Title:  Monoclonal antibodies to the human LDL receptor, their production and use

United States Patent:  6,849,720

Issued:  February 1, 2005

Inventors:  Yonah; Nachum (Gedera, IL); Suissa; Dany (Rehovot, IL); Belzer; Ilana (Rishon le Zion, IL); Antonetti; Francesco (Rome, IT); Smolarsky; Moshe (Rehovot, IL); Dreano; Michel (Collonges-sous-Saleve, FR)

Assignee:  Applied Research Systems ARS Holding N.V. (Curacao, NL)

Appl. No.:  221679

Filed:  January 15, 2003

PCT Filed:  March 8, 2001

PCT NO:  PCT/IL01/00216

371 Date:  January 15, 2003

102(e) Date:  January 15, 2003

PCT PUB.NO.:  WO01/68710

PCT PUB. Date:  September 20, 2001

Abstract

There are provided monoclonal antibodies to the LDL receptor which are useful for the identification and purification of LDL and in treatment of e.g. hepatitis C infection.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to monoclonal antibodies which specifically recognise the human receptor for low-density lipoproteins (LDLR). These antibodies are useful e.g. for the identification and purification of human soluble LDLR (hsLDLR) in production processes as well as in the identification and treatment of diseases such as, hepatitis C infection (HCV).

BACKGROUND OF THE INVENTION

Cholesterol, a component of all eukaryotic plasma membranes, is essential for the growth and viability of cells in higher organisms. However, high serum levels of cholesterol cause disease and death by contributing to the formation of atherosclerotic plaques in arteries throughout the body. The major site of cholesterol synthesis in mammals is the liver. Appreciable amounts of cholesterol are also formed by the intestine. The rate of cholesterol formation by these organs is highly responsive to the amount of cholesterol absorbed from dietary sources. Cells outside of the liver and intestine acquire cholesterol from the plasma rather than by synthesising it de novo. Cholesterol and other lipids are transported in body fluids by lipoproteins, which are classified according to increasing density. A lipoprotein is a particle consisting of a core of hydrophobic lipids surrounded by a shell of polar lipids and apoproteins. These lipoproteins have two roles: they solubilize highly hydrophobic lipids and they contain signals that regulate the movement of particular lipids in and out of specific target cells and tissues. Cholesterol is transported in body fluids by low-density lipoproteins (LDL) which binds to a specific receptor on the plasma membrane of non hepatic cells. The receptor-LDL complex is then internalised into the cells by a transport mechanism known as receptor mediated endocytosis (Goldstein et al. 1979). The low density lipoprotein (LDL) receptor is the prototype of a family of structurally related cell surface receptors that mediate endocytosis of multiple ligands in mammalian cells.

The LDL receptor consists of 822 amino acid residues and exhibits a molecular weight of 164000. It is composed of several domains some of which share sequence homology with other proteins. Its NH₂ -terminal ligand-binding domain consists of 292 residues, arranged in 7 cysteine-rich imperfect repeats. Each repeat contains six cysteine residues which are disulphide bonded in the pattern one to three, two to five, and four to six. (Bieri et al. 1995). This domain is followed by four additional domains: the first consists of 400 amino acid residues and is homologous to the EGF receptor, the second consists of 58 amino acid residues rich in O-linked sugars, the third is a single trans-membrane domain of 22 amino acid residues and the fourth is a cytoplasmic domain of 50 amino acid residues (Sudhof et al. 1985), (Brown et al. 1986).

The physiologic importance of the LDL receptor was revealed by Brown and Goldstein's studies on familial hypercholesterolemia (FH). The disease was found to be due to a molecular genetic defect resulting in the absence or deficiency of functional receptors for LDL (Brown et al. 1976). Several classes of FH mutations have been characterised. (Goldstein et al. 1975).

A soluble form of the sLDLR exhibiting antiviral activity was identified and isolated from the culture supernatant of interferon-induced cells (Fischer et al. 1993) and in body fluids (Fischer et al. 1994). Several interferon-induced proteins have been identified that are instrumental in the induction of the antiviral state by IFNs. One such protein exhibiting antiviral activity was produced and accumulated in the culture supernatant of human amnion WISH cells. This protein was purified to homogeneity and identified as the sLDLR (see EP 0 553 667 and Fischer et al. 1993). The sLDLR was found to be secreted into the medium by mammalian cells that enter an antiviral state in response to interferon. In contrast to interferon, sLDLR does not induce an antiviral state in the cells but is antiviral by itself. It was found that sLDLR apparently has to be present throughout the process of viral replication maturation and budding suggesting it might be involved in a complex process that leads to the inhibition of virus assembly or budding (unpublished data). Endocytosis of the hepatitis C virus has been recently shown to be mediated by LDL receptors on cultured cells (Agnello et al. 1999). These and other findings suggest that the family of LDL receptors may serve as viral receptors. Therefore, antibodies rised against the sLDLR receptor may block the entry and budding of viral particles by binding to the cellular LDL receptor.

The only available monoclonal antibody to LDLR known so far is C7, an antibody to bovine LDLR (Beisiegel et al. 1981, commercially available from Amersham, UK) which was prepared by immunization of mice with the bovine adrenal cortex LDLR purified to homogeneity. Membranes from the bovine adrenal cortex were solubilized and the receptor was partially purified by elution from a DEAE-cellulose column (Beisiegel et al. 1981). The antibody to the bovine LDLR only weakly cross-reacts with human LDLR.

In fact, the C7 Mab to bovine LDLR was found to have significant disadvantages when used for detection and quantitation of recombinant human LDLR:

a) It has very low affinity to the human LDLR

b) It significantly cross reacts with cell culture derived impurities

Specific antibodies to human LDLR were not previously available. This appears surprising since it is very common to raise antibodies against novel proteins, be it for purification, identification or for assay development purposes. It is possible that such antibodies have not been generated so far, since a condition for generating monoclonal antibodies is the availability of sufficiently large amounts of highly purified antigen which allow efficient immunization of mice. A highly purified antigen is one which appears as a single major peak in RP-HPLC. Furthermore methods for identification and quantitation of the antigen during purification processes were not easy to establish. In accordance with the invention, the antiviral activity assay described herein was employed for the identification of LDLR during purification processes.

There exists a need to generate specific Mabs to human soluble LDLR to provide the means for developing an efficient immunoassay (ELISA) and for the identification of the protein in Western blot. These antibodies are required for the monitoring and quantitation of the recombinant human soluble LDLR during development of the production and purification processes of the recombinant protein and for detection of the natural protein.

SUMMARY OF THE INVENTION

The present invention allows the generation of hybridoma cells lines producing monoclonal antibodies capable of specifically recognising and binding the human LDL receptor and fragments thereof.

More specifically the present invention allows the generation of hybridoma cells lines producing monoclonal antibodies capable of specifically recognising and binding the human soluble LDL receptor.

Thus the present invention relates to a monoclonal antibody, chimeric antibody, humanized antibody, anti-anti-Id antibody or fragment thereof which specifically recognises and binds the human LDL receptor and fragments thereof, except monoclonal antibody C7.

The present invention provides such monoclonal antibodies that recognise and bind the human soluble LDLR and meet the following needs:

1. Mabs that can be used as a pair in an ELISA, e.g. a sandwich ELISA (Enzyme Linked Immuno Sorbent Assay) for detection of human soluble LDLR.

2. Mabs that can be used for identification of the LDLR in Western Blot analysis.

3. Mabs that can be used to neutralise the antiviral biological activity of the human soluble LDLR.

4. Mabs that can be used to inhibit virus infection, such as HCV.

The present invention further provides a method for the detection and/or the quantitation of human LDLR which comprises the use of the specific monoclonal antibodies according to the invention in a known manner for that purpose.

The present invention also provides cloned hybridoma comprising a spleen cell from a mammal immunized with recombinant human LDLR and a homogenic or heterogenic lymphoid cell.

A monoclonal antibody according to the invention is prepared in a conventional manner, e.g. by growing a cloned hybridoma comprising a spleen cell from a mammal immunized with hsLDL and a homogenic or heterogenic lymphoid cell in liquid medium or mammalian abdomen to allow the hybridoma to produce and accumulate the monoclonal antibody.

The invention, in yet another aspect, provides a method for purifying the human LDLR which comprises contacting a material containing crude LDLR with a monoclonal antibody according to the invention. A column with adsorbed LDLR specific monoclonal antibody may be used as an affinity purification step, in the purification process of the recombinant protein.

A method for detecting and measuring recombinant human LDLR which comprises using as antibody the monoclonal antibodies of the present invention in an ELISA assay as described in example 5.

As the LDLR or fragment of a LDLR for immunizing animals any LDLR can be used as long as it is the LDLR of a warm-blooded mammal. A mutein of LDLR can be also used. A representative example of such a mammalian human soluble LDLR is the soluble LDLR +291 form which includes the amino acid sequence beginning at amino acid Asp at position +4 and ending with amino acid Glu at position +291 of the sequence of the human LDLR, any other form may be used as well, such as the +292 form etc.

DETAILED DESCRIPTION OF THE INVENTION

Monoclonal antibodies (Mabs) to human soluble LDLR (hsLDLR) were generated. Using these monoclonal antibodies, an ELISA and a Western blotting procedure for the identification of hsLDLR and a neutralising assay to the antiviral activity of hsLDLR were developed.

The Mabs were generated in mice, immunized with the recombinant +291 form of hsLDLR, which consists of the N-terminal ligand binding domain of the human soluble LDLR, from Asp +4 to Glu +291. The recombinant +291 form of hsLDLR, was produced in CHO cells and purified to homogeneity.

The immunized mice produced significant titres of specific antibodies. After screening of hybridomas, five clones (numbers 12, 28, 29, 30 and 50) were identified as the highest antibody producers. These clones were selected for further subcloning. After subcloning, 29 subclones which had high antibody productivity were isolated and ampoules from the parent clones and from the subclones were frozen.

A pair of monoclonal antibodies was chosen for the ELISA to the r-hsLDLR. Mab 28 was selected as the coating antibody, and Mab 29.8, labelled with biotin, was chosen as the second antibody. Mabs 12.6 and 29.8 were found to be suitable for the identification of the native and recombinant hsLDLR in Western blot analysis and Mabs 28 and 30 were found to be suitable for the identification of the recombinant hsLDLR in Western blot analysis. Mabs 12.6 and 50.30 were found to be suitable for inhibiting the antiviral activity of hsLDLR.

It was also found in accordance with the invention that Mabs 12.6, 28 and 29.8 inhibit replication of the viral genome of hepatitis C virus (HCV) in human hepatocytes primary cultures. Thus, these antibodies may be used for the treatment of hepatitis C infection.

The subclass isotype of the Mab produced by the clones was determined. Clones 12.6, 28, 29.8 and 30 were identified as IgG₁ whereas clone 50.30 was found to be IgM.

The Mabs, developed against the +291 form of the hsLDLR recognised also other forms of the hsLDLR, i.e. the +292 form and the +331 form of the r-hsLDLR produced in recombinant CHO cells, in ELISA and in Western blot analysis. The +292 form comprises the N-terminus part of the receptor from amino acid residue Asp +4 to Cys +292 and the +331 form comprises the N-terminus part of the receptor from amino acid residue Asp +4 to Cys +331.

The antigen used to immunize mice for generating monoclonal antibodies was the r-hsLDLR +291 form, which was produced in CHO cells. Production of the r-hsLDLR was performed in bioreactors, using the stationary phase Fibracel matrix system. The r-hsLDLR was purified to homogeneity and used for immunizing mice.

Immune spleen cells from the best mouse responder were used for fusion and generation of hybridomas.

As regards the antibodies mentioned herein throughout, the term "monoclonal antibody" is meant to include monoclonal antibodies, chimeric antibodies, fully humanized antibodies, antibodies to anti-idiotypic antibodies (anti-anti-Id antibody) that can be labeled in soluble or bound form, as well as fragments thereof provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques.

A monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which populations contains substantially similar epitope binding sites. Mabs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature, 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubel et al., eds., Harlow and Lane ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience N.Y., (1992-1996), the contents of which references are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo. Production of high titers of Mabs in vivo or in situ makes this the presently preferred method of production.

Chimeric antibodies are molecules of which different portions are derived from different animal species, such as those having the variable region derived from a murine Mab and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine Mabs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric Mabs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published Nov. 14, 1984); Neuberger et al., Nature 314:268-270 (1985); Taniguchi et al., European Patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO 8601533, (published Mar. 13, 1986); Kudo et al., European Patent Application 184187 (published Jun. 11, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986); Robinson et al., International Patent Application No. WO8702671 (published May 7, 1987); Liu et al., Proc. Natl. Acad. Sci USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988); Riechmann et al., Nature 332:323-327. and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra. These references are entirely incorporated herein by reference.

"Fully humanized antibodies" are molecules containing both the variable and constant region of the human immunoglobulin. Fully humanized antibodies can be potentially used for therapeutic use, where repeated treatments are required for chronic and relapsing diseases such as autoimmune diseases. One method for the preparation of fully human antibodies consist of "humanization" of the mouse humoral immune system, i.e. production of mouse strains able to produce human Ig (Xenomice), by the introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated. The Ig loci are exceedingly complex in terms of both their physical structure and the gene rearrangement and expression processes required to ultimately produce a broad immune response. Antibody diversity is primarily generated by combinatorial rearrangement between different V, D, and J genes present in the Ig loci. These loci also contain the interspersed regulatory elements, which control antibody expression, allelic exclusion, class switching and affinity maturation. Introduction of unrearranged human Ig transgenes into mice has demonstrated that the mouse recombination machinery is compatible with human genes. Furthermore, hybridomas secreting antigen specific hu-mAbs of various isotypes can be obtained by Xenomice immunisation with antigen.

Fully humanized antibodies and methods for their production are known in the art (Mendez et al., Nature Genetics 15:146-156 (1997);Buggemann et al., Eur. J. Immunol. 21:1323-1326 (1991); Tomizuka et al., Proc. Natl. Acad Sci. USA 97:722-727 (2000) Patent WO 98/24893.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the Mab to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Pat. No. 4,699,880, which is herein entirely incorporated by reference.

The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original Mab, which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a Mab, it is possible to identify other clones expressing antibodies of identical specificity.

Accordingly, Mabs generated against LDLR, its isoforms, analogs, fragments or derivatives of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id Mabs. Further, the anti-Id Mabs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original Mab specific for an epitope of the above LDLR protein, or analogs, fragments and derivatives thereof.

The anti-Id Mabs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated. The term "monoclonal antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of the LDLR protein according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

A monoclonal antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody, which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody, which antigen is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with an epitope on its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The antibodies, including fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the LDLR proteins in a sample or to detect presence of cells that express the LDLR proteins of the present invention. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with fluorescence microscopy, flow cytometric, or fluorometric detection.

The antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of the LDLR proteins of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and providing the labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the LDLR proteins but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Such assays for the LDLR proteins of the present invention typically comprises incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a labeled antibody capable of identifying the LDLR proteins, and detecting the antibody by any of a number of techniques well known in the art.

The biological sample may be coupled to a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers followed by treatment with a labeled antibody in accordance with the present invention, as noted above. The solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by conventional means.

By "solid phase support", "solid phase carrier", "solid support", "solid carrier", "support" or "carrier" is intended any support or carrier capable of binding antigen or antibodies. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrier configuration may be spherical, as in a bead, cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know may other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of antibody, of the invention as noted above, may be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.

One of the ways in which an antibody in accordance with the present invention can be labeled is by linking the same to an enzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomeras, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholin-esterase. The detection can be accomplished by calorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactive labeling the antibodies or antibody fragments, it is possible to detect R-PTPase through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S. et al., North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a g counter or a scintillation counter or by autoradiography.

It is also possible to label an antibody in accordance with the present invention with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can be then detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵² E, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined, as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.

Claim 1 of 13 Claims

What is claimed is:

1. A monoclonal antibody expressed by hybridoma clone 12.6 deposited at the CNCM under No.1-2390.

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