WO1996033279A2 - Repertoire cloning process, products derived therefrom and uses for said products - Google Patents

Abstract

A repertoire cloning process for producing mammalian monoclonal antibodies. Also provided are methods for using these monoclonal antibodies, and kits which contain these monoclonal antibodies.

Description

REPERTOIRE CLONING PROCESS. PRODUCTS DERIVED THEREFROM AND USES FOR SAID PRODUCTS

Background of the Invention This invention relates generally to a novel repertoire cloning process for producing monoclonal antibodies, and more particularly, relates to a novel mammalian repetoire cloning process monoclonal antibodies capable of producing monoclonal antibodies in mammals, monoclonal antibodies produced therefrom, and uses for these monoclonal antibodies, Recently, methods have been developed for cloning immunoglobulin gene repertoire libraries and isolating clones encoding antibodies directed against specific antigens. For reviews, see Burton, Trends Biotechnol. 9: 169 (1991); Winter and Milstein, Nature 349: 293 (1991); Pluckthun, Current Opinion in Biotechnology 2:228 (1991). Such techniques could provide an alternative method for producing monoclonal antibodies in mammals. These cloning techniques rely on amplification techniques such as the polymerase chain reaction (PCR) to amplify immunoglobulin (Ig) genes from lymphocyte mRNA for molecular cloning. Clones which produce antibodies specific for the antigen of interest are identified and isolated by screening bacterial colonies (Dreher et al., J. Immunol. Methods 139: 197 [1991], phage plaques (Huse et al, Science 246:1275 [1989]; Persson et al., Proc. Natl. Acad. Sci. USA 88:2432 [1991], or phage-display libraries (Kang et al., Proc. Natl. Acad. Sci. USA 88:4363 [1991]; Clackson et al., Nature 352:624 [1991]). The cloned antibody sequences can then be expressed in E. coli to produce antibody fragments (reviewed by Skerra, Current Opinion in Immunology 5:256 [1993]). Since antibody production and assembly by bacteria is inefficient, however, screening of bacterial expression libraries can be problematic. A particular difficulty is the identification of the proper conditions which will provide adequate sensitivity to detect the small amounts and sometimes low-affinity Fab fragments produced by the repertoire library cells, while maintaining sufficiently low non-specific binding by the detection reagents to avoid excessive false positives.

Mammals such as rabbits have long been used as sources of high-affinity antisera directed against a wide range of antigens for both research and commercial applications. Unfortunately, efforts to generate cell lines producing rabbit monoclonal antibodies have met with limited success. See, for example. Yarmush et al., Proc. Natl. Acad. Sci. USA 77:2988 (1980); Kuo et al., Mol. Immunol. 22:351 (1985); and Raybould and Takahashi, Science 240:1788 (1988). For this reason, rabbit polyclonal antisera remain in widespread use both in research and commercial applications despite the inherent difficulties associated with polyclonal antisera. Rabbits appear to be a particularly suitable species for the repertoire cloning process due to the limited number of VH genes which are utilized for immunoglobulin expression (Knight and Becker, Cell 60:963 [1990]). In contrast to mice and humans in which antibody diversity is generated by combinatorial joining of multiple V, D, and J regions, greater than 80% of rabbit immunoglobulins result from the use of the VH1 gene for VDJ rearrangements

(Knight and Becker, supra). Diversity of antigenic specificity is generated by gene conversion and somatic mutation (Becker and Knight, Cell 63:987 [1990]; Short et al., J. Immunol. 5:256 [1991]). The use of a single VH region in expressed antibodies greatly simplifies the cloning process since only a single set of PCR primers is required for amplification rather than the families of primers required for amplification of mouse or human VH genes (Huse et al., supra).

Researchers recently described a system for cloning and expression of rabbit VDJ regions and obtained single-domain antibodies specific to protein C. Suter et al., Immunology Letters 33:53 (1992). Some authors have observed comparable binding for both Fy and VH domains (see, for example, Ward et al., Nature 341:544 [1989]; Barry and Lee. Molecular Immunology 30:833 [1993]) while others have reported greatly diminished affinities for VH domains compared with Fv or Fab antibodies (see, for example, Borrebaeck et al., Biotechnology 10:697 [1992]; Froyen et al., Molecular Immunology 30:805 [1993]). The role of the light chains in the deteπriination of Ig binding characteristics (i.e., specificity, affinity, avidity) thus appears to vary widely depending on the particular antibody examined. Indeed, Hamars-Casterman et al. Nature 363:446 (1993) recently demonstrated the existence of IgG antibodies lacking light chains in camels with no apparent deficiencies in either antibody diversity or affinity. It would be advantageous to provide mammalian monoclonal antibodies and a system for producing mammalian monoclonal antibodies which avoids some of the difficulties described hereinabove which may occur with either Fy or Fab fragments by generating intact mammalian IgG monoclonal antibodies. It also would Ijø advantageous that such a system allow repeated sampling from a single immunized animal, thus allowing the development of appropriate culture conditions for a specific antigen while using a minimum number of animals. Such a system would be particularly useful for those antigens that produce useful immune responses in only a fraction of the animals immunized or only after extensive booster immunizations.

Summary of Invention

The present invention provides a new method for repertoire cloning of mammalian Ig genes and isolating mammalian cell lines expressing antigen-specific mammalian IgG. A step that increases the number of specific cells of interest is performed. A preferred example of such a step is an in vitro sensitization (IVS) culture procedure for lymphocytes which increased the frequency of antigen- specific B-cells in the lymphocyte population prior to isolation of mRNA for molecular cloning. We have used these methods to isolate anύ-Chlamydia trachomatis (C. trachomatis) lipopolysaccharide (QLPS) IgG's. The method consists of four steps: 1) an enrichment step to increase the frequency of the cells of interest; 2) cloning of the Ig kappa (K) and gamma (γ) genes into eukaryotic expression vectors; 3) introduction of the cloned Ig kappa and gamma genes into mammalian cells capable of secreting IgG; and 4) isolation of cell lines producing antibodies having the desired antigenic specificity. A prefeired enrichment step was in vitro sensitization (IVS) of lymphocytes to increase the frequency of antigen-specific B-cells in the population from which RNA was isolated.

Lymphocytes are obtained from mammals selected from the group consisting of rabbits, goats, humans and sheep. The mammalian cells are selected from the group consisting of Sp2/0, CHO, HeLa, HEK and BHK. The Ig kappa and or Ig gamma genes also may be cloned into eukaryotic viral expression vectors, in particular, eukaryotic retroviral expression vectors. The cloned Ig kappa and Ig gamma genes can be inserted into mammalian cells by infection.

The present invention also provides a monoclonal antibody or fragment thereof which is a member of a specific binding pair and which specifically binds to the other member of the specific binding pair, wherein said monoclonal antibody is produced by a repertoire cloning process. A cell line which secretes a monoclonal antibody or fragment thereof which is a member of a specific binding pair which specifically binds to the other member of the specific binding pair also is provided, wherein said monoclonal antibody is produced by a repertoire cloning process In addition, an immunoassay for an analyte of interest is provided wherein a monoclonal antibody is used to capture said analyte, the improvement comprising the step of adding a known amount of monoclonal antibody or fragment thereof produced by a repertoire cloning process. An assay kit for deteπriining the presence of an analyte of interest in a test sample also is provided which comprises a container containing at least one monoclonal antibody or fragment thereof wherein said monoclonal antibody or fragment thereof is produced by a repertoire cloning process.

A repertoire cloning process to produce monoclonal antibodies having a desired specifity also comprises the steps of increasing the percentage of antibody producing cells approximately 10-fold or greater; cloning the Ig kappa ( ) and Ig gamma (γ) genes into eukaryotic expression vectors or eukaryotic viral expression vectors; introducing cloned Ig kappa and Ig gamma genes into mammalian cells by tranfecting or infecting with viral expression vectors containing cloned Ig kappa and/or Ig gamma genes; and isolating cell lines producing antibodies having the desired specificity. The Ig kappa and/or Ig gamma genes can be cloned into eukaryotic retroviral expression vectors. The mammalian cells are selected from the group consisting of Sp2/0, CHO, HeLa, HEK and BHK.

The present invention also provides that in an immunoassay comprising the steps of binding said analyte which is a member of a specific binding pair and its specific other binding pair member to form a complex, and detecting the presence of said analyte by contacting said complexes with an indicator reagent comprising a monoclonal antibody and a signal generating compound, he improvement comprises utilizing a monoclonal antibody or fragment thereof produced by a repertoire cloning process.

Brief Description of the Drawings FIGURE 1 presents a flow diagram of the repertoire cloning process of the invention which utilizes an in vitro sensitization culture method.

FIGURE 2 presents a schematic showing the relative position of the PCR primers in relation to the Ig gamma and Ig kappa mRNA, wherein "1" designates SEQUENCE ID NO 1, "2" designates SEQUENCE ID NO 2, "3" designates SEQUENCE ID NO 3 and "4" designates SEQUENCE ID NO 4.

FIGURE 3A shows the Ig kappa cloning strategy into a retrovirus cloning vector containing the HygB gene encoding resistance to hygromycin B.

FIGURE 3B shows the Ig gamma cloning strategy into pLCLneo cloning vector, _j FIGURE 4 presents a nucleotide sequence alignment of the V region of gamma gene of C. trachomatis wherein SEQUENCE ID NOS. 5, 6, 7, 8 represent ,,„,„„„ PCT/US96/05114 6/33279

the four different clones, SEQUENCE ID NO 9 is the prototype sequence and SEQUENCE ID NO 15 presents the consensus sequence.

FIGURE 5 presents the nucleotide sequence alignment of the V region of the kappa gene C. trachomatis wherein SEQUENCE ID NOS. 10, 11, 12, 13 represent the four different clones, SEQUENCE ID NO 14 is the prototype sequence and SEQUENCE ID NO 16 presents the consensus sequence.

FIGURE 6 is a bar graph which plots the absorbance vs. antibody reactivity to C. trachomatis lipopolysaccharide (CtLPS) and to Salmonella LPS (SalLPS) as measured by an indirect ELISA methods wherein a filled-in solid bar represents CtLPS and an open bar represents SalLPS.

FIGURE 7 shows a graph of Protein G column of fractions of total kappa IgG of monoclonal antibody B2 wherein absorbance490 is plotted against fractions.

FIGURE 8 shows a graph of Protein A column of fractions of total kappa IgG of monoclonal antibody A8 wherein the amount of IgG (ng/ml) is plotted against fractions.

FIGURE 9 is an SDS-PAGE of the molecular weights of the antigens precipitated by monoclonal antibodies B2 and A8 under reducing and non-reducing conditions.

Detailed Description of the Invention

The system we have discovered avoids several of the potential difficulties which may occur with either Fy or Fab fragments by generating intact mammalian IgG. The procedure we developed includes an enrichment step in which an in vitro sensitization (TVS) procedure was utilized to increase the frequency of antigen-specific B-cells from which mRNA was isolated. IVS has been used to facilitate production of hybridomas by increasing the frequency of antigen-specific B-cells prior to cell fusion (Luben and Mohler, Mol. Immunol. 17:635 [1980]; Borrebaeck, Trends Biotechnol. 4: 147 [1986]; De Boer et al., J. Immunol. Methods 113: 143 [1988]). Unlike most IVS methods, which utilize splenic or lymph node lymphocytes, our procedure utilized peripheral blood lymphocytes (PBL) from immunized rabbits, allowing repeated sampling from a single animal. This facilitates the development of appropriate culture conditions for a specific

using a minimum number of animals. The ability to repeatedly sample from a single animal is particularly useful for those antigens which produce useful immune responses in only a fraction of the animals immunized or only after extensive booster immunizations. Although we performed this procedure in rabbits, the repertoire cloning process described herein can be used for other mammals. Examples of other mammals include goats, sheep, humans, etc. In order for the method described herein to be useful for making human monoclonal antibodies, severe combined immune deficiency (SCID)-human (HU) animals such as mice populated with human cells are preferred to be used to allow stimulation of human antibody without the need to immunize humans with the antigen of interest. See, for example, Duchosal et al., Nature 355:258-262 (1992). The enrichment step also provides the advantage of decreasing the number of screenings required to be performed from at least 1:10,000 to 1:1000, or less. We reasoned and discovered that by using easily-transfectable mammalian cell lines and retroviral vectors it would be possible to construct an antibody expression library directly in mammalian cells. Such a library should have several desirable features. Mammalian cells would produce bivalent IgGs, which would be expected to have higher binding affinities than the Fab fragments produced by bacteria. The antibodies would have intact Fc regions, allowing for purification by protein A- or protein G-affinity chromatography. Screening for clones with appropriate antigenic specificities could be accomplished by the well-characterized ELISA methods used to identify conventional monoclonal antibody-producing hybridoma clones. We discovered that the number of screenings necessary to find appropriate monoclonal antibody decreased significantly, from about at least 1:10,000 with known fusion techniques to 1:1000 or less when the method of the present invention was employed. The details of the procedure we discovered are described herein and outlined ^grammatically in FIGURE 1. Referring to FIGURE 1, the method consists of four steps: 1) an enrichment step to increase the frequency of the cells of interest; 2) cloning of the Ig kappa (K) and gamma (γ) genes into eukaryotic expression vectors; 3) introduction of the cloned Ig kappa and gamma genes into mammalian cells capable of secreting IgG; and 4) isolation of cell lines producing antibodies having the desired antigenic specificity. A preferred enrichment step was in vitro sensitization (IVS) of lymphocytes to increase the frequency of antigen-specific B-cells in the population from which RNA was isolated. However, it is contemplated and within the scope of the invention that other methods for enrichment of specific antibody producing cells can be u ed. Examples of these other methods include but are not limited to cell sorting, affinity binding of antibody producing cells and single cell manipulation by, for example, laser manipulation to enrich for specific antibody producing cells. The present invention contemplates the use of other viral expression vectors not described in the Examples such as but not limited to adenovirus vectors, adeno associated virus vectors and herpes virus vectors, pDEM, baculovirus, SV40 promoters, CMV promoters, etc. Other vectors which are suitable for use in the methods described herein are known to those skilled in the art. It is contemplated and within the scope of the present invention that large scale isolation of mammalian monoclonal antibody by subcloning Ig kappa and Ig gamma genes (as cDNA clones) from isolated specific antibody producing cells obtained from the repertoire cloning process can be performed using well-known protein expression techniques in conjunction with the teachings provided herein. Transfer of antibody cDNAs from one type of antibody producing cells to, for example, Sp 2/0 is well- known to those skilled in the art. See, for example, D.M. Knight, et al., Hum. Antib. Hybrid.3(3):129-136 (1992). Mammalian cell lines available as hosts for expression are known in the art and include many immortahzed cell lines which are available from the American Type Culture Collection. These include HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and others, as well as cell lines such as human embryonyic kidney cells (HEK) and IPBL-Sf21. Still other suitable hosts are known to the routineer.

The present invention details methods for producing monoclonal antibodies from rabbits, but it is within the scope of the present invention that the repetoire cloning process described herein is not dependent upon the mammal used, i.e., the repertoire cloning process described herein can be used in other mammals. The monoclonal antibodies of the present invention are useful in a variety of ways. They can be used in numerous in vitro and in vivo assay methods to detect the presence, if any, of an analyte of interest Examples of such uses are described herein. The monoclonal antibodies produced by the novel repertoire cloning process furthermore can be used for affinity purification of antigens and for the generation of chimeric antibodies. These monoclonal antibodies also can be used therapeutically in a variety of treatment regimes, as well as used prognostically in a variety of ways.

The present invention provides immunoassays which utilizes specific binding members. A "specific binding member," as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules

or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. A specific binding pair member also can include a combination of a conjugate (as defined hereinbelow) and a probe (as defined hereinbelow). Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA molecules. The term "hapten", as used herein, refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein.

"Analyte," as used herein, is the substance to be detected which may be present in the test sample. The analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared. Thus, an analyte is a substance that can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic substances, haptens, antibodies, and combinations thereof. As a member of a specific binding pair, the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B 12, or the use of lectin as a member of a specific binding pair for the determination of a carbohydrate. The analyte can include a protein, a peptide, an amino acid, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances. The details for the preparation of such antibodies and the suitability for use as specific binding members are well known to those skilled in the art

A "capture reagent", as used herein, refers to an unlabeled specific binding member which is specific either for the analyte as in a sandwich assay, for the indicator reagent or analyte as in a competitive assay, or for an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay. The capture reagent can be directly or indirectly bound to a solid phase material before the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample . Test samples which can be tested by the methods of the present invention described herein include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, biological fluids such as cell culture supematants, fixed tissue specimens and fixed cell specimens. It also is within the scope of the present invention that a variety of non-human or non-animal body fluids also can be analyzed for analytes using the monoclonal antibodies of the present invention.

The "solid phase," if used in assays, is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non- magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips and tanned sheep red blood cells are all suitable examples. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. A "solid phase", as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, and other configurations known to those of ordinary skill in the art.

It is contemplated and within the scope of the invention that the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens. Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well. Such useful solid supports include: jjatural polymeric carbohydrates and their synthetically modified, cross- linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyarnides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing polymerization of synthetic polymers on a pre-existing natural polymer. All of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics. The porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents including monoclonal antibodies. Nylon also possesses similar characteristics and also is suitable.

It is contemplated that such porous solid supports described hereinabove are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm. The pore size may vary within wide limits, and is preferably from about 0.025 to 15 microns, especially from about 0.15 to 15 microns.

To change or enhance the intrinsic charge of the solid phase, a charged substance can be coated directly to the material or onto microparticles which then are retained by a solid phase support material. Alternatively, microparticles can serve as the solid phase, by being retained in a column or being suspended in the mixture of soluble reagents and test sample, or the particles themselves can be retained and immobilized by a solid phase support material. By "retained and immobilized" is meant that the particles on or in the support material are not capable of substantial movement to positions elsewhere within the support material. The particles can be selected by one skilled in the art from any suitable type of particulate material and include those composed of polystyrene, polymethylacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials. The size of the particles is not critical, although it is preferred that the average diameter of the particles be smaller than the average pore size of the support material being used. Thus, embodiments which utilize various other solid phases also are contemplated and are within the scope of this invention. For example, ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer, described in co-pending U. S. Patent Application Serial No. 150,278 corresponding to EP Publication No. 0326100, and U. S. Patent Application

Serial No. 375,029 (EP Publication No. 0406473), can be employed according to the present invention to effect a fast solution-phase immunochemical reaction. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged polyaiύon/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including those described in chemiluminescent signal measurements as described in co-pending U.S. Patent Application Serial No. 921,979 corresponding to EPO Publication No. 0273,115. Also, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including automated and semi- automated systems wherein the solid phase comprises a microparticle. Such systems include those described in pending U. S. Patent Application 425,651 and U. S. Patent No. 5,089,424, which correspond to published EPO applications Nos. EP O 425 633 and EP 0424634, respectively, and U.S. Patent No. 5,006,309.

The indicator reagent comprises a signal generating compound (label) which is capable of generating a measurable signal detectable by external means conjugated (attached) to a specific binding memberof the analyte. "Specific binding member" as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to being an antibody member of a specific binding pair for the analyte, the indicator reagent also can be a member of any specific binding pair, including either hapten-anti- hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to the analyte of interest as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay. The various signal generating compounds (labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chenύluminescent compounds such as luminol, dioxetanes, phenanthridinium compounds and acridinium compounds, radioactive elements, and direct visual labels. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.

Other embodiments which utilize various other solid phases also are contemplated and are within the scope of this invention. For example, ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer, described in co-pending U. S. Patent Application Serial No. 150,278 corresponding to EP publication 0326100, and U. S. Patent Application Serial No. 375,029 (EP publication no. 0406473) both of which enjoy common ownership and are incorporated herein by reference, can be employed according to the present invention to effect a fast solution-phase immunochemical reaction. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including those described in cheπiiluiT-inescent signal measurements as described in co-pending U.S. Patent Application Serial No. 921,979 corresponding to EPO Publication No. 0 273,1 15, which enjoys common ownership and which is incorporated herein by reference.

The use of scanning probe microscopy (SPM) for immunoassays also is a technology to which the monoclonal antibodies of the present invention are easily adaptable. In scanning probe microscopy, in particular in atomic force microscopy, the capture phase, for example, at least one of the monoclonal antibodies of the invention, is adhered to a solid phase and a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase. The use of scanning tunneling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. Such a system is described in pending U. S. patent application Serial No. 662,147, corresponding to EP Application No. 92908113.1, which enjoys common ownership and is incorporated herein by reference. The use of SPM to monitor specific binding reactions can occur in many ways. In one embodiment one member of a specific binding partner (analyte specific substance which is the monoclonal antibody of the invention) is attached to a surface suitable for scanning. The attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art Or, covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized. Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece. If the test piece is silicon or glass, the surface must be activated prior to attaching the specific binding partner. Activated silane compounds such as triethoxy amino propyl silane (available from Sigma Chemical Co., St. Louis, MO), triethoxy vinyl silane (Aldrich Chemical Co., Milwaukee, WI), and (3-mercapto-propyl)-trimethoxy silane (Sigma Chemical Co., St. Louis, MO) can be used to introduce reactive groups such as amino-, vinyl, and thiol, respectively. Such activated surfaces can be used to link the binding partner directly (in the cases of amino or thiol) or the activated surface can be further reacted with linkers such as glutaraldehyde, bis (succirrimidyl) suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio] propionate), SMCC (succinimidyl-4-[N- maleimidomethyl] cyclohexane-1-carboxylate), SIAB (succinimidyl [4-iodoacetyl] aminobenzoate), and SMPB (succinimidyl 4-[l-maleimidophenyl] butyrate) to separate the binding partner from the surface. The vinyl group can be oxidized to provide a means for covalent attachment It also can be used as an anchor for the polymerization of various polymers such as poly acrylic acid, which can provide multiple attachment points for specific binding partners. The amino surface can be reacted with oxidized dextrans of various molecular weights (known to those of ordinary skill in the art) to provide hydrophilic linkers of different size and capacity. Also, polyelectrolyte interactions may be used to immobilize a specific

on a surface of a test piece by using techniques and chemistries described by pending U. S. Patent applications Serial No. 150,278, filed January 29, 1988 and Serial No. 375,029, filed July 7, 1989, each of which enjoys common ownership and each of which is incorporated herein by reference. The preferred method of attachment is by covalent means. Following attachment of a specific binding member, the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding. The surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.

The monoclonal antibody of the present invention can be employed in various assay systems to determine the presence, if any, of the antigens of interest or the antibodies of interest in a test sample. Fragments of the monoclonal antibody provided also may be used.

For example, in a preferred assay format, the monoclonal antibody of the invention can be employed as a reagent in a competitive assay for the detection of antibodies to an analyte of interest For example, the analyte of interest previously coated on a solid phase is contacted with a test sample suspected of containing antibody to the analyte of interest and incubated with an indicator reagent comprising a signal generating compound which generates a measurable signal attached to the monoclonal antibody of the invention for a time and under conditions sufficient to form antigen/antibody complexes of the test sample to the solid phase or the indicator reagent to the solid phase. The reduction in binding of the monoclonal antibody of the invention to the solid phase, as evidenced by a reduction in the generated signal, can be quantitatively measured. A measurable reduction in the signal compared to the signal generated from a confirmed negative test sample would indicate the presence of anti-analyte antibody in the test sample. In an alternative assay method.for detection of the analyte of interest, a polyclonal or monoclonal anti-analyte antibody or a fragment thereof, which has been coated on a solid phase, is contacted with a test sample which may contain the analyte of interest, to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antigen antibody complexes. Then, an indicator reagent comprising a monoclonal or a polyclonal antibody or a fragment thereof, which specifically binds to the analyte of interest to which a signal generating compound which generates a measurable signal has been attached, is contacted with the antigen antibody complexes to form a second mixture. This second mixture then is incubated for a time and under conditions sufficient to form antibody/antigen/indicator reagent complexes. The presence of the analyte of interest present in the test sample and captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound. The amount of analyte present in the test sample is proportional to the signal generated.

Alternatively, a polyclonal or monoclonal anti-analyte antibody or fragment thereof which is bound to a solid support, the test sample and an indicator reagent comprising a monoclonal or polyclonal antibody or fragments thereof, which specifically binds to the analyte of interest to which a signal generating co npound which generates a measurable signal is attached, are contacted simultaneously to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antibody/antigen indicator reagent complexes. The presence, if any, of the analyte of interest present in the test sample and captured on the solid phase is deteπnined by detecting the measurable signal generated by the signal generating compound. The amount of analyte present in the test sample is proportional to the signal generated. In this or the assay format described above, the monoclonal antibody of the invention can be employed either as the capture phase or as part of the indicator reagent

In yet another detection method, the monoclonal antibody of the present invention can be employed in the detection of an analyte of interest in fixed tissue sections, as well as fixed cells by immunohistochemical analysis, by standard methods well-known to those skilled in the art.

In addition, the monoclonal antibody can be bound to matrices similar to CNBr-activated sepharose and used for the affinity purification of specific analytes of interest from cell cultures, or biological tissues such as blood and liver. The monoclonal antibody of the invention also can be used for the generation of chimeric antibodies for therapeutic use, or other similar applications. For example, it can be used as one component of a cocktail of antibodies, each having different binding specificities to an identified antigen or agent. Since the cocktail is composed of monoclonal antibodies having different cell and tissue specificity, it is useful for diagnostic applications and therapy, as well as for studyiung cell differentiation and cell-type specificity. For example, a monoclonal antibody can be tagged with a detectable label such as a dye or fluorescent molecules or a radioactive tracer for tumor imaging. Suitable tracers include Iodine¹-^ 1, Indium* ^x 1 or Technetium99. Suitable monoclonal antibodies can be used therapeutically both in conjugated or unconjugated forms in a cocktail of monoclonal antibodies or separately. Suitable conjugates for these monoclonal antibodies include chemotherapeutic drugs, toxins or radioisotopes. Certain radioactive isotopes such as Iodinel31 can be conjugated directly while other radioisotopes such as Indium 111 or Technetium _can be conjugated indirectly to the monoclonal antibodies of the invention though the use of chelators or by other methods known to those of ordinary skill in the art. The conjugated or unconjugated monoclonal antibodies may be admininstered in a cocktail of monoclonal antibodies or in separate dose form. Dosages for humans can be determined by known methods. The monoclonal antibodies described herein are useful for prognostic applications as well such as in staging disease states where the antibodies are tagged as described hereinabove and either administered in vivo to an individual in appropriate predetermined dosages or cells of interest are removed from an individual and assayed in vitro for the analyte of interest.

The monoclonal antibody or fragment thereof can be provided individually to detect analyte of interest Combinations of the monoclonal antibody (and fragments thereof) of the present invention provided herein also may be used in combination with other monoclonal antibodies that have differing specificities for the analyte of interest as components in a mixture or "cocktail" of anti-analyte antibodies, each having different binding specificities. Thus, this cocktail can include the monoclonal antibody of the invention directed to a specific analyte or portion thereof, along with different monoclonal antibodies directed to other regions of the analytes, such as other binding sites on an analyte of interest. This cocktail of monoclonal antibodies would then be used in place of the single monoclonal antibody as described in the assay formats herein.

While the present invention discloses the preference for the use of solid phases, it is contemplated that the monoclonal antibody of the present invention can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invention.

The monoclonal antibody of the invention can be used as a positive control in an assay which is designed to detect the presence of an analyte antibody in a test sample. In an assay which detects the presence of an analyte antibody in a test sample, antibody's specific binding pair member would be used as a capture phase. These antibody's specific binding pair members could be prepared by various means (if microbiological in natue) from viral, yeast or bacterial lysates, synthetic peptides of various immunogenic regions of the antibody's specific binding pair member's genome, and/or recombinant proteins produced by using either synthetic or native antigens or epitopes of antigens. Any substance to which a mammal can produce monoclonal antibodies against is within the scope of the present invention. It also is contemplated that these types of antibody's specific binding pair members could be employed in a variety of assay formats including those described herein as either the capture phase or detection phase. The use of the monoclonal antibody of the invention would ensure that the reagents provided to detect the analyte antibody were performing adequately by being used in place of a test serum in the performance of the assay, according to procedures known to those of ordinary skill in the art.

Additional assay methods for the monoclonal antibodies of the invention include their use in flow cytometric procedures and in particle counting procedures. For example, in particle counting, analytes which are members of specific binding pairs are quantified by mixing an aliquot of test sample with microparticles coated with monoclonal antibody capable of binding to the analyte of interest as the other member of the specific binding pair. If the analyte is present in the test sample, it will bind to some of the microparticles coated with the monoclonal antibody and agglutinates will form. The analyte concentration is inversely proportional to the unagglutinated particle count. See, for example, Rose et al., eds., Manual of Clinical Laboratory Immunology, 3rd edition, Chapter 8, pages 43-48, American Society for Microbiology, Washington, D. C. (1986). Flow cytometry methods that sense electronic and optical signals from cells which are illuminated allows determination of cell surface characteristics, volume and cell size. Monoclonal antibodies to analytes present in a test sample are bound to the analyte and detected with a fluorescent dye which is either directly conjugated to the monoclonal antibody or added via a second reaction. Different dyes, which may be excitable at different wavelengths, can be used with more than one monoclonal antibody specific to different analytes such that more than one analyte can be detected from one sample. In fluorescence flow cytometry, a suspension of particles, typically cells in a blood sample, is transported through a flowcell where the individual particles in the sample are illuminated with one or more focused light beams. One or more detectors detect the interaction between the light beam(s) and the labeled particles flowing through the flowcell. Commonly, some of the detectors are designed to measure fluorescence emissions, while other detectors measure scatter intensity or pulse duration. Thus, each particle hat passes through the flowcell can be mapped into a feature space whose axes are the emission colors, light intensities, or other properties, i.e., scatter, measured by the detectors. In one situation, the different particles in the sample map into distinct and non-overlapping regions of the feature space, allowing each particle to be analyzed based on its mapping in the feature space. To prepare a test sample for flow cytometry analysis, the operator manually pipettes a volume of blood from the sample tube into an analysis tube. A volume of the desired fluorochrome labeled monoclonal antibody of the invention is added. The sample/antibody mixure then is incubated for a time and under conditions sufficient to allow antibody/antigen bindings to take place. After incubation, and if necessary, the operator adds a volume of RNS lyse to destroy any RBCs in the sample. After lysis, the sample is centrifuged and washed to remove any left-over debris from the lysing step. The centrifuge/wash step may be repeated several times. The sample is resuspended in a volume of a fixative and the sample then passes through the fluorescence flow cytometry instrument. A method and apparatus for performing flow automated analysis is described in co-owned U.S. Patent application Serial No. 08/283,379, which is incorporated herein by reference. It is within the scope of the present invention that microsphere can be utilized in conjunction with the antibodies described herein, tagged or labeled, and employed for in vitro diagnostic applications. It also is within the scope of the present invention that other cells or particles, including bacteria, viruses, durocytes, etc., can be tagged or labeled with the monoclonal antibodies of the present invention and used in flow cytometric methods.

It is contemplated that the reagent employed for the in vcitro assays can be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a monoclonal antibody, or a cocktail of monoclonal antibodies, employed in the assay(s). These kits also could contain vials or containers of other reagents needed for performing the assay(s), such as washing, processing and indicator reagents. The same may be feasible for in vivo applications, both for diagnostic and therapeutic uses.

The following examples demonstrate the advantages and utility of the method and monoclonal antibodies of the invention by describing methods for the development characterization, and clinical utility of the monoclonal antibodies.

EXAMPLES Example 1. In vitro sensitization. NZW rabbits immunized with Chlamydia trachomatis (C. trachomatis) were us€d as the source of peripheral blood lymphocytes (PBL), as follows. The antibodies can be produced by employing an antigen common to the prevalent strains of C. trachomatis, in accordance with known techniques. Such antigens are readily ascertained by the routineer and include, for example, C. trachomatis LGV Type II strain Tang, C. trachomatis Trachoma serotype A strain HAR-13, C. trachomatis LGV Type II strain 434 and the like. Although a single antigen may be utilized to produce chlamydial antibody, a pool of antigens from various strains may be employed in the serial immunization of an animal, to produce a chlamydial antibody. See, for example, U.S. Patent No. 4,497,899, which enjoys common ownership and is incoφorated herein by reference. At each immunization period, the total dose per rabbit was 10*^u purified chlamydia particles. Rabbits were injected at four sites subcutaneously and in each footpad with a chlamydia antigen suspension mixed with an equal volume of Freunds adjuvant in two separate inoculations one month apart One week following the second immunization, rabbits were given an intraveneous injection of the chylamydia suspension. Rabbits were given two additional intravenous injections two weeks apart three months later. The immunization protocol has been described by, for example, L. Howard, Infection and Immunity ll(4):698-703 (1975). Animals routinely received booster immunizations seven days prior to bleeding for PBL isolation. All animal manipulations were carried out in accordance with applicable regulations regarding animal care and handling. PBL were isolated from citrated whole blood by centrifugation through Lympholyte-R (Catalog number CL5050, Cedarlane Laboratories, Hornby, Ontario, Canada). Residual red blood cells in the lymphocyte fraction were removed by lysis with 17 mM Tris, pH 7.2/144 mM NH4CI followed by centrifugation of the lymphocytes through fetal calf serum. Viable cell numbers were determined by Trypan blue exclusion. Isolated lymphocytes were suspended at 3 x 10^ cells/ml in Dulbecco's modified Eagle medium (DMEM), 4.5 g/L glucose, supplemented with 2 mM sodium pyruvate, 10 mM non-essential amino acids, 20 mM glutamine, 50 μM 2- mercaptoethanol, 10% normal rabbit serum (GIBCO), 8% human mixed- lymphocyte reaction (MLR) supernate, 8% Jurkat cell culture supernate, 4% human T-cell Polyclone (Becton Dickinson, Bedford, MA), and 0.5 μg/ml C. trachomatis lipopolysaccharide (CtLPS) or no antigen. The cultures were incubated at 37°C, 5% CO2- After three days, the culture was supplemented with 8% Jurkat and 4% Polyclone supernates. Cells were harvested one day later for determ iation of the frequency of antigen specific, antibody-secreting cells. Example 2. Immunogold microplaque assay. The frequency of antigen-specific, antibody-secreting cells was estimated using a microplaque immunoassay (Walker and Dawe, J. Immunol. Methods 104:281 [1987]). 96-well immunoassay plates (Catalog number 3590, Costar, Cambridge, MA) were coated overnight at 4°C with lOOμl per well of 2 μg/ml CtLPS in phosphate-buffered saline (PBS). Plates were blocked with MAB diluent (0.1M Tris, pH 7.2/ 0.1% Tween-20®/ 0.01% Thimerosal®/ 25% phosphate-buffered saline, pH 7.2/ 25% fetal bovine serum) for 20 min at 37°C. Two-fold serial dilutions of washed lymphocytes suspended in serum-free DMEM were plated in the wells and incubated in a vibration-free CO2 incubator for 3 hr. Plates were washed with 10 mM Tris, pH 8.2. Colloidal gold-conjugated goat anti-rabbit IgG (Auroprobe™ LM, Amersham, Arlington Heights, IL) was added and the plate was incubated overnight at 4°C. The plate was washed to remove non-bound antibody and sites of binding of gold-conjugated antibody were visualized by development with Intense™ silver stain solution (Amersham,

Arlington Heights, IL). Plaques were counted by microscope. The frequency of antigen-specific, antibody-secreting cells was calculated as the number of plaques per well divided by the number of lymphocytes plated in the well ("IGSS frequency"). As the data in TABLE 1 show, IVS increased by 10- to 600-fold the frequency of antigen-specific B-cells in the PBL population. For subsequent cloning procedures, we utilized mRNA isolated from an IVS reaction which yielded a 30-fold increase in the frequency of cells secreting antibody to CtLPS IgG. The pre-IVS frequency of antibody to CtLPS cells was 0.036%. Following IVS, the frequency of antibody to CtLPS cells was 1.1% and the frequency of antibody to Salmonella LPS cells (SalLPS) was 0.46%.

TABLE 1

IVS Response of PBL from Chlamydia Immunized Rabbits

Rabbit Pre-IVS* CtLPS SalLPS

7718 0.01 0.16 0.08

7718 <0.001 0.05 ND

7714 0.003 0.03 0.03

7447 0.036 1.1 0.46

7447 <0.001 0.6 0.5

7081 ND 0.4 0.98

7428 0.003 0.6 3

7917 <0.001 0.05 0.05

7081 <0.001 0.4 0.02

^♦Average IGSS frequency v. CtLPS and SalLPS.

Example 3. PCR primers.

DNA amplification primers specific for rabbit Ig gamma and kappa genes were designed based on the rabbit Ig gene sequences reported in Kabat et al., 5th ed., U.S. Department of Health and Human Services, Washington, D. C. (1991) and GenBank®. The GenBank® accession numbers were as follows for gamma- specific sequences: M21260, K00752, J00665; the GenBank® accession number for the kappa-specific sequence was K01359. See, Nucleic Acid Res. 22:3441- 3444 (1994. The 5'- and 3 '-gamma- specific primers had the sequences GGTCTAGAATGGAGACTGGGCTGCGCT (SEQUENCE ID NO. 1) and GGGAGCTCGCTGGGTGCTTTATTTGTGT (SEQUENCE ID NO. 2), respectively. The 5'- and 3 '-kappa-specific primers had the sequences

CCGGATCCATGGACACGAGGGCCCCCA (SEQUENCE ID NO. 3) and GGGTCGACGCGGCCGCCTAGGGTCGCTGGGAGTGC (SEQUENCE ID NO. 4), respectively. These primers include restriction sites which allow with directional cloning into expression vectors for the gamma (Xbal-Sacl) and kappa (BamHl-Notl) libraries. FIGURE 2 schematically shows the primer sequences and their positions in relation to the Ig gamma and kappa mRNAs.

Example 4. Isolation of mRNA From Rabbit Lymphocytes. The Dynabead mRNA Purification Kit (catalog number 610.01, DYNAL, Lake Success, NY) was used to isolate mRNA from in v/trø-sensitized lymphocytes. Briefly, 1 x 10⁶ to 3 x 10⁶ cells were washed with cold PBS (pH 7.1). The pellet was resuspended in 100 μl 10 mM Tris-HCl, pH7.5/0.14 M NaCl 5 mM KC1 1% Triton X-100® and placed on ice for 1 min. Nuclei were then pelleted by centrifugation and the supernate transferred to a new tube containing Oligo (dT)25 Dynabeads (500μg per 1 x 10⁶ cells) in 100 μl 20 mM Tris-HCl, pH7.5/l M LiCl/2 mM EDTA/1% SDS. The mixture was incubated for 5 min at room temperature to allow binding of mRNA to the magnetic beads. The beads were then trapped using the MPC-E-1 magnetic particle concentrator (DYNAL) and the supernate removed. The beads were washed three times with 200 μl 10 mM Tris-HCl, pH7.5/0.15 M LiCl/1 mM EDTA/0.2% SDS using the MPC-E- 1. RNA was eluted from the beads by incubation at 65°C for 2 min in 2 mM EDTA, pH 7.5 (20 μl/ 1 x 10⁶ cells) .

Example 5. Ig Gene Amplification and Cloning. cDNA was synthesized and Ig genes were amplified using Perkin-Elmer-

Cetus' RNA PCR Kit (Catalog number N808-0017, Perkin-Elmer-Cetus, Norwalk, CT). Oligo (dT) l DNA was used as the primer used for synthesis of Ig kappa cDNA. Both oligo (dT)i6 DNA and random DNA hexamers were used for priming Ig gamma cDNA synthesis. Synthesis was carried out for 30 min at 42°C followed by denaturation at 99°C for 5 min and quenching at 4°C. Ig genes were amplified by PCR in the same tubes after the addition of 4 μl M MgCl2, 8 μl 10X PCR Buffer fl, 1 μl Perfect Match (Stratagene, La Jolla, CA), 0.5 μl AmpliTaq DNA polymerase, 2 μl primers, and 64.5 μl sterile distilled water to give a volume of 100 μl per reaction. DNA was denatured by heating to 95°C for 2 min. AmpUfication was carried out by 40 cycles of denaturation at 94°C for 1.5 min and elongation at 64°C for 3 min. A final cycle at 64°C for 10 min was performed to ensure complete synthesis of amplified strands. The amplified DNA was then stored at 4°C until used for cloning. The kappa-specific primers (SEQUENCE ID NOS. 3 and 4) yielded DNA fragments of approximately 700 bp and the gamma-specific primers (SEQUENCE ID NOS 1 and 2) yielded fragments of approximately 1400 bp. The amplified gene products were cloned into eukaryotic expression vectors using standard techniques. The Ig kappa DNA fragments were digested with Sail and BamHl and ligated into a retroviral vector containing the hygromycinB (hygB) gene encoding resistance to hygromycin B to construct the Ig kappa library. The ligated plasmids were used to transform E. coli. Plasmid DNA was prepared from these bacterial populations for transfection into mammalian cells. FIGURE 3A shows the Ig kappa cloning strategy into a retrovirus cloning vector containing the HygB gene encoding resistance to hygromycin B. FIGURE 3B shows the Ig gamma cloning strategy into pLCLneo cloning vector. The Ig gamma fragment DNA was digested with Sacl and Xbal and ligated into pLCLneo vector to construct the Ig gamma libraries. The ligated plasmids were used to transform E. coli. Ampicillin-resistant bacterial colonies resulting from these transformations were pooled to generate kappa and gamma sequence libraries. These data are presented in TABLE 2. Plasmid DNA was isolated from these libraries for transfection into mammalian cells.

TABLE 2

Kappa and gamma sequence libraries prepared in E. coli.

Number of colonies

Library Gene pooled

106 Gamma 230

107 Gamma 136

108 Gamma 1200

123 Kappa 1000

124 Kappa 1500

Example 6. Transfection of mammalian cells.

The cloned DNAs described hereinabove were introduced into mammalian cells using Lipofectin (Catalog number 18292-037, Gibco BRL, Gaithersburg, MD) according to the manufacturer's recommendations. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and gentamicin (50μg/ml) and the appropriate selective agents. Plasmid DNA isolated from gamma library 107 was introduced into mouse L-cells (available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 as A.T.C.C. deposit number CC11) and the cells selected for resistance to genteticin (600 μg/ml) (Gibco BRL, Gaithersburg, MD). Approximately 2200 genteticin-resistant colonies were obtained. These colonies, designated RHC (Recomb ant Heavy Chain) cells, were pooled and stored in liquid nitrogen (LN2). The kappa library was introduced into the PA317 retrovirus packaging cells and the cells selected for resistance to hygromycin B (400 μg/ml) (Calbiochem, La Jolla, CA). Approximately 1500 colonies were obtained following selection. Virus stocks were prepared from RLC (Recombinant Light Chain) cells (hygromycin-resistant cells) and stored at -80°C. The hygromycin-resistant cells were pooled and stored in LN2-

The recombinatorial antibody-producing cell line library was produced by infecting RHC cells with RLC virus at a multiplicity of infection (MOI) > 5 to ensure infection of all RHC cells. The infected cells were selected for resistance to both genteticin (600 μg/ml) and hygromycin B (400 μg/ml). Since the cells were infected at an MOI of ~5, little cell killing resulted from this selection step. Aliquots of the genteticin/hygromycin-resistant cells were stored in LN2 prior to screening for cells secreting antibody specific for CtLPS.

Example 7. Screening Recombinatorial Library for aCtLPS Clones. Clones expressing antibody to CtLPS were identified by screening culture supemates from cell populations using an indirect ELISA, as follows. Recombinatorial cells were plated at 1000 cells per well in 96-well plates in selection media. When cells reached near-confluence, the selective media was replaced with DIFTI^'S media (DMEM:Ham's F12, 1:1, supplemented with 1.2 g/L glycine, 8 g/L glucose, 10 ml/L ITS+™ (Catalog number 40352, Beckton Dickinson, Bedford, MA), 10 ml/L non-essential amino acids, and 2 mM L- glutamine) and incubated for four days. Culture supemates from these cell populations were diluted 1 :2 in MAB diluent (0.1M Tris, pH 7.2, 0.1 %

Tween20®, 0.01% Thimersol®, 50% PBS, pH 7.2, 50% fetal bovine serum) and assayed for antibody to CtLPS and antibody to SalLPS, a common cross-reacting antigen for CtLPS antibodies, using an indirect ELISA. Briefly, ELISA microtiter plates (COS TAR) were coated with CtLPS or SalLLS suspended at 1 μg/ml in Chlamidiazyme™ specimen dilution buffer (available from Abbott Laboratories, Abbott Park, IL). Rabbit Ig captured onto these plates was detected using peroxidase-conjugated anti-rabbit IgG (Fc-specific) (available from Jackson ImmunoResearch, West Grove, PA) and o-phenylenediamine (OPD) (available from Abbott Laboratories, Abbott Park, IL) and quantitated by measuring absorbance at 490 nm (A490).

Samples exhibiting A490 values > two times the background level (=0.085 in this case) were considered positive in the primary screen. Supemates from 11 of 96 wells exhibited binding to CtLPS-coated plates. Supemates from four of 96 wells exhibited binding to SalLPS-coated plates and three of these four supemates were also positive for binding to CtLPS. Cells from positive wells were replated at 20 cells per well and reassayed for production of antibodies to CtLPS and SalLPS. Cell populations which were deterrnined to be positive at this secondary screening level were subjected to single cell cloning. Single cell clonal populations which produced antibodies having the desired specificities were isolated and expanded for characterization of the antibodies and gene sequences.

Example 8. Re-isolation of antibody genes for sequence analysis. Rabbit gamma and kappa sequences were isolated from 681.1B2, 681.1B5, 71.4A8, and 71.4B4 cell populations by PCR as described above. The sequences of the V-regions of the gamma and kappa genes were deterrnined using standard DNA sequencing techniques. The aligned sequences for the gamma and kappa V-regions are shown in FIGURES 4 and 5, respectively. Cell line 681.1B2's gamma V-region also is presented as SEQUENCE ID NO. 5 and its kappa v-region is presented as SEQUENCE ID NO 10; cell line 71.4B4's gamma V-region also is presented as SEQUENCE ID NO. 6 and its kappa v-region is presented as SEQUENCE ID NO 11; cell line 681.1B5's gamma V-region also is presented as SEQUENCE ID NO. 7 and its kappa V-region is presented as SEQUENCE ID NO 12; cell line 71.4A8's gamma V-region also is presented as SEQUENCE ID NO. 8 and its kappa V-region is presented as SEQUENCE ID NO 13. The prototype sequence for the gamma V-region is presented as SEQUENCE ID NO 9 and the consensus sequence for the gamma V-region is presented as SEQUENCE ID NO 15. The prototype sequence for the kappa V-region is presented as SEQUENCE ID NO 14 and the consensus sequence for the kappa V- region is presented as SEQUENCE ID NO 16.

Example 9. ELISA. Total Ig and antibody to LPS production were assayed by ELISA. Briefly, ELISA microtiter plates (COSTAR) were coated with anti-rabbit IgG (Fab'2- specific) (available from Jackson ImmunoResearch, West Grove, PA) to measure total Ig, or with antibody to C/LPS or antibody to SalLPS, as described above, to measure antibody to LPS Ig. Rabbit Ig captured onto these plates was detected using peroxidase-conjugated anti-rabbit IgG (Fc-specific) (available from Jackson ImmunoResearch, West Grove, PA) and O-phenylenediamine (OPD) (available from Abbott Laboratories, Abbott Park, EL) and quantitated by measuring absorbance at 490 nm (A490). Example 10. Antibody production and characterization. Cloned cell populations were grown to confluence in selective media following methods known in the art. The growth media was replaced with D TS media to produce antibody-containing culture supemates. Antibody- containing media was stored at -20°C prior to assay. The concentration of rabbit IgG in the culture supernate remained relatively constant from day 3 to day 7 of this production period, as assayed by ELISA methods described hereinabove. Antibody production by 4 L-cell lines, designated 681.1 B2 (which expressed SEQUENCE ID. NO. 5 and SEQUENCE ID. NO. 10), 681.1 B5 (which expressed SEQUENCE ID NO. 7 and SEQUENCE ID NO 12), 71.4A8 (which expressed SEQUENCE ID NO. 8 and SEQUENCE ID NO 13) and 71.4B4 (which expressed SEQUENCE ID NO. 6 and SEQUENCE ID NO 11) was characterized. Total IgG secretion into serum-free culture media was measured by ELISA as described hereinabove. All cell lines produced similar levels of IgG in 4-day culture supemates, ranging between 50 ng/ml and 100 ng/ml. These data are summarized graphically in FIGURE 6, wherein binding to CtLPS and SalLPS of polyclonal antibody to C. trachomatis (designated as "anti- C. trach") and four monoclonal antibodies produced by the methods described hereinabove (E1C5B2, E1G4B5, G9F11A8 and G9F11B4) are plotted against A490. Referring to FIGURE 6, the data demonstrated that all antibodies exhibited binding to CtLPS comparable to that of the polyclonal rabbit anti-CtLPS. The 681.1 monoclonal antibodies and polyclonal sera also exhibited binding to Salmonella LPS (SalLPS), a common cross-reacting antigen for anti-Chlamydia antisera (see FIGURE 6). These cell lines have been deposited at the A.T.C.C. as of , 1995, under the terms of the Budapest Treaty.

The affinity ligands Protein A and Protein G frequently are used as convenient means both to purify and concentrate IgG. Amicon MAC protein A (Catalog number 11035) or protein G (Catalog number 11039) capsules (Amicon, Beverly, MA) were used for this purpose following manufacturer's directions. Filtered culture supemates were loaded onto the capsule and the capsule washed with PBS. IgG was eluted with 0.15 M glycine (pH 2.3), 1 ml fractions collected, and the fractions neutralized with 1 M Tris (pH 8). As expected for rabbit IgG, both Protein A and Protein G could be used successfully to concentrate and purify IgG from culture supemates These data are presented in TABLE 3 and in

FIGURES 7 and 8. FIGURE 7 shows a graph of Protein G column of fractions of total kappa IgG of monoclonal antibody B2 wherein absorbance490 is plotted against fractions. FIGURE 8 shows a graph of Protein A column of fractions of total kappa IgG of monoclonal antibody A8 wherein the amount of IgG (ng/ml) is plotted against fractions.

TABLE 3

Specificity for Chlamydia and Salmonella LPS by 681.1 and 71.4 monoclonal antibodies from culture supemates and affinity-purified IgG*

Source Plate Coating

Antibody SalLPS anti-Fab CtLPS SalLPS

B2 supernate 2.67 0.61 2.51

A8 supernate 1.63 0.21 0

B2 Protein G 2.04 0.406 1.9

A8 Protein A 1.737 0.314 0 *Assayed by ELISA. Values = A490.

As the data of TABLE 3 and FIGURES 7 and 8 demonstrate, no IgG was detected in the column flow-through or wash fractions, indicating that the IgG material detected in the culture supemates represented intact IgG. Antibody was also concentrated by ultrafiltration using a stirred cell with an

Amicon YM100 membrane or an Amicon Centriprep 100 concentrator. In both devices, the ultrafiltration membrane retains molecules with molecular weights greater than 100 kd in the concentrated material. IgG was detected only in the concentrated material as expected for intact IgG (MW=150 kd). The molecular weight of the affinity-purified antibodies also was assessed by Western blotting. Protein A- or Protein G-purified IgG was suspended in Laemli buffer either with or without dithiothreitol (DTT), separated on 4% - 20% polyacrylamide gels (Bio-Rad, Hercules, CA), and transferred to nitrocellulose by electroblotting. Blots were blocked with MAB diluent. Rabbit Ig bands were detected using a mixture of goat-anti-rabbit IgG, Fab'2-specific and goat-anti- rabbit IgG, heavy- and light-chain-specific followed by horseradish peroxidase- conjugated, mouse anti-goat IgG (available from Jackson ImmunoResearch, West Grove, _^RA). Antibody binding was visualized using 4-chloronaphthol. When electrophoresed under non-reducing conditions, a single band was obtained which co-migrated with rabbit IgG. Under reducing conditions, two bands of were detected, corresponding to the kappa and gamma polypeptide chains. FIGURE 9 shows the bands obtained under reducing or non-reducing conditions, where lane 1 contains blank, lane 2 contains control MW markers of (Catalog number 72807 A, BioRad, Hercules, CA) of myosin, β-galactosidase, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme and aprotinin, lane 3 contains rabbit IgG (Jackson ImmunoResearch, West Grove, PA) (reduced), lane 4 contains monoclonal antibody obtained from cell line 681.1B2 (reduced), lane 5 contains monoclonal antibody obtained from cell line 71.4A8 (reduced), lane 6 contains blank, lane 7 contains monoclonal antibody obtained from cell line 71.4A8 (non-reduced), lane 8 contains monoclonal antibody obtained from cell line 681.1B2 (non-reduced), lane 9 contains rabbit IgG (Jackson ImmunoResearch, West Grove, PA) (non-reduced) and lane 10 contains blank.

The mammalian monoclonal antibodies made by the novel repertoire cloning process described herein provide several advantages over known monoclonal antibodies and their production methods. The process described herein avoids some of the difficulties which may occur with either Fy or Fab fragments by generating intact mammahan IgG monoclonal antibodies. It also provides a system which allows repeated sampling from a single immunized animal, thus allowing the development of appropriate culture conditions for a specific antigen while using a minimum number of animals. Monoclonal antibodies against those antigens that produce useful immune responses in only a fraction of the animals immunized or only after extensive booster immunizations thus can be produced. Further, screenings required to identify monoclonal antibodies of interest are significantly reduced.

The cell lines produced by the methods described herein, as well as the plasmids described herein, have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, as of , under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit or for five (5) years after the last request for the deposit or for the enforceable period of the U.S. patent whichever is longer. The deposits and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. The sequences in all of the deposited materials are incorporated herein by reference. The plasmids associated with monoclonal antibody produced by cell line 681.1B2 designated as p681.1B2κ and p681.1B2γ were accorded A.T.C.C. deposit nos. , plasmids associated with monoclonal antibody produced by cell line 681.1B5 designated as p681.1B5 and p681.1B5γwere accorded A.T.C.C. deposit nos. , plasmids associated with monoclonal antibody produced by cell line 71.4A8 designated as p71.4A8κ and p71.4A8γ were accorded A.T.C.C. deposit nos. and plasmids associated with monoclonal antibody produced by cell line 71.4B4 designated as p71.4B4 and p71.4B4γ were accorded the following A.T.C.C. deposit numbers: . The cell lines were accorded the following A.T.C.C. deposit numbers: cell line 681.1 B2 (termed AL681.1B2) was accorded A.T.C.C. deposit number , cell line 681.1 B5 (termed AL681.1B5) was accorded

A.T.C.C. deposit number , cell line 71.4A8 (termed AL71.4A8) was accorded A.T.C.C. deposit number and cell line 71.4B4 (termed

AL71.4B4)was accorded A.T.C.C deposit number .

Other modifications and variations of the specific embodiments of the invention as set forth herein will be apparent to those skilled in the art Accordingly, the invention is intended to be limited in accordance with the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: OKASINSKI, GREGORY F. FRY, DENNIS G.

(ii) TITLE OF INVENTION: REPETOIRE CLONING PROCESS, PRODUCTS DERIVED

THEREFROM AND USES FOR SAID PRODUCTS

(iii) NUMBER OF SEQUENCES: 16

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: ABBOTT LABORATORIES

(B) STREET: ONE HUNDRED ABBOTT PARK ROAD

(C) CITY: ABBOTT PARK

(D) STATE: IL

(E) COUNTRY: USA

(F) ZIP: 60064-3500

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: 5695.US.01

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNE /AGENT INFORMATION:

(A) NAME: Pore bski, Priscilla E.

(B) REGISTRATION NUMBER: 33,207

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 708-937-6365

(C) TELEX: 708-938-2623

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEC^' NCE DESCRIPTION: SEQ ID Nθ:l: f GGTCTAGAAT GGAGACTGGG CTGCGCTG 28 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGGAGCTCGC TGGGTGCTTT ATTTGTGT 28

(2) INFORMATION FOR SEQ ID NO.-3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (geno ic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCGGATCCAT GGACACGAGG GCCCCCA 27

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : GGGTCGACGC GGCCGCCTAG GGTCGCTGGG AGTGC 35

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 606 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) /TOPOLOGY : linear

( ii ) MOLECULE TYPE : DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GGATCCCTGA CACTCACCTG CACAGTTTCT GGATTCTCCT TCAGTAGCGG CTACTGGATA 60

TGCTGGGTCC GCCAGGCTCC AGGGNAAGGG CTTGGAGTGG ATCGCATGCA TCTATACTGG 120

TGATAGTAGT GGTAGCACTG ACTACGCGAA CTGGGCGAAA GGCCGATTCA CCAGCTCCAA 180

AAGCTCGTCG ACCACGGTGA CTCTGCAAAT GACCAGTCTG ACAGCCGCGG ACACGGNCAC 240

CTATTTCTGT GCGAGAGCCC CGTCTTACGT TGTTTATTCT ACTGCTGGTT ATACTTATGC 300

TTCCTACTTG GATTTGTGGG GSCCAGGCAC CCTGGTCACC GTCTCCTCGG GGNAACCTAA 360

GGSTCCATCA GTCTTCCCAC TGGCCCCCTG CTGNGGGGAC ACACCCAGCT CCACGGTGAC 420

CCTGGGCTGN CTGGTCAAAG GCTACCTCCC GGAGCCAGTG ACCGTGACCT GGAACTCGGG 480

CACCCTCACC AATGGGGTAC GCACCTTCCC GTCCGTCCGG NAGTCCTCAG GCCTCTACTC 540

GCTGAGCAGA GTGGTGAGCG TGACCTCAAG CAGGCAGNCC GTCACCTGNA ACGTGGNCCA 600

CCCAGN 606

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 616 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

GGATCCCTGA CACTCACCTG CACAGTTTCT GGATTCTCCT TCAGTAGCGG CTACTGGATA 60

TGCTGGGTCC GCCAGGCTCC AGGGNAAGGG CTTGGAGTGG ATCGCATGCA TCTATACTGG 120

TGATAGTAGT GGTAGCACTG ACTGCGCGAA CTGGGCGAAA GGCCGATTCA CCAGCTCCAA 180

AAGCTCGTCG ACCACGGTGA CTCTGCAAAT GACCAGTCTG ACAGCCGCGG ACACGGCCAC 240

CTATTTCTGT GCGAGAGCCC CGTCTTACGT TGTTTATTCT ACTGCTGGTT ATACTTATGC 300

TTCCTACTTG GATTTGTGGG GSCCAGGCAC CCTGGTCACC GTCTCCTCGG GGNAANCTAA 360

GGCTCCATCA GTCTTCCCAC TGGCCCCCTG CTGNGGGGAC ACACCCAGCT CCACGGTGAC 420

CCTGGGCTGC CTGGTCAAAG GCTACCTCCC GGAGCCAGTG ACCGTGACCT GGAACTCGGG 480

CACCCTCACC AATGGGGTAC GCACCTTCCC GTCCGTCCGG NAGTCCTCAG GCCTCTACTC 540 GCTGAGCAGA GTGGTGAGCG TGACCTCAAG CAGNCAGNCC GTCACCTGNA NCGTGGNCCA 600 CCCAGNCACC AACAAC 616

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 683 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii ) MOLECULE TYPE : DNA (genomic)

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 7 :

GGATCCATGG AGACTGGGCT GCGCTGGCTT CTCCTGGTCG CTGTGCTCAA AGGTGTCCAG 60

TGTCAGGAGC AGCTGGAGGA GTCCGGAGGA GGCCTGGTCA AGCCTGGGGC ATCCCTGACA 120

CTCACCTGCA AAGCCTCTGG ATTCTCCTCT GGTAGAGGCG ACGACGTGTG CTGGGTCCGC 180

CAGGCTCCGG GGAAGGGGCT GGAGTGGATC GCATGCATTG ACCTTGATAG GCGCAACACA 240

GTCTATGCGA CCTGGGCGAA AGGNCGATTC TCCATCTCCA AGACCTCGCC GACCACGGTG 300

GATCTGAAAA TGACCAGTCT GACAGACGCG GACACGGSCA CCTATTTCTN GTGCGAGACA 360

GATTTATGCT GCGACTTGTA ATTTGTGGGG CCCAGGCACC CTGGTCACCG TCTCCTCAGG 420

GCAACCTAAG GCTCCATCAG TCTTCCCACT GGCCCCCTGC TGNGGGGACA CACCCAGCTC 480

CACGGTGACC CTGGGCTGNC TGGTCAAAGG CTACCTCCCG GAGCCAGTGA CCGTGACCTG 540

GAACTCGGGC ACCCTCACCA ATGGGGTACG CACCTTCCCG TCCGTCCGGG AGTCCTCAGG 600

NCTCTACTCG CTGAGCAGNG TGGTGAGCGT GACCTCAAGC AGGCAGCCCG TCACCTGTAA 660

NGTGGNCCAC CCAGCCACCA ACA 683

(2 ) INFORMATION FOR SEQ ID NO : 8 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 663 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY : linear

( ii ) MOLECULE TYPE : DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGATCCATGG AGACTGGGCT GCGCTGGCTT CTCCTGGTCA CTGTGCTCAA AGGTGTCCAG 60

TGTCAGTCGT TGGAGGAGTC CGGGGGAGGC CTGGTCAAGC CTGGGGCCTC CCTGAAACTC 120

GCCTGCACAG CCTCTGGATT CTCCCTCAGT GGCAAGCCAA TGTGTTGGGT CCGCCAGGCT 180

CCAGGGAAGG GGCTGGAGTG GATCGCATGC CTTCGGCCTA GTGGCAGTAT TTATTATAGG 240

AGTTGGGCGA AAGGNCGATT CACCATCTCC AAGACCTCGT CGACCACGAT GACTCTCCAA 300

ATGACCAGTC TNACAGGCGC GGACACGGGC ACGTATTTTT KTGGGAGAAG TGATTATGAT 360

GGTGATGATA GTGCCTTTGA CTTGTGGGGC CCAGGCACCC TGGTCACCGT CTCCTCAGGG 420

CAACCTAAGG CTCCATCAGT CTTCCCACTG GCCCCCTGCT GGGGGGACAC ACCCAGCTCC 480

ACGGTGACCC TGGGCTGNCT GGTCAGAGGC TACCTCCCGG AGCCAGTGAC CGTGACCTGG 540

AACTCGGGCA CCCTCACCAA TGGGGTACGN ACCTTCCCGT CCGTCCGGGA GTCCTCAGGN 600

CTCTNCTCGN TGAGCAGNGT GGTGAGCGTN ANCTCAAGCA GGCAGNCCGT CACCTGCAAC 660

GTG 663

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1455 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : ATGGAGACTG GGCTGCGCTG GCTTCTCCTG GTCGCTGTGC TCAAAGGTGT CCAGTGTCAG 60

TCGGTGGAGG AGTCCGGGGG TCGCCTGGTC ACGCCTGGGA CACCCCTGAC ACTCACCTGC 120

AAAGCCTCTG GATTCTCCCT CGGTAGCTAT ACAGTGATGA GCTGGGTCCG ACAGGCTCCA 180

GGGAAGGAGC TGGAGTGGAT CGGATACATT AGTTATGGTG GTAGTGCATA CTACGCGAGC 240

TGGGCGAAAA GCCGATCCAC CATCACCAGA AACACCAACG AGAACACGGT GACTCTGAAA 300

ATGACCAGTC TGACAGCCGC GGACACGGCC ACCTATTTCT GTGCGAGACA TTGGGGCATC 360

TGGGGCCCAG GCACCCTGGT CACCGTCTCC TCAGGGCAAC CTAAGGCTCC ATCAGTCTTC 420

CCACTGGCCC CCTGCTGCGG GGACACACCC AGCTCCACGG TGACCCTGGG CTGCCTGGTC 480

AAAGGCTACC TCCCGGAGCC AGTGACCGTG ACCTGGAACT CGGGCACCCT CACCAATGGG 540

GTACGCACCT TCCCGTCCGT CCGGCAGTCC TCAGGCCTCT ACTCGCTGAG CAGCGTGGTG 600 35 AGCGTGACCT CAAGCAGCCA GCCCGTCACC TGCAACGTGG CCCACCCAGC CACCAACACC 660 AAAGTGGACA AGACCGTTGC ACCCTCGACA TGCAGCAAGC CCACGTGCCC ACCCCCTGAA 720 CTCCTGGGGG GACCGTCTGT CTTCATCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC 780 TCACGCACCC CCGAGGTCAC ATGCGTGGTG GTGGACGTGA GCCAGGATGA CCCCGACGTG 840 CAGTTCACAT GGTACATAAA CAACGAGCAG GTGCGCACCG CCCGGCCGCC GCTACGGGAG 900 CAGCAGTTCA ACAGCACGAT CCGCGTGGTC AGCACCCTCC CCATCACGCA CCAGGACTGG 960

CTGAGGGGCA AGGAGTTCAA GTGCAAAGTC CACAACAAGG CACTCCCGGC CCCCATCGAG 1020

AAAACCATCT CCAAAGCCAG AGGGCAGCCC CTGGAGCCGA AGGTCTACAC CATGGGCCCT 1080

CCCCGGGAGG AGCTGAGCAG CAGGTCGGTC AGCCTGACCT GCATGATCAA CGGCTTCTAC 1140

CCTTCCGACA TCTCGGTGGA GTGGGAGAAG AACGGGAAGG CAGAGGACAA CTACAAGACC 1200

ACGCCGGCCG TGCTGGACAG CGACGGCTCC TACTTCCTCT ACAACAAGCT CTCAGTGCCC 1260

ACGAGTGAGT GGCAGCGGGG CGACGTCTTC ACCTGCTCCG TGATGCACGA GGCCTTGCAC 1320

AACCACTACA CGCAGAAGTC CATCTCCCGC TCTCCGGGTA AATGAGCGCT GTGCCGGCGA 1380

GCTGCCCCTC TCCCTCCCCC CCACGCCGCA GCTGTGCACC CCGCACACAA ATAAAGCACC 1440

CAGCTCTGCC CTGAA 1455

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 800 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY : linear

(ii ) MOLECULE TYPE : DNA (genomic)

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 10 :

GGTACCCGGG GATCCATGGA CACGAGGGCC CCCACTCAGC TGCTGGGGCT CCTGCTGCTC 60

TGGCTCCCAG ATGCCAGATG TGCCCTTGTG ATGACCCAGA CTCCATCCCC TGTGTCTGCA 120

GCTGTGGGAG GCACAGTCAC CATCAACTGC CAGGCCAGTC AGAGTGTTTA TAAGAACAAC 180

TACTTATCCT GGTATCAGCA GAAACCAGGG NAGCCTCCCG AGCTTCTGAT CTACAAGGCT 240

TCCACTCTGG CATCTGGGGT CCCATCGCGG TTCAAAGGCA GTGGATCTGG GACACAGTTC 3 00

ACTCTCACCA TTAGCGACGT GCAGTGTGAC GATGCTGCCA CTTACGCCTG TGCAGGCTAT 360 AAAAGTCTCA CTAATGATGA GTTTGCTTTC GGCGGAGGGA CCGAGGTGGT GGTCAAGGGT 420

GATCCAGTTG CACCTACTGT CCTCATCTTC CCACCAGCTG CTGATCAGGT GGCAACTGGA 480

ACAGTCACCA TCGTGTGTGC GGCGAATAAA TACTTTCCCG ATGTCACCGT CACCTGGGAG 540

GTGGATGGCA CCACCCAAAC AACCGGCATC GAGAACAGTA AAACACCGCA GAATTCTGCA 600

GATTGTACCT ACAACCTCAG CAGCACTCTG ACACTGACCA GCACACAGTA CAACAGCCAC 660

AAAGAGTACA CCTGCAAGGT GACCCAGGGC ACGACCTCAG TCGTCCAGAG CTTCAATAGG 720

GGTGACTGTT AGAGCGAGAC GCCTGCCAGG GCACTGCCAG CGACCCCGAG CGNCCGCTCG 780

ACCTGCAGGC ATGCAAGCTT 800

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 801 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CGGTACCCGG GGATCCATGG ACACGAGGGC CCCCACTCAG CTGCTGGGGC TCCTGCTGCT 60

CTGGCTCCCA GATGCCAGAT GTGCCCTTGT GATGACCCAG ACTCCATCCC CTGTGTCTGC 120

AGCTGTGGGA GGCACAGTCA CCATCAACTG NCAGGCCAGT CAGAGTGTTT ATAAGAACAA 180

CTACTTATCC TGGTATCAGC AGAAACCAGG GNAGGCTCCC GAGCTTCTGA TCTACAAGGC 240

TTCCACTCTG GCATCTGGGG TCCCATCGCG GTTCAAAGGC AGTGGATCTG GGACACAGTT 300

CACTCTCACC ATTAGCGACG TGCAGTGTGA CGATGCTGCC ACTTACGCCT GTGCAGGCTA 360

TAAAAGTCTC ACTAATGATG AGTTTGCTTT CGGCGGAGGG ACCGAGGTGG TGGTCAAGGG 420

TGATCCAGTT GCACCTACTG TCCTCATCTT CCCACCAGCT GCTGATCAGG TGCCAACTGG 480

AACAGTCACC ATCGTGTGTG CGGCGAATAA ATACTTTCCC GATGTCACCG TCACCTGGGA 540

GGTGGATGGC ACCACCCAAA CAACCGGCAT CGAGAACAGT AAAACACCGC AGAATTCTGC 600

AGATTGTACC TACAACCTCA GCAGCACTCT GACACTGACC AGCACACAGT ACAACAGCCA 660

CAAAGAGTAC ACCTGCAAGG TGACCCAGGG CACGACCTCA GTCGTCCAGA GCTTCAATAG 720

GGGTGACTGT TAGAGCGAGA CGCCTGCCAG GGCACTGCCA GCGACCCCGA GCGGCCGCTC 780

GACCTGCAGG CATGCAAGCT T 801 (2) INFORMATION FOR SEQ ID NO:12 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 805 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : DNA (genomic)

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 12 :

AGCTTGCATG CCTGCAGGTC GAGCGGCCGC TCAGGGTCGC TGGCAGTGCC CTGGCAGGCG 60

TCTCGCTCTA ACAGTCACCC CTATTGAAGC TCTGGACGAC TGAGGTCGTG CCCTGGGTCA 120

CCTTGCAGGT GTACTCTTTG TGGCTGTTGT ACTGTGTGCT GGTCAGTGTC AGAGTGCTGC 180

TGAGGTTGTA GGTACAATCT GCAGAATTCT GCGGTGTTTT ACTGTTCTCG ATGCCAGTTG 240

TTTGGGTGGT GCCATCCACC TCCCAGGTGA CGGTGACATC GGGAAAGTAT TTATTCGCCG 300

CACACACGAT GGTGACTGTT CCAGTTGNCA CCTGATCAGC AGCTGGTGGG AAGATGAGGA 360

CAGTAGGTGC AACTGGATCA CCCTTGACCA CCACCTCGGT CCCTCCGCCG AAAGCAAACT 420

CATCATTAGT GAGACTTTTA TAGCCTGCAC AGGCGTAAGT GGCAGCATCG TCACACTGCA 480

CGTCGCTGAT GGTGAGAGTG AACTGTGTCC CAGATCCACT GCCTTTGAAC CGCGATGGGA 540

CCCCAGATGC CAGAGTGGAA GCCTTGTAGA TCAGAAGCTT GGGAGNCTGC CCTGGTTTCT 600

GCTGATACCA GGATAAGTAG TTGTTCTTAT AAACACTCTG ACTGGCCTGG CAGTTGATGG 660

TGACTGTGCC TCCCACAGCT GCAGACACAG GGGATGGAGT CTGOGTCATC ACAAGGGCAC 720

ATCTGGCATC TGGGAGCCAG AGCAGCAGGA GCCCCAGCAG CTGAGTGGGG GCCCTCGTGT 780

CCATGGATCC CCGGGTACCG AGCTC 805

(2 ) INFORMATION FOR SEQ ID NO : 13 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 808 base pairs

(B ) TYPE : nucleic acid

( C) STRANDEDNESS : double

(D) TOPOLOGY : linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AAGCTTGCAT GNCTGCAGGT CGAGCGGCCG CTCAGGGTCG CTGGCAGTGC CCTGGCAGGC 60 GTCTCGCTCT AACAGTCACC CCTATTGAAG CTCTGGACGA CTGAGGTCGT GCCCTGGGTC 120

ACCTTGCAGG TGTACTCTTT GTGGCTGTTG TACTGTGTGC TGGTCAGTGT CAGAGTGCTG 180

CTGAGGTTGT AGGTACAATC TGCAGAATTC TGCGGTGTTT TACTGTTCTC GATGCCAGTT 240

GTTTGGGTGG TGCCATCCAC CTCCCAGGTG ACGGTGACAT CGGGAAAGTA TTTATTCGCC 300

ACACACACGA TGGTGACTGT TCCAGTTGNC ACCTGATCAG CAGCTGGTGG GAAGATGAGG 360

ACAGTAGGTG CAACTGGATC ACCTTTGACC ACCACCTCGG TCCCTCCGGC GAAAGCATTT 420

CCATCATTAT TGTCACTTTN ATATCCTGCA CAGTAGTAAG TGGCAGCATC GTCACACACC 480

ACATCGCTTA TGGTGAGAGT AAACTGTTTC CCAGATCCAC TGCCTTTGAA CCGCGATGGG 540

ACCCCAGATA CCAGAGTGGA TGCAAGATAG ATCAGGAGCT TGGGAGGCTG CCCTGGTTTC 600

TGCTGATACC AGGCTAAACG GTTATTTTTA TAAACACTCT CACTGGCCTG GCAATTGATG 660

GTGACTGTGT CTCCCTCAGG GACAGACTTG GAAGATGGAG TCTGGGTCAT CACGATGNCA 720

AATGTGGCAC CTGGGAGCCA GAGCAGCAGG AGCCCCAGCA GCTGAGTGGG GGCCCTCGTG 780

TCCATGGATC CCCGGGTACC GAGCTCGC 808

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 898 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14 :

ATGGACACGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC TGCTCTGGCT CCCAGGTGCC 60

AGATGTGCCC TCGTGATGAC CCAGACTCCA GCCTCCGTGT CTGCAGCTGT GGGAGGCACA 120

GTCACCATCA AGTGCCAGGC CAGTGAGAAC ATTTACAGCT CTTTAGCCTG GTATCAGCAG 180

AAACCAGGGC AGCCTCCCAA GCTCCTGATC TATGGTGCAT CCACTCTGGC ATCTGGGGTC 240

CCATCGCGGT TCAAAGGCAG TAGATCTGGG ACAGAGTACA CTCTCACCAT CAGCGGCGTG 300

CAGCGTGAGG ATGCTGCCAC CTACTACTGT CTAGGCAGTG ATAGTAGTAG CGATACTGCT 360

TTCGGCGGAG GGACCGAGCT GGAGATCCTA TGTGATCCTC CAATTGCGCC TACTGTCCTC 420

CTCTTCCCAC CATCTGCTGA TCAGCTGACA ACTGAAACAG TCACCATCGT GTGCGTGGCA 480 AATAAATTCC GTCCCAATGA CATCACCGTC ACCTGGAAGG TGGATGACGA AATCCAACAG 540

AGCGGCATCG AGAACAGTAC AACACCGCAG AGCCCCGAAG ACTGTACCTA CAACCTCAGC 600

AGCACTCTGT CACTGACCAA AGCACAGTAC AACAGCCACA GCGTGTACAC CTGCGAGGTG 660

GTCCACAACT CGGGCTCAGC GATCGTCCAG AGCTTCAATA GGGGTGACTG TTAGAGTGAG 720

ACGCCTGCCA GGGCACTGCC AGCGACCCTG AGGCCCAGCC TCGCCCCTCC CTCCCCTCAG 780

TGGACCCATT CCCATCACAG TCCTCCAGCC CCTCCCCTCC CGGCCCTCAC CCCCTCCTTG 840

GCTTTAACCG TGCGAATGTT GGTGAGATGG ATGAATAAAG TGAATCGTTG CACTTGTG 898

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 740 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GGATCCATGG AGACTGGGCT GCGCTGGCTT CTCCTGGTCG CTGTGCTCAA AGGTGTCCAG 60

TGTCAGTCGT GGAGGAGTCC GGGGGAGGCC TGGTCAAGCC TGGGGCATCC CTGACACTCA 120

CCTGCACAGC CTCTGGATTC TCCTTCGTAG CGGCACGGAT GTGCTGGGTC CGCCAGGCTC 180

CAGGGAAGGG GCTTGGAGTG GATCGCATGC ATCTATACTG GTTATAGTGG TAGCACTGAC 240

TACGCGACTG GGCGAAAGGC CGATTCACCA TCTCCAAACC TCGTCGACCA CGGTGACTCT 300

GCAAATGACC AGTCTGACAG CCGCGGACAC GGCCACCTAT TTCTGTGGAG AGCCCCGTCT 360

TACGTTGTTT ATTCTACTGT GGTTATACTT ATGCTCCTAC TTGGATTTGT GGGGCCCAGG 420

CACCCTGGTC ACCGTCTCCT CAGGGCAACC TAAGGCTCCA TCAGTCTTCC CACTGGCCCC 480

CTGCTGNGGG GACACACCCA GCTCCACGGT GACCCTGGGC TGNCTGGTCA AAGGCTACCT 540

CCCGGAGCCA GTGACCGTGA CCTGGAACTC GGGCACCCTC ACCGGAGCCA GTGACCGTGA 600

CCTGGAACTC GGGCACCCTC ACCAATGGGG TACGCACCTT CCCGTCCGTC CGGAGTCCTC 660

AGGCCTCTAC TCGCTGAGCA GGTGGTGAGC GTGACCTCAA GCAGGCAGNC CGTCACCTGA 720

ACGTGGNCCA CCCAGCACCA 740

(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 805 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

GAGCTCGGTA CCCGGGGATC CATGGACACG AGGGCCCCCA CTCAGCTGCT GGGGCTCCTG 60

CTGCTCTGGC TCCCAGATGC CAGATGTGCC CTTGTGATGA CCCAGACTCC ATCCCCTGTG 120

TCTGCAGCTG TGGGAGGCAC AGTCACCATC AACTGCCAGG CCAGTCAGAG TGTTTATAAG 180

AACAACTACT TATCCTGGTA TCAGCAGAAA CCAGGGCAGC CTCCCAAGCT TCTGATCTAC 240

AAGGCTTCCA CTCTGGCATC TGGGGTCCCA TCGCGGTTCA AAGGCAGTGG ATCTGGGACA 300

CAGTTCACTC TCACCATAGC GACGTGCAGT GTGACGATGC TGCCACTTAC GCCTGTGCAG 360

GCTATAAAAG TCTCACTAAT GATGAGTTTG CTTTCGGCGG AGGGACCGAG GTGGTGGTCA 420

AGGGTGATCC AGTTGCACCT ACTGTCCTCA TCTTCCCACC AGCTGCTGAT CAGGTGNCAA 480

CTGGAACAGT CACCATCGTG TGTGCGGCGA ATAAATACTT TCCCGATGTC ACCGTCACCT 540

GGGAGGTGGA TGGCACCACC CAAACAACCG GCATCGAGAA CAGTAAAACA CCGCAGAATT 600

CTGCAGATTG TACCTACAAC CTCAGCAGCA CTCTGACACT GACCAGCACA CAGTACAACA 660

GCCACAAAGA GTACACCTGC AAGGTGACCC AGGGCACGAC CTCAGTCGTC CAGAGCTTCA 720

ATAGGGGTGA CTGTTAGAGC GAGACGCCTG CCAGGGCACT GCCAGCGACC GTGAGCGGCC 780

GCTCGACCTG CAGGCATGCA AGCTT 805

Claims

WHAT IS CLAIMED IS:

1. A monoclonal antibody or fragment thereof which specifically binds to a specific binding pair member wherein said monoclonal antibody is produced by a repertoire cloning process.

2. A cell line which secretes a monoclonal antibody or fragment thereof which specifically binds to a specific binding pair member wherein said monoclonal antibody is produced by a repertoire cloning process.

3. In an immunoassay for an analyte of interest comprising the steps of contacting a test sample suspected of containing an analyte of interest with a monoclonal antibody and detecting the presence of the analyte in the test sample, wherein the improvement comprising the step of adding a known amount of an anti- analyte monoclonal antibody or fragment thereof produced by a repertoire cloning process.

4. An assay kit for determining the presence of an analyte in a test sample comprising a container containing at least one monoclonal antibody or fragment thereof wherein said monoclonal antibody or fragment thereof is produced by a repertoire cloning process.

5. A repertoire cloning process comprising: a. increasing the frequency of the cells of interest by performing an enrichment step; b. cloning the Ig kappa (K) and Ig gamma (γ) genes into eukaryotic expression vectors; c. introducing the cloned Ig kappa and Ig gamma genes into mammalian cells capable of secreting IgG; and d . isolating cell lines producing antibodies having the desired antigenic specificity.

6. The repertoire cloning process of claim 5 wherein step (a) further comprises the in vitro sensitization (TVS) of lymphocytes to increase the frequency of antigen- specific B-cells in the population from which RNA is isolated.

7. The repertoire cloning process of claim 6 wherein said lymphocytes are obtained from mammals selected from the group consisting of rabbits, goats and sheep.

8. The repertoire cloning process of claim 5 wherein said lymphocytes are obtained from rabbits.

9. The repertoire cloning process of claim 5 wherein said mammalian cells are selected from the group consisting of Sp2/0, CHO, HeLa, HEK and BHK.

10. The repertoire cloning process of claim 5 wherein the Ig kappa and/or Ig gamma genes are cloned into eukaryotic viral expression vectors.

1 1. The repertoire cloning process of claim 10 wherein the Ig kappa and/or Ig gamma genes are cloned into erkaryotic retroviral expression vectors.

12. The repertoire cloning process of claim 10 wherein the cloned Ig kappa and Ig gamma genes are inserted into mammalian cells.

13. The repertoire cloning process of claim 12 wherein the cloned Ig kappa and Ig gamma genes are inserted into mammalian cells by infection.

14. A repertoire cloning process to produce monoclonal antibodies having a desired specifity comprising: a. increasing the percentage of antibody producing cells approximately 10-fold or greater; b . cloning the Ig kappa (K) and Ig gamma (γ) genes into eukaryotic expression vectors or eukaryotic viral expression vectors; c. introducing cloned Ig kappa and Ig gamma genes into mammalian cells by tranfecting or infecting with viral expression vectors containing cloned Ig kappa and/or Ig gamma genes; and d . isolating cell lines producing antibodies having the desired specificity.

15. The repertoire cloning process of claim 14 wherein the Ig kappa and/or Ig gamma genes are cloned into eukaryotic retroviral expression vectors.

16. The repertoire cloning process of claim 14 wherein said mammalian cells are selected from the group consisting of Sp2/0, CHO, HeLa, HEK and BHK.

17. In an immunoassay for an analyte of interest wherein said immunoassay comprises binding said analyte and its specific binding pair member to form a complex and detecting the presence of said analyte by contacting said complexes with an indicator reagent comprising a monoclonal antibody and a signal generating compound, wherein the improvement comprising utilizing a monoclonal antibody or fragment thereof produced by a repertoire cloning process.