14 1990-09-20 Board Of Regents Of The University Of Nebraska Anticorps monoclonaux pour cations metalliques sur petites molecules
WO1994018220A1 (fr) * 1993-02-02 1994-08-18 The Scripps Research Institute Procedes de production de sites de liaison de metaux sur des polypeptides et compositions necessaires
WO1995018156A1 (fr) * 1993-12-30 1995-07-06 Abbott Laboratories Complexes metalliques de coordination ion-ligand, anticorps diriges contre ceux-ci, et dosages effectues avec ces anticorps

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990010709A2 (fr) * 1989-03-14 1990-09-20 Board Of Regents Of The University Of Nebraska Anticorps monoclonaux pour cations metalliques sur petites molecules
WO1994018220A1 (fr) * 1993-02-02 1994-08-18 The Scripps Research Institute Procedes de production de sites de liaison de metaux sur des polypeptides et compositions necessaires
WO1995018156A1 (fr) * 1993-12-30 1995-07-06 Abbott Laboratories Complexes metalliques de coordination ion-ligand, anticorps diriges contre ceux-ci, et dosages effectues avec ces anticorps

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL entry MM23047; 16 April 1995 (1995-04-16), JANG Y ET AL: "Heavy chain dominance in the binding of native DNA by a lupus mouse monoclonal autoantibody", XP002013728 *
DUVAL G ET AL: "Preliminary purification and characterization studies of a low molecular weight, high affinity cytosolic lead-binding protein in rat brain.", BIOCHEM BIOPHYS RES COMMUN, FEB 28 1989, 159 (1) P177-84, UNITED STATES, XP000601647 *
DZIERZAK E A ET AL: "Molecular characterization of antibodies bearing Id-460", JOURNAL OF IMMUNOLOGY, vol. 136, 1986, BALTIMORE US, pages 1864 - 1870, XP000601650 *
KUBOTA T ET AL: "A monoclonal anti-double stranded DNA antibody from an autoimmune MRL/Mp-lpr/lpr mouse: specificty and idiotype in serum immunoglobulins", IMMUNOLOGY LETTERS, vol. 14, 1986 - 1987, pages 53 - 58, XP000601123 *
WYLIE DE ET AL: "Monoclonal antibodies specific for mercuric ions.", PROC NATL ACAD SCI U S A, MAY 1 1992, 89 (9) P4104-8, UNITED STATES, XP000601648 *

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7511122B2 (en) 1997-06-16 2009-03-31 Henry M. Jackson Foundation For The Advancement Of Military Medicine Humanized and chimeric antibodies specific for lipoteichoic acid of GRAM positive bacteria
US6939543B2 (en) 1997-06-16 2005-09-06 Henry M. Jackson Foundation For The Advancement Of Military Medicine Opsonic and protective monoclonal and chimeric antibodies specific for lipoteichoic acid of gram positive bacteria
US8372958B2 (en) 1997-06-16 2013-02-12 Biosynexus Incorporated; Opsonic and protective monoclonal and chimeric antibodies specific for lipoteichoic acid of gram posiive bacteria
EP2357198A1 (fr) * 1997-06-16 2011-08-17 Henry M. Jackson Foundation for the Advancement of Military Medicine Anticorps monoclonaux et chimériques protecteurs et opsoniques spécifiques de l'acide lipotéichoïque de bactéries à gram positif
US7884198B2 (en) 1997-06-16 2011-02-08 The Henry M. Jackon Foundation for the Advancement of Military Medicine Polynucleotides encoding humanized and chimeric antibodies specific for lipoteichoic acid of gram positive bacteria
US7250494B2 (en) 1998-06-15 2007-07-31 Biosynexus Incorporated Opsonic monoclonal and chimeric antibodies specific for lipoteichoic acid of Gram positive bacteria
US7777017B2 (en) 1998-06-15 2010-08-17 Biosynexus Incorporated Nucleic acids encoding opsonic monoclonal and chimeric antibodies specific for lipoteichoic acid of gram positive bacteria
WO2000023082A1 (fr) * 1998-10-19 2000-04-27 Yeda Research And Development Co. Ltd. Traitement du lupus erythemateux systemique par regulation negative de la reponse auto-immune a des autoantigenes
EP1302477A1 (fr) * 2000-07-14 2003-04-16 Matsushita Electric Industrial Co., Ltd. Fructose peptidique et complexe de ce dernier associe a une proteine de liaison
EP1302477A4 (fr) * 2000-07-14 2005-06-01 Matsushita Electric Ind Co Ltd Fructose peptidique et complexe de ce dernier associe a une proteine de liaison
WO2002034788A1 (fr) * 2000-10-20 2002-05-02 Inhibitex, Inc. Anticorps monoclonaux diriges contre la proteine map et procede d'utilisation de ceux-ci dans le traitement ou la prevention des infections
WO2003050143A1 (fr) * 2001-12-11 2003-06-19 Expressive Constructs, Inc. Detection et elimination du plomb au moyen de proteines de liaison du plomb
WO2003059260A3 (fr) * 2001-12-21 2004-01-15 Biosynexus Inc Anticorps monoclonaux et chimeriques opsoniques specifiques de l'acide lipoteichoique de bacteries gram-positif
US8133981B2 (en) 2003-05-08 2012-03-13 Abbott Biotherapeutics Corp. Therapeutic use of anti-CS1 antibodies
US9175081B2 (en) 2003-05-08 2015-11-03 Abbvie Biotherapeutics Inc. Therapeutic use of anti-CS1 antibodies
US10442859B2 (en) 2003-05-08 2019-10-15 Abbvie Biotherapeutics Inc. Therapeutic use of anti-CS1 antibodies
US8008450B2 (en) 2003-05-08 2011-08-30 Abbott Biotherapeutics Corp. Therapeutic use of anti-CS1 antibodies
US8088898B2 (en) 2003-05-08 2012-01-03 Abbott Biotherapeutics Corp. Therapeutic use of anti-CS1 antibodies
US7709610B2 (en) 2003-05-08 2010-05-04 Facet Biotech Corporation Therapeutic use of anti-CS1 antibodies
US8461306B2 (en) 2003-05-08 2013-06-11 Abbvie Biotherapeutics Inc. Therapeutic use of anti-CS1 antibodies
US8349330B2 (en) 2003-05-08 2013-01-08 Abbott Biotherapeutics Corp. Therapeutic use of anti-CS1 antibodies
US8445646B2 (en) 2003-05-08 2013-05-21 Abbvie Biotherapeutics Inc. Therapeutic use of anti-CS1 antibodies
US8436146B2 (en) 2003-05-08 2013-05-07 Abbvie Biotherapeutics Inc. Therapeutic use of anti-CS1 antibodies
US8444980B2 (en) 2003-05-08 2013-05-21 Abbvie Biotherapeutics Inc. Therapeutic use of anti-CS1 antibodies
WO2005102387A3 (fr) * 2004-03-29 2006-03-09 Protein Design Labs Inc Utilisation therapeutique des anticorps anti-cs1
AU2011200999B2 (en) * 2004-03-29 2012-05-31 Abbvie Biotherapeutics Inc. Therapeutic use of anti-CS1 antibodies
CN1960750B (zh) * 2004-03-29 2011-06-29 艾伯特生物治疗学公司 抗cs1抗体的治疗用途
AU2004318675B2 (en) * 2004-03-29 2011-06-16 Abbvie Biotherapeutics Inc. Therapeutic use of anti-CS1 antibodies
US9309305B2 (en) 2011-06-10 2016-04-12 National Research Council Of Canada Anti-ricin antibodies and uses thereof
US10233249B2 (en) 2013-03-19 2019-03-19 Beijing Shenogen Pharma Group Ltd. Antibodies and methods for treating estrogen receptor-associated diseases
EP2976362A4 (fr) * 2013-03-19 2017-03-22 Beijing Shenogen Pharma Group Ltd. Anticorps et procédés permettant de traiter des maladies associées aux récepteurs des strogènes
JP2016520533A (ja) * 2013-03-19 2016-07-14 ベイジン シェノゲン ファーマ グループ リミテッド エストロゲン受容体関連疾患を処置するための抗体及び方法
US9567404B2 (en) 2014-09-16 2017-02-14 Ovascience, Inc. Anti-vasa antibodies, and methods of production and use thereof
US9403913B2 (en) * 2014-09-16 2016-08-02 Ovascience, Inc. Anti-VASA antibodies, and methods of production and use thereof
US9862764B2 (en) 2014-12-19 2018-01-09 Novartis Ag Compositions and methods for antibodies targeting BMP6
US10683346B2 (en) 2014-12-19 2020-06-16 Novartis Ag Compositions and methods for antibodies targeting BMP6
US11530261B2 (en) 2014-12-19 2022-12-20 Novartis Ag Compositions and methods for antibodies targeting BMP6
US11434269B2 (en) 2016-06-15 2022-09-06 Novartis Ag Methods for treating disease using inhibitors of bone morphogenetic protein 6 (BMP6)

Also Published As

Publication number Publication date
AU6094696A (en) 1996-12-24

Similar Documents

Publication Publication Date Title
CA2047717C (fr) Anticorps monoclonaux contre de petites molecules et methodes pour les preparer
Jemmerson Antigenicity and native structure of globular proteins: low frequency of peptide reactive antibodies.
US5639624A (en) Monoclonal antibodies specific for metallic cations and method therefor
US5217896A (en) Monoclonal antibodies recognizing parathyroid hormone-like protein
WO1996039518A1 (fr) Polypeptides fixant le plomb et nucleotides codants afferent
JPH04228068A (ja) トリオーマ細胞およびその製造方法
US4762706A (en) Peptide antibodies and their use in detecting oncogene products
US6111079A (en) Lead binding polypeptides and nucleotides coding therefore
JP3107225B2 (ja) Ｐａｃａｐに対する抗体およびその用途
KR101736420B1 (ko) 파툴린 특이적인 항체 및 이를 이용한 파툴린의 검출 방법
Schifferli et al. Use of monoclonal antibodies to probe subunit-and polymer-specific epitopes of 987P fimbriae of Escherichia coli
US8344109B2 (en) Anti-ricin antibody
US5972656A (en) Mercury binding polypeptides and nucleotides coding therefore
EP0741749B1 (fr) Polypeptides de fixation au mercure et nucleotides codant pour lesdits polypeptides
CN111424018B (zh) 一种分泌抗CLas转运蛋白Ctp4单克隆抗体的杂交瘤细胞株4A12及应用
US5332665A (en) Species specific, high affinity monoclonal antibodies
US5330909A (en) Monoclonal antibody specifically recognizing N-terminal portion of human calcitonin
CN105440137B (zh) 抗莱克多巴胺的抗体及其应用
EP0229794B1 (fr) Anticorps monoclonaux pour l'interferon gamma, leur production et utilisation
KR100188245B1 (ko) 면역원성 물질의 고도로 보존된 아미노산 서열에 대한 항체, 이의 제조 방법 및 이를 함유하는 면역 검정물
US5665559A (en) Production of monoclonal antibodies to bacteroides gingivalis by hybridoma BGII, VF9/2D
Odya et al. Enzyme-linked immunosorbent assays for kinins using high-affinity monoclonal kinin antibodies
KR100493932B1 (ko) 레지스틴에 대한 단클론 항체, 이의 제조 방법, 및 용도
KR102760674B1 (ko) 중증열성혈소판감소증후군 바이러스 Gc 항원에 특이적으로 결합하는 항체 및 이의 용도
GB2113713A (en) Forming monoclonal antibodies to carbohydrate hapten antigens

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 08767128

Country of ref document: US

AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载