WO2005047903A1 - A method for generating antibody conjugates that preserves antigen recognition - Google Patents

Abstract

The present invention relates to methods of conjugating an antibody or antigen-binding fragment to a conjugating molecule wherein the antibody or antigen-binding fragment retains an ability to bind to a target molecule for which the antibody or antigen-binding fragment has binding specificity.

Description

A METHOD FOR GENERATING ANTIBODY CONJUGATES THAT PRESERVES ANTIGEN RECOGNITION

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 60/518,066, filed on November 7, 2003, the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION In assays in which antibodies are used as a detection component, anti-species secondary antibodies bearing a visualization tag are also often used. These secondary antibodies offer tremendous versatility because the same secondary antibody can be used to detect any antibody that is generated in that same animal host. Secondary antibodies also provide the benefit of signal amplification as numerous secondary antibodies can bind to the same primary antibody. However, when an indirect detection system is not feasible or a single antibody has the potential for use in a variety of applications which employ disparate detection systems, direct tagging of the primary antibody becomes the more convenient option. A common approach is to covalently couple the primary antibody to biotin, which allows the conjugated antibody to take advantage of the very strong interaction that occurs between biotin and avidin (e.g., streptavidin). The biotinylated antibody can then be labeled with one of several streptavidin conjugates to generate a variety of one-step visualization reagents. Biotinylation of antibodies can be achieved, for example, by conjugating the biotin moieties to primary amine groups present in the antibody molecule. However, biotin labeling of antibodies can result in a dramatic loss of antigen binding if there are primary amine groups susceptible to biotin labeling located within the antigen- binding recognition site(s) of the antibody. Thus, there is a need to develop new, improved and effective methods of labeling antibodies without decreasing antigen recognition and binding. SUMMARY OF THE INVENTION In one aspect, the present invention is a method of conjugating an antibody or antigen-binding fragment to a conjugating molecule, wherein the antibody or antigen-binding fragment retains an ability to bind to an immunogen for which it has binding specificity. In another aspect, the invention is a method of producing a conjugated antibody or conjugated antigen-binding fragment that retains an ability to bind to an immunogen for which the antibody or antigen-binding fragment has binding specificity. In another aspect, the invention is a conjugated antibody or conjugated antigen-binding fragment that is produced by the methods of the invention. In one embodiment, the invention is a method of conjugating an antibody or antigen-binding fragment to a conjugating molecule, wherein the antibody or antigen-binding fragment retains an ability to bind to an immunogen for which it has binding specificity. In the method, the antibody or antigen-binding fragment is contacted with the immunogen under conditions in which it will bind to the immunogen, thereby producing an antibody-immunogen complex or an antigen- binding fragment-immunogen complex. A conjugating molecule is then conjugated to the antibody-immunogen complex or the antigen-binding fragment-immunogen complex. This method allows the conjugated antibody or conjugated antigen- binding fragment to retain its ability to bind to the immunogen. In another embodiment, the invention is a method of conjugating an antibody or antigen-binding fragment to a conjugating molecule through a primary amine linkage, wherein the antibody or antigen-binding fragment retains an ability to bind to an immunogen for which it has binding specificity. In the method, amine- containing molecules are removed from the antibody or antigen-binding fragment. The antibody or antigen-binding fragment is contacted with the immunogen under conditions in which it will bind to the immunogen, thereby producing an antibody- immunogen complex or an antigen-binding fragment-immunogen complex. The conjugating molecule is then conjugated to the antibody-immunogen complex or the antigen-binding fragment-immunogen complex through a primary amine linkage. This method allows the conjugated antibody or conjugated antigen-binding fragment to retain its ability to bind to the immunogen. In another embodiment, the invention is a method of conjugating an antibody or antigen-binding fragment to a conjugating molecule through a primary amine linkage, wherein the antibody or antigen-binding fragment retains an ability to bind to an immunogen for which it has binding specificity. In the method, amine- containing molecules are removed from the antibody or antigen-binding fragment. The antibody or antigen-binding fragment is contacted with the immunogen, which is conjugated to a support matrix, under conditions in which the antibody or antigen- binding fragment will bind to the immunogen, thereby producing an antibody- immunogen complex or an antigen-binding fragment-immunogen complex. The conjugating molecule is then conjugated to the antibody-immunogen complex or the antigen-binding fragment-immunogen complex through a (one or more; at least one) primary amine linkage. This method allows the conjugated antibody or conjugated antigen-binding fragment to retain its ability to bind to the immunogen. In another embodiment, the invention is a method of producing a conjugated antibody or conjugated antigen-binding fragment, wherein the conjugated antibody or conjugated antigen-binding fragment retains an ability to bind to an immunogen for which it has binding specificity. In the method, the antibody or antigen-binding fragment is contacted with the immunogen under conditions in which it will bind to the immunogen, thereby producing an antibody-immunogen complex or an antigen- binding fragment-immunogen complex. A conjugating molecule is then conjugated to the antibody-immunogen complex or the antigen-binding fragment-immunogen complex. This method allows the conjugated antibody or conjugated antigen- binding fragment to retain its ability to bind to the immunogen. In another embodiment, the invention is a method of producing a conjugated antibody or conjugated antigen-binding fragment, wherein the conjugated antibody or conjugated antigen-binding fragment retains an ability to bind to an immunogen. In the method, amine-containing molecules are removed from the antibody or antigen-binding fragment. The antibody or antigen-binding fragment is contacted with the immunogen under conditions in which the antibody or antigen-binding fragment will bind to the immunogen, thereby producing an antibody-immunogen complex or an antigen-binding fragment-immunogen complex. The conjugating molecule is then conjugated to the antibody-immunogen complex or the antigen- binding fragment-immunogen complex through a primary amine linkage. This method allows the conjugated antibody or conjugated antigen-binding fragment to retain its ability to bind to the immunogen. In the methods of producing a conjugated antibody or conjugated antigen-binding fragment, the immunogen can be conjugated to a support matrix. In other embodiments, the invention is a conjugated antibody or conjugated antigen-binding fragment that is produced by the methods of the invention. Since the conjugating step occurs while the antibody or antigen-binding fragment is bound to the immunogen, the methods of the invention have the advantage that the conjugated antibody or conjugated antigen-binding fragment retains an ability to bind to an immunogen. The methods described herein prevent the conjugating molecule from conjugating to the reactive groups of the antibody or antigen-binding fragment that are important for binding to the immunogen (e.g., the antigen-binding recognition site(s) of the antibody or antigen-binding fragment). Thus, the antigen-binding recognition sites of the antibody or antigen-binding fragment are protected during conjugation, and as a result, the conjugated antibody or conjugated antigen-binding fragment has the same, or a similar, binding affinity for the antigen (immunogen), as opposed to a diminished binding affinity which occurs when the antigen-binding recognition sites are not protected during conjugation.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 A is a table summarizing the experimental parameters of the corresponding Western blot (FIG. IB) in which the unconjugated antibody (anti- phospho-HSP27, lanes 2 and 3) is compared with different concentrations of the biotin conjugate (16X Biotin, lanes 4 through 9) on lysates of either untreated MCF- 7 cells (MCF-7) or treated MCF-7 cells (MCF-7 +). The following abbreviations are used: LMWM = low molecular weight markers; MCF-7 = lysate from MCF-7 cells; MCF-7 + = lysate from MCF-7 cells treated with 200 μM arsenite for 30 minutes 16 hours after a 30-minute heat shock at 42°C; anti-phospho-HSP27 = unconjugated anti-phospho HSP27 mAb; 16X Biotin = biotin-conjugated anti-phospho HSP27 mAb (containing 2.26 mole biotin/mole antibody); Anti-Mouse HRP = anti-mouse HRP (Horseradish Peroxidase) secondary antibody; Streptavidin HRP = Streptavidin HRP secondary conjugate. FIG. IB is a Western blot. The blot was incubated with the secondary antibody/conjugate indicated in FIG. 1 A. ECL (Amersham; Piscataway, NJ) was used as the detection reagent. FIG. 2 A is a table summarizing the experimental parameters of the corresponding Western blot (FIG. 2B) in which the unconjugated antibody (anti- phospho-HSP27, lanes 2 and 3) is compared with different concentrations of the biotin conjugate (56X Biotin, lanes 4 through 7) on lysates of either untreated MCF- 7 cells (MCF-7) or treated MCF-7 cells (MCF-7 +). The following abbreviations are used: LMWM = low molecular weight markers; MCF-7 = lysate from MCF-7 cells; MCF-7 + = lysate from MCF-7 cells treated with 200 μM arsenite for 30 minutes 16 hours after a 30 minute heat shock at 42°C; anti-phospho-HSP27 = unconjugated anti-phospho HSP27 mAb; 56X Biotin = biotin-conjugated anti-phospho HSP27 mAb (containing 12 mole biotin/mole antibody); Anti-Mouse HRP = anti-mouse HRP (Horseradish Peroxidase) secondary antibody; Streptavidin HRP = Streptavidin HRP secondary conjugate. FIG. 2B is a Western blot demonstrating that increasing the molar biotin content of an antibody conjugate can decrease antigen recognition. The blot was incubated with the secondary antibody/conjugate indicated in FIG. 2A. ECL (Amersham; Piscataway, NJ) was used as the detection reagent. FIG. 3 A is a table summarizing the experimental parameters of the corresponding Western blot (FIG. 3B) in which the unconjugated antibody (anti- phospho-HSP27, lanes 6 and 7) is compared with different concentrations of the third biotin conjugate (30X Biotin) in combination with either the Streptavidin-HPR conjugate (lanes 2 through 5) or the anti-mouse HRP conjugate used with anti- phospho-HSP27 (lanes 8 and 9) on lysates of either untreated MCF-7 cells (MCF-7) or treated MCF-7 cells (MCF-7 +). The following abbreviations are used: LMWM = low molecular weight markers; MCF-7 = lysate from MCF-7 cells; MCF-7 + = lysate from MCF-7 cells treated with 200 μM arsenite for 30 minutes 16 hours after a 30-minute heat shock at 42°C; anti-phospho-HSP27 = unconjugated anti-phospho HSP27 mAb; 3 OX Biotin = biotin-conjugated anti-phospho HSP27 mAb (containing 6.8 mole biotin/mole antibody); Anti-Mouse HRP = anti-mouse HRP (Horseradish Peroxidase) secondary antibody; Streptavidin HRP = Streptavidin HRP secondary conjugate. FIG. 3B is a Western blot demonstrating that biotinylation of anti-phospho

HSP27 mAb in solution decreases antigen recognition. The blot was incubated with the secondary antibody/conjugate indicated in FIG. 3A. ECL (Amersham, Piscataway, NJ) was used as the detection reagent. FIG. 4 A is a table summarizing the experimental parameters of the corresponding Western blot (FIG.4B) in which the unconjugated antibody (anti- phospho-HSP27, lanes 2 and 4) is compared with different concentrations of the biotin conjugate prepared using the column-immobilization technique (3 OX Col. Biotin, lanes 4 through 7) on lysates of either untreated MCF-7 cells (MCF-7) or treated MCF-7 cells (MCF-7). The following abbreviations are used: HMWM = high molecular weight markers; LMWM = low molecular weight markers; MCF-7 = lysate from MCF-7 cells; MCF-7 + = lysate from MCF-7 cells treated with 200 μM arsenite for 30 minutes 16 hours after a 30-minute heat shock at 42°C; anti-phospho- HSP27 = unconjugated anti-phospho HSP27 mAb; 30X Col. Biotin = biotin- conjugated anti-phospho HSP27 mAb prepared using the column-antibody- immobilization technique (containing 1.94 mole biotin/mole antibody); Anti-Mouse HRP = anti-mouse HRP secondary antibody; Streptavidin HRP = Streptavidin HRP secondary conjugate. FIG. 4B is a Western blot demonstrating that biotinylation of column- immobilized antibodies retains antigen recognition. The blot was incubated with the secondary antibody/conjugate indicated in FIG. 4A. ECL (Amersham, Piscataway, NJ) was used as the detection reagent. FIG. 5 A is a table summarizing the experimental parameters of the corresponding Western blot (FIG. 5B) to determine if the original conjugate

(Original Col. Conj., lanes 1 and 2) perfonns as well as the conjugate prepared on the once used column (Once used Col., lane 3 and 4) or the conjugate prepared with a fresh column (Fresh Col., lanes 6 and 7) on lysates of either untreated MCF-7 cells (MCF-7) or treated MCF-7 cells (MCF-7 +). The following abbreviations are used: LMWM = low molecular weight markers; MCF-7 = lysate from MCF-7 cells; MCF- 7 + = lysate from MCF-7 cells treated with 200 μM arsenite for 30 minutes 16 hours after a 30-minute heat shock at 42°C; Original Col. Conj. = original biotin- conjugated anti-phospho HSP27 mAb prepared using the column-antibody- immobilization technique (depicted in FIGS. 4A and 4B); Once Used Col. = biotin- conjugated anti-phospho HSP27 mAb prepared using the column-antibody- immobilization technique and a previously once-used and regenerated column; Fresh Col. = biotin-conjugated anti-phospho HSP27 mAb prepared using the column- antibody-immobilization technique and a fresh newly made column; Streptavidin HRP = Streptavidin HRP secondary conjugate. FIG. 5B is a Western blot demonstrating that biotinylation columns are reusable. The blot was incubated with the secondary antibody/conjugate indicated in FIG. 5A. ECL (Amersham, Piscataway, NJ) was used as the detection reagent. FIG. 6A is a table summarizing the experimental parameters of the corresponding Western blot (FIG. 6B) performed with the various anti-polyHis-Tag biotin conjugates (lanes 2, 3, 5-8, 10 and 11) and the corresponding unconjugated antibody (lane 1). For each of lanes 1-3, 5-8, 10 and 11, 50ng of His-DFF45/ICAD protein was resolved by electrophoresis, transferred to nitrocellulose and probed with the indicated anti-polyHis-Tag biotinylated antibody (i.e., anti-polyHis-Tag biotinylated antibodies immobilized on a peptide affinity column and biotinylated with either 15- or 40-fold molar excess of Sulfo-NHS-LC-LC-Biotin at room temperature or on ice). The following abbreviations are used: HMWM = high molecular weight markers; LMWM = low molecular weight markers; DFF45/ICAD = DFF45/ICAD protein; anti-polyHis-tag = unconjugated anti-polyHis-tag antibody; 15X RT = anti-polyHis-Tag biotinylated antibodies immobilized on a peptide affinity column and biotinylated with 15-fold molar excess of Sulfo-NHS-LC-LC- Biotin at room temperature; 40X RT = anti-polyHis-Tag biotinylated antibodies immobilized on a peptide affinity column and biotinylated with 40-fold molar excess of Sulfo-NHS-LC-LC-Biotin at room temperature; 15X ICE = anti-polyHis-Tag biotinylated antibodies immobilized on a peptide affinity column and biotinylated with 15-fold molar excess of Sulfo-NHS-LC-LC-Biotin on ice; 40X ICE = anti- polyHis-Tag biotinylated antibodies immobilized on a peptide affinity column and biotinylated with 40-fold molar excess of Sulfo-NHS-LC-LC-Biotin on ice; Anti- Mouse HRP = anti-mouse HRP secondary antibody; Streptavidin HRP = Streptavidin HRP secondary conjugate. FIG. 6B is a Western blot demonstrating that the column-antibody- immobilization technique can be applied to additional antibodies. The blot was incubated with the secondary antibody/conjugate indicated in FIG. 6A. ECL (Amersham, Piscataway, NJ) was used as the detection reagent. DETAILED DESCRIPTION OF THE INVENTION In one aspect, the present invention is a method of conjugating an antibody or antigen-binding fragment to a conjugating molecule (e.g., a conjugating molecule comprising a label) wherein the antibody or antigen-binding fragment retains an ability to bind to an immunogen for which it has binding specificity. In another aspect, the invention is a method of producing a conjugated antibody or conjugated antigen-binding fragment that retains an ability to bind to an immunogen for which the antibody or antigen-binding fragment has binding specificity. In another aspect, the invention is a conjugated antibody or conjugated antigen-binding fragment that is produced using the methods of the invention. As described and exemplified herein, the methods and compositions of the invention have the advantage of producing conjugated antibodies and conjugated antigen-binding fragments that retain an ability to bind to an immunogen. As described herein, the conjugating step is performed while the antibody or antigen- binding fragment is bound to an immunogen. In this way, the conjugating molecule is prevented from conjugating to the reactive groups of the antibody or antigen- binding fragment that are involved in binding to the immunogen (e.g., the antigen- binding recognition site(s) (e.g., a complementarity-determining region (CDR) of the antibody or antigen-binding fragment)). In one embodiment, the invention is a method of conjugating an antibody or antigen-binding fragment to a conjugating molecule, wherein the antibody or antigen-binding fragment retains an ability to bind to an immunogen for which it has binding specificity. In the method, the antibody or antigen-binding fragment is contacted with the immunogen under conditions in which it will bind to the immunogen, thereby producing an antibody-immunogen complex or an antigen- binding fragment-immunogen complex. A conjugating molecule is then conjugated to the antibody-immunogen complex or the antigen-binding fragment-immunogen complex. This method allows the conjugated antibody or conjugated antigen- binding fragment to retain its ability to bind to the immunogen. The methods of conjugation described herein can be used with antibodies or antigen-binding fragments. As used herein, the term "antibody" encompasses both polyclonal and monoclonal antibodies (e.g., IgG, IgM, IgA, IgD and IgE antibodies). The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production. Any antibody or antigen-binding fragment to which a conjugating molecule can be conjugated can be used in the methods of the invention. For example, single chain antibodies, chimeric antibodies, mammalian (e.g., human) antibodies, humanized antibodies, CDR-grafted antibodies (e.g., primatized antibodies), veneered antibodies, multivalent antibodies (e.g., bivalent antibodies) and bispecifϊc antibodies are encompassed by the present invention and the term "antibody". Chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, are also encompassed by the present invention and the term "antibody". The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al, U.S. Patent No. 4,816,567; Cabilly et al, European Patent No. 0,125,023 Bl; Boss et al, U.S. Patent No. 4,816,397; Boss etal, European Patent No. 0,120,694 Bl; Neuberger, M.S. et al, WO 86/01533; Neuberger, M.S. et al, European Patent No. 0,194,276 Bl; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen et al, European Patent No. 0 451 216 Bl; and Padlan, E.A. et al, EP 0 519 596 Al. See also, Newman, R. et al, BioTechnology, 10: 1455-1460 (1992) regarding primatized antibody, and Ladner et al, U.S. Patent No. 4,946,778 and Bird, R.E. et al, Science, 242: 423-426 (1988)) regarding single chain antibodies. As used herein, the phrase "mammalian antibody" includes an antibody in which the variable and constant regions (if present) have amino acid sequences that are encoded by nucleotide sequences derived from mammalian germline immunoglobulin genes. A "mammalian antibody" can include sequences that are not encoded in the germline (e.g., due to N nucleotides, P nucleotides, and mutations that can occur as part of the processes that produce high-affinity antibodies, such as somatic mutation, affinity maturation, clonal selection) that occur as a result of biological processes in a suitable in vivo expression system (e.g., a human, a transgenic animal expressing a human antibody). In one embodiment, the antibody is a human antibody in which the variable and constant regions (if present) have amino acid sequences that are encoded by nucleotide sequences derived from human (Homo sapiens) germline immunoglobulin genes. Antibodies, antigen-binding fragments of antibodies, and portions or regions of antibodies can be produced, for example, by expression of a nucleic acid of non-human origin (e.g., a synthetic nucleic acid) that has the requisite nucleotide sequence. As used herein, the term "CDR-grafted antibody" includes an antibody that comprises a complementarity-determining region (CDR) that is not naturally associated with the framework regions of the antibody. Generally the CDR is from an antibody from a first species and the framework regions and constant regions (if present) are from an antibody from a different species. The CDR-grafted antibody can be a "humanized antibody". As used herein, the term "humanized antibody" includes an antibody comprising a CDR that is not of human origin and framework and/or constant region(s) that are of human origin. For example, a humanized antibody can comprise a CDR derived from an antibody of nonhuman origin (e.g., a natural antibody, such as a murine (e.g., mouse, rat) antibody, an artificial antibody), and framework and constant regions (if present) of human origin (e.g., a human framework region, a human consensus framework region, a human constant region (e.g., C_L, C_H1, hinge, C_H2, C_H3, C_H4)). CDR-grafted single chain antibodies containing a CDR of non-human origin and framework and/or constant region(s) (if present) of human origin (e.g., CDR-grafted scFv) are also encompassed by the term humanized antibody. Humanized antibodies can be produced using synthetic or recombinant DNA technology using standard methods or other suitable techniques. Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see, e.g., Kamman, M., et al, Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al, Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al, Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P. and J.S. Crowe, Gene, 101: 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see, e.g., Krebber et al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213). ^* As used herein, the term "chimeric antibody" includes an antibody comprising portions of immunoglobulins from different origin. None of the portions of immunoglobulins that comprise a chimeric antibody need to be of human origin. For example, a chimeric antibody can comprise an antigen-binding region of nonhuman region (e.g., rodent) and a constant region of non-human primate origin (e.g., a chimpanzee framework region, a chimpanzee constant region (e.g., C_L, C_H1, hinge, C_H2, C_H3, Cg4)). Antibodies used in the methods of the invention can be single chain antibodies (e.g., a single chain Fv (scFv)) and can include a linker moiety (e.g., a linker peptide) not found in native antibodies. For example, an scFv can comprise a linker peptide, such as about two to about twenty glycine residues or other suitable linker, which connects a heavy chain variable region to a light chain variable region. In addition, antibodies used in the methods of the invention can be bispecific antibodies. As used herein, a "bispecific antibody" includes an antibody that binds two different types of antigen. Bispecific antibodies can be secreted by triomas and hybrid hybridomas. Generally, triomas are formed by fusion of a hybridoma and a lymphocyte (e.g., an antibody-secreting B cell) and hybrid hybridomas are formed by fusion of two hybridomas. Each of the cells that are fused to produce a trioma or hybrid hybridoma produces a monospecific antibody. However, triomas and hybrid hybridomas can produce an antibody containing antigen-binding sites that recognize different antigens. The supernatants of triomas and hybrid hybridomas can be assayed for bispecific antibody using a suitable assay (e.g., ELISA), and bispecific antibodies can be purified using conventional methods (see, e.g., U.S. Patent No. 5,959,084 (Ring et al), U.S. Patent No. 5,141,736 (Iwasa et al), U.S. Patent Nos. 4,444,878, 5,292,668 and 5,523,210 (Paulus et al) and U.S. Patent No. 5,496,549 (Yamazaki et al)). Antigen-binding fragments encompass functional fragments of antibodies including, e.g., fragments of single chain antibodies, chimeric antibodies, human antibodies, humanized antibodies, CDR-grafted antibodies (e.g., primatized antibodies), veneered antibodies and bispecific antibodies. Antigen-binding fragments further encompass Fv, Fab, Fab' and F(ab')₂ fragments. Antigen-binding fragments, such as Fv, Fab, Fab' and F(ab')₂ fragments, can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')₂ fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')₂ fragments. Antigen-binding fragments can also be produced recombinantly using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding an F(ab')₂ heavy chain portion can be designed to include DNA sequences encoding the CH_j domain and hinge region of the heavy chain. In certain embodiments, an isolated antibody or isolated antigen-binding fragment is contacted with an immunogen, under conditions in which the antibody or antigen-binding fragment binds to the immunogen. As used herein, the terms "isolated antibody" and "isolated antigen-binding fragment" refer to an antibody or antigen-binding fragment, respectively, which is substantially free of other proteins. Methods of isolating antibodies or antigen-binding fragments include, e.g., using affinity matrices, chromatography (e.g., affinity chromatography), selection (e.g., selection from a library (e.g., a phage display library)), affinity binding assays (e.g., using an antigen). In one embodiment, an affinity matrix that protects the antigen- binding site of the antibody or antigen-binding fragment is used to isolate the antibody or antigen-binding fragment. Such affinity matrices include, e.g., a matrix that is conjugated to an immunogen for which the antibody or antigen-binding fragment has specificity and a matrix that is conjugated to an immunogen mimetic or a related compound. The antibodies or antigen-binding fragments used in the methods of the invention can be obtained from a variety of sources. For example, antibodies or antigen-binding fragments from a host animal (e.g., a mammal (e.g., a human, a primate, a rat, a mouse, a rabbit, a camel, a llama)) can be used in the methods of the invention. The antibody or antigen-binding fragment can also be obtained from a commercial source. The antibody or antigen-binding fragment can be present, e.g., in serum, in ascites, in a cell culture medium (e.g., containing an in v tro-produced antibody or antigen-binding fragment), and in a cell lysate. In addition, the antibody or antigen-binding fragment can be produced using techniques known to those of skill in the art. For example, a variety of methods for preparing and using an immunizing antigen, and for producing polyclonal and monoclonal antibodies are known in the art (see, e.g., Kohler et al, Nature, 256: 495-497 (1975) and Eur. J. Immunol 6: 511-519 (1976); Milstein et al, Nature 266: 550-552 (1977); Koprowski et al, U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.M. et al. , Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). Generally, for monoclonal antibodies, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line, such as SP2/0, P3X63Ag8.653 or a heteromyloma) with antibody-producing cells. Antibody- producing cells can be obtained from the peripheral blood, or preferably the spleen or lymph nodes of humans or other suitable animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA). Other suitable methods of producing or isolating antibodies (e.g., human antibodies or antigen-binding fragments) include, for example, methods which select recombinant antibody from a library (e.g., a phage display library). Transgenic animals capable of producing a repertoire of human antibodies (e.g., Xenomouse^® (Abgenix, Fremont, CA)) can be produced using suitable methods (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al, Nature, 362: 255-258 (1993)). Additional methods that are suitable for production of transgenic animals capable of producing a repertoire of human antibodies have been described (e.g., Lonberg et al, U.S. Patent No. 5,545,806; Surani et al, U.S. Patent No. 5,545,807; Lonberg et al, WO97/13852). In the methods of the invention, the antibody or antigen-binding fragment is conjugated to a conjugating molecule. As used herein, the term "conjugating molecule" is intended to encompass any moiety that would be desired to be linked or conjugated to an antibody or antigen-binding fragment. Such conjugating molecules include, but are not limited to, e.g., one or more of a protein moiety, a glycoprotein moiety, a peptide moiety, a peptidomimetic moiety, an organic moiety (e.g., a small organic molecule), a lipid moiety, a phospholipid moiety, a glycolipid moiety, a nucleic acid moiety, a carbohydrate moiety and an antigenic moiety. For example, the conjugating molecule can be one or more of a drug, toxin, and/or enzyme. The conjugating molecule can also be a detection agent (e.g., a fluorescent agent, a chemiluminescent agent, or any suitable agent that allows for detection and/or imaging of the antibody or antigen-binding fragment). In certain embodiments, the conjugating molecule can be a cell-permeable protein (e.g., a protein or peptide that comprises a translocation sequence). Other suitable conjugating molecules include, e.g., a liposome, a particle (e.g., gold particle) and a virus. Any suitable fragments of the conjugating molecules described herein can also be used in the methods of the invention. Drugs and toxins that can be conjugated to antibodies and antigen-binding fragments include, e.g., chemotherapeutic agents (e.g., mitomycin C, paxlitaxol, methotrexate, 5-fluorouracil, cisplatin, cyclohexaniide) and toxins (e.g., diphtheria toxin, ricin, gelonin). Enzymes that can be conjugated to antibodies and antigen-binding fragments include, e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), β- galactosidase (β-gal), glucose oxidase (GO), maltose binding protein and glutathione-S-transferase (see, e.g., Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, CA (1996); the entire teachings of which are incorporated herein by reference). Other suitable enzymes, proteins and/or peptides that possess one or more properties that are suitable for detection and/or imaging of the antibody or antigen-binding fragment can also be used as conjugating molecules. In certain embodiments, the conjugating molecule comprises a tag or label.

As used herein, "a tag" or "label" is an agent that can be used to label an antibody or antigen-binding fragment. Suitable labels include, e.g., an affinity label, a spin label, an enzyme label, a fluorescent group, a chemiluminescent group, a radioactive label. Suitable fluorescent labels include, but are not limited to, fluorescein (e.g., fluorescein isothiocyanate (FITC), NHS-fluorescein), rhodamine, coumarin, Texas red (e.g., Texas red sulfonyl chloride), BODIPY fluorophores, Cascade Blue^™ fluorophores, Lucifer Yellow fluorophores, phycobiliproteins, (e.g., B- phycoerythrin, R-phycoerythrin) and derivatives of any of the foregoing (see, e.g., Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, CA (1996), p. 298-364). Suitable radioactive labels include, but are not limited to, iodine-131, iodine- 125, bismuth-212, yttrium-90, yttrium-88, technetium-99m, copper-67, rhenium- 188, rhenium-186, galium-66, galium-67, indium-111, indium-114m, indium-115 and boron- 10 (see, e.g., Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, CA (1996), p. 365 et seq.). Antibodies and antigen-binding fragments can be labeled by direct labeling (e.g., attaching a radioactive atom to a functional group of the antibody) or indirect labeling (e.g., utilizing a bifunctional chelating agent containing a chemical-reactive group for co plexing a radioactive metal) (Hermanson, Id.). In a particular embodiment, the label is a detectable moiety that possesses a specifically identifiable physical property which allows it to be distinguished from other .molecules that are present in a heterologous mixture. In one embodiment, the conjugating molecule comprises an affinity label

(e.g., biotin, avidin (e.g., streptavidin)), chitin, amylase, glutathione, an epitope tag (e.g., hemagglutinin (HA), FLAG) and a peptide affinity label). In another embodiment, the conjugating molecule comprises a solvent soluble dye (e.g., a solvent soluble laser dye, such as an infrared dye or a near infrared dye). In a particular embodiment, the conjugating molecule is biotin or is a molecule that comprises biotin. Biotin, a water-soluble vitamin, is used extensively in biochemistry and molecular biology for a variety of purposes including macromolecular detection, purification and isolation, and in cytochemical staining (see, e.g., U.S. Patent No. 5,948,624; the entire teachings of which are incorporated herein by reference). Biotin also has important applications in medicine in the areas of clinical diagnostic assays, tumor imaging and drug delivery, and is used extensively in the field of affinity cytochemistry for the selective labeling of cells, subcellular structures and proteins. The utility of biotin arises from its ability to bind strongly to the tetrameric protein avidin, found in egg white and the tissues of birds, reptiles and amphibians, or to its chemical cousin, streptavidin, which is slightly more specific for biotin than is avidin. The biotin interaction with avidin is among the strongest non-covalent affinities known, exhibiting a dissociation constant of about 1.3 x 10^"ls M (Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, CA (1996), p. 570). Generally, biotin is first bound to a target molecule (e.g., an antibody, an antigen-binding fragment) using specific chemical linkage. The subsequent interaction of the linked biotin with avidin can then be used to isolate the target molecule. Often, avidin is immobilized on a surface (e.g., a membrane, a gel, a filter, a microtiter well, magnetic beads, sepharose beads, a column (e.g., an avidin- containing affinity column)) to aid in the isolation of the target molecule. Alternatively, or additionally, avidin can be conjugated to an enzyme (e.g., horseradish peroxidase (HRP)) which can catalyze a chromogenic reaction and thereby allow for detection of the target molecule through its interaction with biotin. In other embodiments, the conjugating molecule is biocytin, a biotin analog (e.g., biotin amido caproate N-hydroxysuccinimide ester, biotin-PEO₄-N- hydroxysuccinimide ester, biotin 4-amidobenzoic acid, biotinamide caproyl hydrazide) and/or a biotin derivative (e.g., biotin-dextran, biotin-disulfide-N- hydroxysuccinimide ester, biotin-6 amido quinoline, biotin hydrazide, d-biotin-N hydroxysuccimmide ester, biotin maleimide, d-biotin p-nitrophenyl ester, biotinylated nucleotides, a biotinylated amino acid, such as N.epsilon.-biotinyl-l- lysine) (see, e.g., U.S. Patent No. 5,948,624). In another embodiment, the conjugating molecule is avidin or is a molecule that comprises avidin (an avidinylated molecule). Avidin is a glycoprotein found in egg whites that contains four identical subunits, each of which possesses a binding site for biotin (Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, CA (1996), p. 570). Streptavidin and/or other avidin analogs can also be used in the methods of the present invention. Such avidin analogs include, e.g., avidin conjugates, streptavidin conjugates, highly purified and/or fractionated species of avidin or streptavidin, non or partial amino acid variants of avidin or streptavidin (e.g., recombinant or chemically-synthesized avidin analogs with amino acid or chemical substitutions that still allow for high-affinity, multivalent or univalent binding of the avidin analog to biotin). Streptavidin is another biotin- binding protein that is isolated from Streptomyces avidinii (Hermanson, Id.) As used herein, the term "conjugating" encompasses any attachment (e.g., covalent, non-covalent) of a conjugating molecule to the molecule with which it is desired to be conjugated. Antibodies and antigen-binding fragments include a number of functional groups to which a conjugating molecule can be attached. For example, lysine ε-amine and N-terminal α-amine groups of antibodies or antigen- binding fragments may be used for attachment of the conjugating molecule. Carboxylate groups of antibodies or antigen-binding fragments (e.g., C-terminal carboxylate groups, aspartic acid carboxylate groups, glutamic acid carboxylate groups), sulfnydryl groups and carbohydrate groups may also be used for attachment of the conjugating molecule (Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, CA (1996), p. 459 etseq.). For example, as described and exemplified herein, random conjugation of a conjugating molecule (e.g., biotin) to an antibody or antigen-binding fragment can occur through conjugation to particular reactive groups of the antibody or antigen-binding fragment (e.g., amine-containing lysine residues of the antibody or antigen-binding fragment). In certain embodiments, the conjugating molecule is conjugated to the antibody or antigen-binding fragment through a primary amine linkage. Such "conjugating" includes attachment by covalent and non-covalent means and can be direct or indirect (e.g., via a linker or cross-linker). In certain embodiments, conjugation of the conjugating molecule to the antibody or antigen-binding fragment is accomplished using a linker or cross-linker. Suitable linkers include, e.g., zero-length linkers, homobifunctional cross-linkers, heterobifunctional cross-linkers, trifunctional cross-linkers and cleavable cross- linking reagents (see, e.g., Hermanson, Id.). Zero-length linkers that can be used to conjugate the conjugating molecule to the antibody or antigen-binding fragment include, but are not limited to, carbodiimides, Woodward's reagent (N-ethyl-3-phenylisoxazolium-3 '-sulfonate) and N,N'-carbonyldiimidazole (GDI). Homobifunctional cross-linkers that can be used to conjugate the conjugating molecule to the antibody or antigen-binding fragment include, but are not limited to, N-hydroxysuccinimide (NHS) esters (e.g., Lomant's reagent (dithiobis(succinimidylpropionate) (DSP)), disuccinimidyl suberate (DSS), disuccinimidyl tartarate (DST), homobifunctional imidoesters (e.g., dimethyl adipimiidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), homobifunctional sulfhydryl-reactive cross-linkers (e.g., l,4-di-[3'-2 - pyridyldithio)propionamido]butane (DPDPB), bismaleimidohexane (BMH)), difluorobenzene derivatives, homobifunctional photoreactive cross-linkers, homobifunctional aldehydes (e.g., formaldehyde, gluteraldehyde), bis-epoxides (e.g., 1,4-butanediol diglycidyl ether), homobifunctional hydrazides (e.g., adipic acid dihydrazide, carbohydrazide), bis-diazonium derivatives (e.g., o-Tolidine, bis- diazotized benzidine), and bis-alkylhalides. Heterobifunctional cross-linkers that can be used to conjugate the conjugating molecule to the antibody or antigen-binding fragment include, but are not limited to, amine-reactive and sulfhydryl-reactive cross-linkers (e.g., N- succinimydyl 3-(2-pyridyldithio)propionate (SPDP), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), carbonyl-reactive and sulfhydryl-reactive cross- linkers (e.g., 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), sulfhydryl-reactive and photoreactive cross-linkers (e.g., l-(p-azidosalicylamido)-4-(iodoacetamido)butane (ASIB), carbonyl-reactive and photoreactive cross-linkers (e.g., p-azidobenzoyl hydrazide (ABH)), carboxylate-reactive and photoreactive cross-linkers (e.g., 4-(p- azidosalicylamido(butylamine (ASBA)), arginine-reactive and photoreactive cross- linkers (e.g., p-azidophenyl glyoxal (APG)). Trifunctional cross-linkers that can be used to conjugate the conjugating molecule to the antibody or antigen-binding fragment include, but are not limited to, 4-azido-2-nitrophenylbiocytin-4-nitrophenyl ester (ABNP) and sulfosuccinimidyl-2- [6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethyl-l,3'-dithiopropionate (sulfo-SBED). Cleavable cross-linking reagents that can be used to conjugate the conjugating molecule to the antibody or antigen-binding fragment include, but are not limited to, disulfide-containing cross-linking agents, periodate-cleavable glycols, dithionite-cleavable diazo agents, hydroxylamine-cleavable esters and base labile sulfones. Other suitable cleavable cross-linking reagents include, e.g., light- activated cleavable cross-linking reagents and UV-activated cleavable cross-linking reagents. Cleavable cross-linking agents can be advantageous in that they allow the conjugation reaction to be verified through identification of the cross-linked molecules after conjugation and purification. As used herein, the terms "immunogen", "target immunogen", "antigen",

"target antigen", "target molecule", and "epitope" (e.g., T cell epitope, B cell epitope) refer to a substance for which an antibody or antigen-binding fragment has binding specificity. Unless otherwise specified, the terms "immunogen", "target immunogen", "antigen", "target antigen" and "target molecule" are interchangeable. The antibodies and antigen-binding fragments for use in the methods of the invention have binding specificity for a variety of immunogens (e.g., polypeptides). In one embodiment, the antibodies and antigen-binding fragments have binding specificity for an antigenic mimic (e.g., a mimetic). In another embodiment, the antibody or antigen-binding fragment has binding specificity for a heat shock protein. In a further embodiment, the antibody or antigen-binding fragment has binding specificity for a poly-His tag. In another embodiment, the antibody or antigen-binding fragment has binding specificity for an epitope containing a particular modification (e.g., an acetylation moiety, a methylation moiety, a ribosylation moiety, a phosphorylation moiety, a ubiquitination moiety). In still another embodiment, the antibody or antigen-binding fragment has binding specificity for an epitope containing a rare and/or unconventional amino acid (e.g., citrulline, omithine). As used herein, an antibody or antigen-binding fragment has binding specificity for an immunogen if it binds to the immunogen. The antigen-binding properties of an antibody or antigen-binding fragment can be expressed in terms of binding specificity, which may be determined as a comparative measure relative to other known substances that bind to the immunogen (e.g., using dissociation constants (K_d) or association constants (K ). Standard assays for quantitating binding and determining binding affinity are known in the art and include, e.g., equilibrium dialysis, equilibrium binding, gel filtration, surface plasmon resonance, BIACORE^®, microbalances, the use of a labeled immunogen and indirect binding assays (e.g., competitive inhibition assays) (Paul, W.E., Fundamental Immunology, Second Ed. , Raven Press, New York, pp. 315-352 (1989); the entire teachings of which are incorporated herein by reference). For example, as is well known in the art, the dissociation constant of an antibody or antigen-binding fragment can be determined by contacting the antibody or antigen-binding fragment with the immunogen and measuring the concentration of bound and free antibody or antigen- binding fragment as a function of its concentration. Formulaically, this can be represented as:

[Bound] = N x [Free]/ ((K_d) + [Free])

where [Bound] = the concentration of bound antibody or antigen-binding fragment; [Free] = the concentration of unbound antibody or antigen-binding fragment; N = the concentration of binding sites on the antibody or antigen-binding fragment; and K_d = the dissociation constant (a quantitative measure of the binding affinity). The association constant, K_a, is the reciprocal of the dissociation constant, K_d. A detailed description of binding affinities and the kinetics of antibody-immunogen interactions can be found in Paul, W.E., ed., Fundamental Immunology, 4^th Ed., Lippincott- Raven, Philadelphia (1999), p. 75-110. In one embodiment, the immunogen binds to an antibody or antigen-binding fragment with a particular affinity (e.g., a K_a of at least 10⁵ M^"1, a K_a of at least 10⁷ M^"1 or a K_a of at least 10⁹ M^"1). In particular embodiments, the conjugated antibodies and conjugated antigen-binding fragments produced by the methods of the invention have a K_a in the range of about 5 x 10⁴to as high as about 10ⁿ liters/mole. In other embodiments, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_a which is equal to or greater than about 1 x 10⁵ liters/mole, 1 x 10⁶ liters/mole, 1 x 10⁷ liters/mole, 1 x 10⁸ liters/mole, 1 x 10⁹ liters/mole or 1 x 10¹⁰ liters/mole. In other embodiments, the conjugated antibodies and conjugated antigen-binding fragments produced by the methods of the invention have a K_d in the range of about 1.0 x 10^"5 M to about 1.0 x 10^"11 M. In other embodiments, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d which is equal to or less than about 1 x 10^'5 M, 1 x 10^'6 M, 1 x 10^'7 M, 1 x 10^'8 M, 1 x 10^"9 M or 1 x 10"¹⁰ M. As described and exemplified herein, the methods of the invention allow for the conjugation of an antibody or antigen-binding fragment to a conjugating molecule, wherein the antibody or antigen-binding fragment "retains an ability to bind to an immunogen for which the antibody or antigen-binding fragment has binding specificity". That is, the methods allow for the conjugation of an antibody or antigen-binding fragment to a conjugating molecule without negatively affecting, or only minimally affecting, the ability of the antibody or antigen-binding fragment to bind to a target immunogen. By contacting the antibody or antigen-binding fragment with an immunogen prior to conjugating the conjugating molecule to the antibody or antigen-binding fragment, the method prevents the conjugating molecule from conjugating to chemical groups that are involved in binding the immunogen (e.g., chemical groups present in the antigen-binding region of the antibody or antigen-binding fragment). In this way, the method produces a conjugated antibody or antigen-binding fragment that retains its binding specificity for its target immunogen. As described above, the association constant (K and dissociation constant (K_d) of an antibody or antigen-binding fragment can be determined, e.g., using the formula described above. The more stable the interaction between the antibody or antigen-binding fragment and the immunogen, the smaller the K_d of the antibody or antigen-binding fragment. In one embodiment, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d for a particular immunogen that is about equal to the K_d of the corresponding non- conjugated antibody or antigen-binding fragment. In another embodiment, the methods of the invention produce a conjugated antibody or conjugated antigen- binding fragment that has a K_d for a particular immunogen that is higher than the K_d of the corresponding non-conjugated antibody or antigen-binding fragment (the methods of the invention-produce a conjugated antibody or conjugated antigen- binding fragment that has a lower binding affinity for the particular immunogen than the corresponding non-conjugated antibody or antigen-binding fragment). In certain embodiments, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d for a particular immunogen that is about 150% of or less than, about 140% of or less than, about 130% of or less than, about 120% of or less than, about 110% of or less than, or about 105% of or less than, the K_d of the corresponding unmodified (i.e., non-conjugated) antibody or antigen-binding fragment. For example, if a particular non-conjugated antibody has a K_d for a particular immunogen that is 1.55 x 10^"8 M, the same antibody conjugated using the methods of the invention having a K_d of 1.86 x 10^"8 M (i.e., 1.55 x 10^"8 x 1.2 (i.e., 120% ) = 1.86 x 10^"8 M) would have a K_d that is 120% of that of the corresponding non-conjugated antibody (i.e., 120% of the K_d value of the corresponding non-conjugated antibody). In a particular embodiment, the conjugated antibody or conjugated antigen-binding fragment that is produced by the method of the invention has a K_d for a particular immunogen that is about 103% of or less than, or about 101% of or less than, the K_d of the corresponding unmodified (i.e., non-conjugated) antibody or antigen-binding fragment for that immunogen. In another embodiment, the conjugated antibody or conjugated antigen-binding fragment that is produced by the method of the invention has a K_d for a particular immunogen that is about equal to or less than that of the corresponding unmodified (i.e., non-conjugated) antibody or antigen-binding fragment. That is, the conjugated antibody or conjugated antigen-binding fragment produced by the methods of the invention has a greater binding affinity for the immunogen than the binding affinity of the non-conjugated antibody or antigen-binding fragment (e.g., a K_d that is about 10% lower, 20% lower, 30% lower, 40% lower or 50% lower). In another embodiment, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d for a particular immunogen that is 2 times or greater than the K_d of the corresponding unmodified (i.e., non-conjugated) antibody or antigen-binding fragment (and thus, the conjugated antibody or conjugated antigen-binding fragment produced by the methods described herein has a lower binding affinity than the binding affinity of the corresponding non-conjugated antibody or antigen-binding fragment). For example, if a non-conjugated antibody has a K_d for a particular immunogen that is 1.55 x 10^"8 M, the same antibody conjugated using the methods of the invention having a K_d of 3.1 x 10^"8 M (i.e., 1.55 x 10^"8 M x 2 = 3.1 x 10^'8 M) would have a K_d which is 2 times that of the corresponding non-conjugated antibody. In other embodiments, the methods of the invention produce a conjugated antibody or conjugated antigen- binding fragment that has a K_d for an immunogen which is about 4 times or greater, 8 times or greater, 10 times or greater, or 20 times or greater, than that of the K_d of the corresponding non-conjugated antibody or non-conjugated antigen-binding fragment. As described above, suitable assays for determining and comparing dissociation constants are well known in the art. In other embodiments, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d for a particular immunogen that is about equal to the K_d of a corresponding conjugated antibody or conjugated antigen-binding fragment that is produced using methods other than the methods of the present invention (e.g., using a random conjugation method such as exemplified herein). In another embodiment, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d for a particular immunogen that is less than the K_d of a corresponding conjugated antibody or conjugated antigen-binding fragment that is produced using another method. That is, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a higher affinity for the immunogen than does the corresponding conjugated antibody or conjugated antigen-binding fragment produced using another method. In particular embodiments, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d for an immunogen which is about 95% of or less than, about 90% of or less than, about 80% of or less than, about 70% of or less than, about 60% of or less than, or about 50% of or less than, the K_d of a corresponding conjugated antibody or conjugated antigen-binding fragment that is produced using another method. For example, if a conjugated antibody produced using a method other than the method of the present invention (e.g., random conjugation) has a K_d for a particular immunogen that is 1.55 x 10^"8 M, the same antibody conjugated using the methods of the invention having a K_d of 1.09 x 10^"8 M (i.e., 1.55 x 10^"8 M x 0.7 (i.e., 70%) = 1.09 x 10-⁸ M) would have a K_d that is 70% of that of the corresponding conjugated antibody produced by the other method (i.e., 70% of the K_d value of the corresponding antibody produced using another method). In another embodiment, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d for an immunogen which is one half of (i.e., Vi) or less than the K_d of a corresponding conjugated antibody or conjugated antigen-binding fragment produced using methods other than the methods of the present invention (and thus, the conjugated antibody or conjugated antigen-binding fragment produced by the methods described herein has a higher binding affinity than the binding affinity of the corresponding conjugated antibody or antigen-binding fragment produced using another method). For example, if a conjugated antibody produced using a method other than the method of the present invention (e.g., random conjugation) has a K_d for a particular immunogen that is 1.55 x 10^"8 M, the same antibody conjugated using the methods of the invention having a K_d of 7.75 x 10^"9 M (i.e., 1.55 x 10^"8 M x 0.5 = 7.75 x 10^'9 M) would have a K_d which is one half of that of the corresponding conjugated antibody produced by the other method (i.e., 50% or Vz of the K_d value of the antibody produced by the other method). In other embodiments, the methods of the invention produce a conjugated antibody or conjugated antigen-binding fragment that has a K_d for an immunogen which is about 1/4 of or less than, 1/8 of or less than, 1/10 of or less than, or 1/20 of or less than, the K_d of a corresponding conjugated antibody or conjugated antigen-binding fragment produced using methods other than the methods of the present invention. As described above, methods for conjugating a conjugating molecule to an antibody or antigen-binding fragment are well known in the art (e.g., random conjugation of a conjugating molecule to reactive groups of the antibody or antigen-binding fragment). In one embodiment, the conjugating molecule is biotin or a molecule comprising biotin. As described above, biotin (including biotin analogs and derivatives) may be conjugated to the antibody or antigen-binding fragment directly or indirectly (e.g., using a linker). The conjugating molecule (e.g., biotin) may also be conjugated to the antibody or antigen-binding fragment through intervening chemical groups (e.g., an alkane spacer molecule or other hydrocarbon spacer). The use of spacer molecules between the antibody or antigen-binding fragment and conjugating molecule is not required but may be useful for particular applications. For example, the use of a spacer molecule between the antibody or antigen-binding fragment and biotin may aid in rendering the biotin molecule more accessible for binding to avidin. Contact of the antibody or antigen-binding fragment with the immunogen can be performed using any suitable method. Suitable solutions for contacting the antibody or antigen-binding fragment with the immunogen include any solution that does not damage the antibody or antigen-binding fragment and is compatible with the conjugation step of the method (e.g., water, a buffered solution (e.g., Phosphate Buffered Saline (PBS), HEPES, MOPS, Tris(hydroxymethyl)aminomethane (Tris)). Suitable conditions in which the antibody or antigen-binding fragment binds to the immunogen, thereby producing an antibody-immunogen complex or an antigen- binding fragment-immunogen complex, are known to those of ordinary skill in the art and are encompassed by the methods of the invention. In a particular embodiment, conjugation of the conjugating molecule to the antibody-immunogen complex or the antigen-binding fragment-immunogen complex is accomplished through a primary amine linkage. For example, primary amine linkage of biotin to an antibody or antigen-binding fragment can be achieved through conjugation of the biotin molecule to a primary amine group of the antibody or antigen-binding fragment (e.g., a primary amine group of a lysine residue). Methods of conjugating a biotin moiety to an antibody and/or antigen-binding fragment are well known in the art. Moreover, the availability of biotin derivatives has further expanded the possibility of biotin conjugation to a desired substrate (e.g., antibody, antigen-binding fragment). For example, biotin derivatives have been prepared with functionalities that are reactive towards particular chemical groups (e.g., amines, phenols, imidazoles, aldehydes, carboxylic acids, thiols) and this had aided in providing opportunities for conjugation to various molecules (e.g., modified antibodies, modified antigen-binding fragments). In particular embodiments, where conjugating of the conjugating molecule to the antibody-immunogen complex or the antigen-binding fragment-immunogen complex occurs through a primary amine linkage, amme-containing molecules are removed from the antibody or antigen-binding fragment prior to the conjugating step. Removal of excess amine-containing molecules from the antibody or antigen- binding fragment (e.g., from a solution containing the antibody or antigen-binding fragment) facilitates conjugating of the conjugating molecule (e.g., biotin) to the antibody or antigen-binding fragment through a primary amine linkage, hi one embodiment, the amine-containing molecules are removed using dialysis. For example, as exemplified herein, amine-containing molecules were removed from the antibody-containing solution by dialyzing this solution against PBS. Dialysis with other suitable non-amine-containing solutions is also encompassed by the methods of the invention, as is removal of amine-containing molecules using other methods (e.g., antibody or antigen-binding fragment affinity columns). Other suitable methods for removing excess amine-containing molecules from the antibody or antigen-binding fragment include, for example, chromatographical methods (e.g., column chromatography, reverse phase chromatography, gel filtration). In the methods described herein, the immunogen can be attached to a support matrix. Attachment of the immunogen to a support matrix can facilitate the conjugating and/or separation step(s) of the methods of the invention. Any suitable support matrix to which the immunogen can be attached, and which allows the immunogen to be accessible for binding to the antibody or antigen-binding fragment, can be used. Suitable support elements include, but are not limited to, acrylamide derivatives, methacrylate derivatives, polystyrene derivatives (e.g., 96 well plates), magnetic beads, agarose, resins and Sepharose^™. In a particular embodiment, the support matrix is an affinity column (e.g., a peptide affinity column). Suitable methods of attaching an immunogen to a support matrix are known in the art and include, e.g., the methods described herein for conjugating an antibody or antigen- binding fragment to a conjugating molecule, cyanogen bromide attachment, glutathione attachment, chitin, amylase, metal chelated support (e.g., nickel, cobalt). In one embodiment, the immunogen contains a histidine-rich region (e.g., a histidine tag) and is bound to a metal chelated support. In other embodiments, the methods of the invention comprise separating the conjugated antibody or conjugated antigen-binding fragment from the immunogen. Separation of the conjugated antibody or conjugated antigen-binding fragment from the immunogen can be performed using any suitable method, e.g., selective elution, non-selective elution. As used herein, selective elution refers to elution of the conjugated antibody or conjugated antigen-binding fragment using, for example, a competitive inhibitor of the antibody-immunogen or antigen-binding fragment- immunogen interaction. Non-selective elution refers to elution of the conjugated antibody or conjugated antigen-binding fragment using, for example, altered conditions (e.g., ionic strength, pH) which disfavor the interaction of the conjugated antibody or conjugated antigen-binding fragment and the immunogen. As described herein, non-selective elution of the conjugated antibody or conjugated antigen- binding fragment can be performed using a low pH solution (e.g., 50 mM of glycine, pH 2.7, 50 mM of glycine, pH 1.9). Other methods for eluting antibodies and antigen-binding fragments from an immunogen are known in the art and are encompassed by the methods of the invention. Additionally, as exemplified herein, elution fractions can be collected and assayed for the presence of protein (e.g., using a Bradford assay). Fractions containing protein can then be pooled to concentrate and/or obtain desired quantities of antibody or antigen-binding fragment. In other embodiments, the immunogen is conjugated to a support matrix prior to contacting the antibody or antigen-binding fragment, and the antibody- immunogen complex or antigen-binding fragment-immunogen complex is separated from the solid support. The antibody or antigen-binding fragment can then be separated from the immunogen in a subsequent step. In other embodiments, the invention is a method of producing a conjugated antibody or conjugated antigen-binding fragment, wherein the conjugated antibody or conjugated antigen-binding fragment retains an ability to bind to an immunogen. In the method, the antibody or antigen-binding fragment is contacted with the immunogen under conditions in which it will bind to the immunogen, thereby producing an antibody-immunogen complex or an antigen-binding fragment- immunogen complex. A conjugating molecule is then conjugated to the antibody- immunogen complex or the antigen-binding fragment-immunogen complex. This method allows the conjugated antibody or conjugated antigen-binding fragment to retain its ability to bind to the immunogen. As described herein, the methods of producing a conjugated antibody or conjugated antigen-binding fragment can further comprise removing amine- containing molecules from the antibody or antigen-binding fragment (e.g., from a solution containing the antibody or antigen-binding fragment), if the antibody or antigen-binding fragment is to be conjugated to the conjugating molecule through a primary amine linkage. The methods of producing a conjugated antibody or conjugated antigen-binding fragment can further comprise separating the conjugated antibody or conjugated antigen-binding fragment from the immunogen and/or conjugating the immunogen to a support matrix prior to contacting the antibody or antigen-binding fragment. In other embodiments, the invention is a conjugated antibody or conjugated antigen-binding fragment that is produced by the methods described herein. The invention also encompasses pharmaceutical compositions comprising a conjugated antibody or conjugated antigen-binding fragment produced by the methods of the invention. Such pharmaceutical compositions can comprise further agents, such as agents that aid in stabilizing the conjugated antibody or conjugated antigen-binding fragment and suitable pharmaceutical carriers (e.g., if the conjugated antibody or conjugated antigen-binding fragment is to be administered to a subject). Such suitable agents include, e.g., proteins (e.g., BSA, gelatin), sugars (e.g., sucrose, glycerol) and salts. In another embodiment, the invention is a kit that comprises a conjugated antibody or conjugated antigen-binding fragment produced by the methods of the invention. The kits of the invention can comprise, e.g., a conjugated antibody or conjugated antigen-binding fragment produced by the methods of the invention and one or more ancillary reagents (e.g., ancillary reagents suitable for detecting the presence of the conjugated antibody or conjugated antigen-binding fragment). The conjugated antibody or conjugated antigen-binding fragment can be provided in lyophilized form, either alone or in combination with additional antibodies (e.g., secondary antibodies for detecting the conjugated antibody or conjugated antigen- binding fragment). As described herein, the conjugated antibodies or conjugated antigen-binding fragments can be unlabeled or labeled (e.g., using the labels described herein). In addition, the kits of the invention can further comprise adjunct ingredients (e.g., buffers, such as Tris (Tris(hydroxymethyl)aminomethane), phosphate and carbonate, stabilizers, excipients, biocides and/or inert proteins (e.g., bovine serum albumin)). For example, the conjugated antibodies or conjugated antigen-binding fragments can be provided as a lyophilized mixture with the adjunct ingredients, or the adjunct ingredients can be separately provided for combination by the user. Generally these adjunct materials will be present in less than about 5% by weight based on the amount of conjugated antibody or conjugated antigen-binding fragment, and usually will be present in a total amount of at least about 0.001% by weight based on the concentration of the conjugated antibody or conjugated antigen- binding fragment. Where a secondary antibody or antigen-binding fragment capable of binding to the conjugated antibody or conjugated antigen-binding fragment is employed, such antibody or fragment can be provided in the kit, for instance in a separate vial or container. The conjugated antibodies, conjugated antigen-binding fragments and/or ancillary reagent of the kit can be packaged separately or together within suitable containment means (e.g., bottle, box, envelope, tube). When the kit comprises a plurality of individually packaged components, the individual packages can be contained within a single larger containment means (e.g., bottle, box, envelope, tube). The kits of the invention can also optionally include a set of instructions, which can describe any of the embodiments herein. As demonstrated herein, the methods of the invention involve contacting the antibody or antigen-binding fragment with an immunogen prior to conjugating a conjugating molecule to the antibody or antigen-binding fragment, thereby preventing the conjugating molecule from conjugating to chemical groups which are involved in binding the target immunogen. In this way, the methods produce a conjugated antibody or antigen-binding fragment which retains its binding specificity for its target immunogen. The present invention is further illustrated by the following example, which is not intended to be limiting in any way. EXEMPL ICATION This example describes a method for conjugating biotin to an antibody in such a way that the antigen-recognition sites of the antibody is protected from conjugation of biotin. The antibody is allowed to bind to a column containing an immunogen (e.g., a peptide, a protein) prior to conjugation of biotin through a primary amine linkage. As a result, only the primary amine-containing amino acid residues of the antibody (e.g., lysine) that are not involved in binding to the immunogen will be available for conjugation to biotin. The result is a biotinylated antibody that retains full capacity for antigen recognition.

Materials and Methods Column Preparation A peptide affinity column was prepared in advance by coupling 5 mg of an antibody immunogen to 2.5 ml SulfoLink Coupling Gel (Pierce, Rockford, IL) following the manufacturer's instructions. The antibody immunogen was a synthetic peptide containing the following amino acid sequence: YSRALSRQLSSC (SEQ ID NO:l). Ser⁶ of SEQ ID NO:l (which is in bold text) was phosphorylated. In addition, the C- terminal cysteine residue was added to facilitate coupling. A 10 ml Econo-Column Chromatography Column (Biorad Laboratories, Hercules, CA) was used. The column was then stored until use in phosphate buffered saline (PBS; Fisher Scientific, Chicago, IL) with 0.05% azide (PBS/Z) under argon.

^■Dialysis In order to remove amine-containing molecules, the antibody solution was dialyzed at 4°C with gentle stirring in Tube-O-Dialyzer™ (Geno Technology, St. Louis, MO) against 1 liter of PBS. The PBS was replaced with fresh PBS after 1 hour, 2 hours, and 19 hours, and dialysis was completed at 19.5 hours. Following dialysis, antibody concentration was quantitated using a Bradford Assay (Coomassie Plus Protein Assay Reagent; Pierce, Rockford, IL) and Bovine gamma globulin as a standard. Absorbance at 600 nm was measured and the standards were used to calculate antibody concentration.

Biotin Conjugation The affinity column was flushed with PBS to remove all traces of azide, a component of the column storage buffer. One column volume (CV) of PBS was then added to the column to create a 50% bead slurry, which was subsequently transferred to a 15 ml conical tube. Transfer to the conical tube was only performed if the volume of the antibody solution to be applied exceeded the column volume. 2-10 mg of the antibody was then added and allowed to bind to the affinity resin overnight at 4°C with constant rotation. Following the overnight incubation, the slurry was returned to the original column. The conical tube was rinsed with 2-3 ml of PBS to collect any bead residue, and then transferred to the original column, where the slurry was allowed to settle. Unbound antibody was drained and reserved for quantitation of protein content. The column was then flushed with 20 CV of PBS. The amount of antibody retained on the column was estimated by determining the amount of unbound antibody present in the flow through using a Bradford assay (as described above). With the column flow stopped, sufficient PBS was added to the resin to create a slurry in which the antibody concentration approximated 2 mg/ml. Sulfo-NHS-LC-LC-Biotin (Pierce, Rockford, IL) was reconstituted to 10 mg/ml in water according to the manufacturer's instructions and immediately added as indicated (15-60 fold molar excess) to the affinity column containing bound antibody. Calculations were performed with the aid of an Excel Spreadsheet (Microsoft) containing the requisite formulae (Table 1). For conjugations performed on ice, the column was placed in a re-sealable plastic bag filled with ice and the conjugation was allowed to proceed for two hours with constant rotation to prevent the beads from settling. Following incubation, the column was drained of the excess unreacted biotin, approximately 2 CV of PBS was added, and the bead slurry restored by inverting the column to thoroughly remove unreacted biotin. Beads were allowed to settle at room temperature, the column was drained, and then was washed with an additional 5 CV of PBS. Biotin-conjugated antibody was then eluted from the column with 50 mM of glycine, pH 2.7, or 50 mM of glycine, pH 1.9 (if the antibody did not elute at the higher pH). 1 ml fractions were collected into tubes containing 50 μl of Neutralization buffer (2 M Tris-Base, 1.5 M NaCl, 1 mM EDTA, 0.5% azide(w/v)), inverted twice to mix, and then stored on ice. The antibody elution profile was traced using the Bradford assay scaled down for use with microtiter plates, and protein containing fractions (OD₆₀₀ > 0.15) were pooled. The pH of the pooled fractions was adjusted to 7.5 using Neutralization buffer and colorpHast Indicator Strips, pH 0-14 (EM Science, Gibbstown, NJ). The biotin-conjugate antibody was stored at 4°C. The molar coupling efficiency of biotin to the antibody was calculated using 2-(4'-hydroxyazobenzene) benzoic acid (HABA, Pierce, Rockford, IL) in accordance with the manufacturer's instructions.

Table 1. Calculation of Required Amount of NHS-Biotin Ester.

Column Regeneration

Following completion of antibody elution, the column was washed with PBS/Z until no protein was detected using the Bradford assay. The column was then regenerated by adding 3-5 CV of 3 M NaCl, followed by 5 CV of PBS/Z. The column was subsequently flushed with argon, sealed with parafilm and stored at 4°C. The affinity column can be reused 2-4 times. Biotinylation of the column support matrix was undetectable as determined using the HABA assay. Results In assays in which antibodies are used as a detection component, anti-species secondary antibodies bearing a visualization tag are also often used. These secondary antibodies offer tremendous versatility because the same secondary antibody can be used to detect any antibody that is generated in that same animal host. Secondary antibodies also provide the benefit of signal amplification as numerous secondary antibodies can bind to the same primary antibody. However, when an indirect detection system is not feasible or a single antibody has the potential for use in a variety of applications which employ disparate detection systems, direct tagging of the primary antibody becomes the more convenient option. One common approach is to covalently couple the primary antibody to biotin, which allows the conjugated antibody to take advantage the very strong interaction that occurs between biotin and avidin (e.g., streptavidin). Indeed, the interaction of biotin and avidin has been characterized as one of the strongest known non-covalent biological interactions (Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, CA (1996), p. 570). The biotinylated antibody can then be labeled with one of several streptavidin conjugates to generate a variety of one-step visualization reagents.' Biotinylation of antibodies can be achieved using one of several chemistries. In this example, the amine groups of a mouse monoclonal antibody specific for Heat Shock Protein 27 (HSP27) that is phosphorylated at serine residue 27 (hereafter referred to as anti-phospho-HSP27 mAb; Upstate Biotechnology, Inc., Lake Placid, NY, Catalog No. 05-645) were targeted for biotinylation. Targeting of amine groups for biotinylation was chosen because it allowed for random higher molar incorporation of biotin than is achievable using other chemistries. 5 mg of the mouse monoclonal antibody (anti-phospho-HSP27 mAb) was incubated for two hours on ice in a solution containing a 16.6-fold molar excess of Sulfo-NHS-LC-LC- Biotin (Pierce, Rockford, IL) following the manufacturer's recommended protocol. Unreacted biotin was removed by dialysis. Analysis using the HABA assay (Pierce, Rockford, EL) indicated that the conjugate contained 2.26 mole biotin/mole antibody. In order to test the functionality of the conjugate, immunoblot analysis was performed on lysates of control or heat-shocked MCF-7 cells (a human breast tumor cell line; available from the ATCC). FIGS. 1A and IB depict a table summarizing experimental parameters and a corresponding Western blot probed with unconjugated anti-phospho HSP27 mAb (labeled anti-phospho-HSP27; lanes 2 and 3) and different concentrations (0.05 μg/ml, 0.2 μg/ml and 0.7 μg/ml) of the biotin- conjugated anti-phospho HSP27 mAb (labeled 16X Biotin; lanes 4 through 9), respectively. As depicted in FIGS. 1A and IB, although 4- to 14-fold more of the biotin-conjugate was used to probe the Western blot (as compared to the unconjugated antibody), the signal that was detected was a fraction of the signal observed when the blot was probed with the unconjugated antibody (FIG. IB, compare lanes 3 and 9). With unconjugated antibodies, the use of a secondary HRP conjugate amplifies the primary signal, as numerous secondary HRP conjugate molecules can bind to each primary antibody. Given the weak signal that was observed using the biotin-conjugated anti-phospho HSP27 mAb (with a biotin: antibody ratio slightly greater than two), it was possible that this reduced signal was a result of insufficient signal amplification because the molar incorporation of biotin was too low. To address this possibility, an additional 5mg of anti-phospho-HSP27 was subjected to biotinylation with a 56 molar excess of Sulfo-NHS-LC-LC-Biotin (Pierce, Rockford, IL) for two hours on ice according to the manufacturer's recommended protocol. Unreacted biotin was removed by dialysis. This second biotinylated conjugate antibody was found to contain 12 mole biotin/mole antibody, approximately 5- to 6- fold higher than the original biotinylated conjugate antibody. The functionality of this second biotinylated conjugate antibody was also examined using Western blot analysis. FIGS. 2 A and 2B depict a table summarizing experimental parameters and a corresponding Western blot probed with unconjugated anti-phospho HSP27 mAb (labeled anti-phospho-HSP27; lanes 2 and 3) and different concentrations (0.05 μg/ml, 0.25 μg/ml and 1.0 μg/ml) of this second biotin-conjugated anti-phospho HSP27 mAb (labeled 56X Biotin; lanes 4 through 7), respectively. As depicted in FIGS. 2A and 2B, despite the higher molar biotin content of this second conjugated antibody, antigen recognition was undetectable (FIGS. 2 A and 2B, lanes 4 through 7). Concluding that this second biotinylated conjugate antibody (i.e., 56XBiotin) was over-biotinylated, a third biotinylated conjugate antibody was prepared. This third biotinylated conjugate antibody was prepared using a 30-fold molar excess of Sulfo-NHS-LC-LC-Biotin (Pierce, Rockford, EL) in order to isolate a conjugate with an intermediate level of biotinylation (as compared to the first and second biotinylated conjugate antibodies), using the same methodology that was used to prepare the first two conjugate antibodies. This third biotinylated conjugate antibody was found to contain 6.8 mole biotin/mole antibody, as determined using the HABA assay (Pierce, Rockford, EL). FIGS. 3 A and 3B depict a table summarizing experimental parameters and a corresponding Western blot probed with unconjugated anti-phospho HSP27 mAb (labeled anti-phospho-HSP27; lanes 6 and 7) and different concentrations (0.05 μg/ml, 0.25 μg/ml and 1.0 μg/ml) of this third biotin-conjugated anti-phospho HSP27 mAb (labeled 3 OX Biotin; lanes 2-5 and 8-9), respectively. As depicted in FIG. 3B, antigen recognition was restored with this intermediately biotinylated antibody, suggesting that the second biotinylated antibody (i.e., 56X Biotin), which contained 12 mole biotin/mole antibody, was over-biotinylated and therefore could not recognize antigen (FIG. 2B). A comparison of the signals detected by the first and third biotinylated- conjugated antibodies relative to the unconjugated antibody (compare FIG. IB, lanes 9 and 3, with FIG. 3B, lanes 3 and 7) reveals that the signal detected with these two biotinylated-conjugated antibodies is surprisingly similar. Thus, a three-fold increase in biotin content in the third biotinylated-conjugated antibody did not result in a three-fold increase in the signal that was detected. One possibility is that not all of the biotin molecules are accessible for binding with the streptavidin-HRP conjugate used to visualize primary antibody binding. A second possibility to explain the lack of signal amplification is that because the antibody sites of biotinylation are random, it is possible that a greater percentage of antibodies in the third biotinylated-conjugated antibody, relative to the first biotinylated-conjugated antibody, are unable to recognize the target antigen. In order to address the question of how much of the biotinylated-conjugated antibody was bound to the membrane, a goat anti-mouse HRP conjugate was used as the secondary antibody in lieu of the streptavidin-HRP conjugate (FIGS. 3 A and 3B, lanes 8 and 9). Although five-fold more of the biotinylated-conjugated antibody was used to probe the membrane relative to the unconjugated antibody (compare lanes 7 and 9 of FIG. 3B), the signal detected using the anti-mouse-HRP conjugate was two- to four-fold weaker than that observed with the unconjugated antibody. These results support the hypothesis that the biotinylated-conjugated antibody is less effective in antigen recognition that its unconjugated counterpart. In order to address the issue of random biotinylation affecting the ability of the antibody to recognize antigen, the anti-phospho-HSP27 antibody was first immobilized on a column containing the immunizing peptide, YSRALSRQLSSC (SEQ ID NO:l), wherein Ser⁶ of SEQ ID NO:l (which is in bold text) was phosphorylated. Biotinylation of this column-immobilized antibody was then performed by incubating the column-immobilized antibody with a 30-fold molar excess of Sulfo-NHS-LC-LC-Biotin (Pierce, Rockford, EL) for two hours on ice. Unreacted biotin was removed by flushing the column with PBS, and the biotinylated antibody was eluted using low pH glycine. This column-immobilized biotinylated-conjugated antibody was found to contain 1.94 mole biotinmole antibody, as determined using the HABA assay (Pierce, Rockford, EL). FIGS. 4 A and 4B depict a table summarizing experimental parameters and a corresponding Western blot probed with unconjugated anti-phospho HSP27 mAb (labeled anti-phospho-HSP27; lanes 2 and 4) and different concentrations (0.5 μg/ml and 1.0 μg/ml) of the column-immobilized biotin-conjugated anti-phospho HSP27 mAb (labeled 30X Biotin; lanes 4 through 7), respectively. As depicted in FIG. 4B, biotin conjugation of anti-phospho HSP27 mAb using the antibody-immobilization technique resulted in a biotinylated-conjugated antibody with improved antigen recognition, relative to biotinylated-conjugated antibodies prepared in solution (FIG. 4B, lanes 4 through 7) . The first biotinylated-conjugated antibody prepared in solution and the biotinylated-conjugated antibody prepared by the antibody- immobilization technique have comparable biotin/antibody ratios of 1.9 and 2.3 respectively. However, when utilized at similar concentrations, the signal detected with the first biotinylated-conjugated antibody prepared in solution, relative to the corresponding unconjugated antibody (used throughout all experiments as a reference), is strikingly weaker than the signal detected with the biotinylated- conjugated antibody prepared by the antibody-immobilization technique (where the antigen-binding region was protected from biotinylation) (compare FIG. 1, lanes 3 (unconjugated) and 9 (conjugated in solution) with FIG. 4, lanes 3 (unconjugated) and 5 (conjugated using antibody immobilization)). Relative to the unconjugated anti-phospho-HSP27 antibody, the signal detected with the biotinylated-conjugated antibody generated with the antibody-immobilization technique is significantly stronger and indicates that the antigen-recognition sites of the antibody were successfully protected by immobilization. The antibody-immobilization technique described above requires that the biotin-NHS ester be added when the antibody is immobilized (e.g., immobilized on a peptide column). Thus, it was tested whether repeated use of the column was possible. The HABA assay (Pierce, Rockford, IL) was performed on a small aliquot of the column residue and no incorporation of biotin was detected. In addition, a side-by-side functional comparison of the previously used (and regenerated) column and fresh column was performed. Antibodies on both columns were incubated for two hours on ice in the presence of a 30-fold molar excess of Sulfo-NHS-LC-LC- Biotin (Pierce, Rockford, IL). Unreacted biotin was removed by flushing the column with PBS and the biotinylated antibody was eluted using low pH glycine. The biotimantibody ratio of the biotinylated-conjugated antibody prepared on the once- used column was 2.92 while the biotimantibody ratio for the biotinylated-conjugated antibody prepared on the fresh column was 2.39. This indicates that the columns can be regenerated and reused as described herein. FIGS. 5 A and 5B depict a table summarizing experimental parameters and a corresponding Western blot probed with the original conjugated biotinylated antibody (labeled as Original Col. Conj.'; lanes 1 and 2), conjugated biotinylated antibody prepared on the once used column (labeled as 'Once used Col.'; lanes 3 and 4) and conjugated biotinylated antibody prepared on a fresh column (labeled as 'Fresh Col.'; lanes 6 and 7), respectively. As can be seen from FIG. 5B, all three conjugated biotinylated antibodies prepared using the antibody-immobilization technique have similar biotin/antibody ratios and function equally well in antigen detection. These results demonstrate that the columns can be regenerated and reused, and that conjugation efficiency is reproducible. In order to demonstrate the versatility of the antibody-immobilization technique, a second mouse monoclonal antibody was tested. As demonstrated herein, biotinylation of an anti-polyHis-tag antibody in solution with biotin-NHS esters greatly diminished the ability of the antibody to detect proteins containing the His-tag. In order to compare the biotinylated anti-polyHis-tag antibody (in solution) with the biotinylated anti-polyHis-tag antibody prepared using the antibody- immobilization technique, a corresponding peptide affinity column was prepared. This peptide affinity column contained a peptide containing the sequence His-His- His-His-His-His-Gly-Ser-Gly-Gly-Cys (SEQ ID NO:2). As with the previous peptide, the C-terminal cysteine residue was included to enable coupling to the SulfoLink Coupling Gel (Pierce, Rockford, IL). Anti-His Tag antibodies that were secreted into the growth medium were immobilized on the above-described peptide column. Biotinylation was allowed to proceed as described herein, either on ice or at room temperature, in the presence of either 15- or 40-fold molar excess of Sulfo- NHS-LC-LC-Biotin (Pierce, Rockford, IL) (in order to examine the effects that temperature and the amount of biotin-NHS ester had on conjugation efficiency). Umeacted biotin was removed by passing PBS over the column. Molar ratios of biotimanfibody for the various conjugates were determined using the HABA assay and are depicted in Table 2.

Table 2. Molar Biotin: Antibody Ratios For Anti-PolyHis-Tag Antibody-Biotin Conjugates.

Based on the molar biotin content of the four biotinylated-conjugated antibodies depicted in Table 2, it is clear that the molar excess of biotin-NHS ester used in the conjugation reaction is a more significant variable than temperature in determining coupling efficiency. These results are analogous to the results obtained with the anti-phospho-HSP27-biotin conjugate antibodies that were prepared in solution, wherein increasing the molar excess of the biotin-NHS ester increased the biotin content of the conjugate antibody. In order to examine the functionality of the biotinylated anti-polyHis-tag antibody conjugates, Western blot analysis was performed with a recombinant protein containing the poly-His-Tag (His-DFF45/ICAD). FIGS. 6A and 6B depict a table summarizing experimental parameters and a corresponding Western blot probed with various anti-polyHis-Tag biotinylated antibodies (lanes 2, 3, 5-8, 10 and 11) and corresponding unconjugated antibody (lane 1), respectively. For each of lanes 2, 3, 5-8, 10 and 11, 50 ng of His-DFF45/ICAD protein was resolved by gel electrophoresis, transferred to nitrocellulose and probed with the indicated anti- polyHis-Tag biotinylated antibody (i.e., anti-polyHis-Tag biotinylated antibodies immobilized on a peptide affinity column and biotinylated with either 15- or 40-fold molar excess of Sulfo-NHS-LC-LC-Biotin at room temperature or on ice; labeled as 15X RT, 40X RT, 15X ICE or 40X ICE, respectively). As depicted in FIG. 6B, all four of the anti-polyHis-tag antibody-biotinylated conjugate antibodies detected the DFF45/ICAD-His-Tagged protein. The signal that was detected with the conjugates when streptavidin-HRP was used (FIGS. 6A and 6B, lanes 2, 6, 8 and 10) is directly correlated to the molar biotin: antibody ratio, which indicates that as the biotin content of the conjugate increased, more streptavidin-HRP bound. The strongest signal is detected with the biotinylated-conjugated antibody with the highest biotin: antibody molar ratio (FIGS. 6A and 6B, lane 10). In addition, the anti-mouse HRP conjugate was used as a secondary antibody with same panel of biotinylated- conjugated antibodies (FIGS. 6 A and 6B, lanes 3, 5, 7 and 11) and the resulting signal was compared to that of the unconjugated antibody (FIGS. 6A and 6B, lane 1). The strength of the signal that was detected using the secondary anti-mouse HRP conjugate was the same with all of the biotinylated-conjugated primary antibodies and the unconjugated antibody. These results demonstrate that the incorporation of biotin into the anti-His-Tag monoclonal antibody did not affect the ability of the anti-mouse-HRP secondary conjugate to bind the biotinylated antibody, and further demonstrate that biotinylation of antibodies using the immobilization technique successfully protects antigen-recognition sites of the antibody and allows the antibodies to retain their ability to bind target antigens.

Discussion The results described herein demonstrate that for many antibodies and antigen-binding fragments, the presence of biotinylation-susceptible amine- containing groups within the antigen-recognition site(s) of the antibody or antigen- binding fragment can negatively affect its ability to bind target antigens when biotinylated. The results further demonstrate that the antibody-immobilization method described herein, wherein the antigen-recognition site(s) of the antibody or antigen-binding fragment are protected (e.g., protected from amine crosslinking agents) by first binding the antibody to a resin containing the immunogen, is an effective method to generate labeled antibodies or antigen-binding fragments that retain their ability to recognize target antigens. In addition, the results demonstrate that repeated use of an affinity column is possible, and that the conjugation efficiency between batches is reproducible. This approach is also suitable for labeling any other protein-protein interaction (e.g., ligahd-receptor interaction; protein-protein interaction). While this invention has been particularly shown and described with references to particular embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAEMSWhat is claimed is:

1. A method of conjugating an antibody or antigen-binding fragment to a conjugating molecule, wherein said antibody or antigen-binding fragment retains an ability to bind to an immunogen for which said antibody or antigen-binding fragment has binding specificity, comprising: a) contacting said antibody or antigen-binding fragment with said immunogen for which said antibody or antigen-binding fragment has specificity, under conditions in which said antibody or antigen- binding fragment binds to said immunogen, thereby producing an antibody-immunogen complex or an antigen-binding fragment- immunogen complex; and b) conjugating said conjugating molecule to said antibody-immunogen complex or said antigen-binding fragment-immunogen complex, thereby conjugating said antibody or antigen-binding fragment to said conjugating molecule, wherein said antibody or antigen-binding fragment retains an ability to bind to said immunogen for which said antibody or antigen-binding fragment has binding specificity.

2. The method of Claim 1 further comprising separating said conjugated antibody or conjugated antigen-binding fragment from said immunogen.

3. The method of Claim 1 wherein said conjugating molecule comprises one or more of a protein moiety, a peptide moiety, a peptidomimetic moiety, an organic moiety, a lipid moiety, a nucleic acid moiety and a carbohydrate moiety.

4. The method of Claim 1 wherein said conjugating molecule comprises a label selected from the group consisting of an affinity label, a spin label, an enzyme label, a fluorescent group, a chemiluminescent group, a radioactive label and a solvent soluble dye.

5. The method of Claim 1 wherein said conjugating molecule comprises an affinity label selected from the group consisting of: biotin and avidin.

6. The method of Claim 1 wherein said conjugating of said conjugating molecule to said antibody-immunogen complex or said antigen-binding fragment-immunogen complex is through a primary amine linkage.

7. The method of Claim 6 wherein said conjugating molecule is biotin.

8. The method of Claim 6 further comprising removing amine-containing molecules from said antibody or antigen-binding fragment prior to contacting said antibody or antigen-binding fragment with said immunogen.

9. The method of Claim 8 wherein said amine-containing molecules are removed using dialysis.

10. The method of Claim 1 wherein said immunogen is conjugated to a support matrix prior to contacting said antibody or antigen-binding fragment.

11. The method of Claim 10 wherein said support matrix is an affinity column.

12. The method of Claim 1 wherein said antibody or antigen-binding fragment has binding specificity for a heat shock protein or a poly-His tag.

13. The method of Claim 1 wherein said antibody or antigen-binding fragment has binding specificity for an epitope containing a modification selected from the group consisting of an acetylation moiety, a methylation moiety, a ribosylation moiety, a phosphorylation moiety and a ubiquitination moiety.

14. The method of Claim 1 wherein said conjugating of said conjugating molecule to said antibody-immunogen complex or antigen-binding fragment- immunogen complex is accomplished using a crosslinking agent.

15. A method of conjugating an antibody or antigen-binding fragment to a conjugating molecule through a primary amine linkage, wherein said antibody or antigen-binding fragment retains an ability to bind to an immunogen for which said antibody or antigen-binding fragment has binding specificity, comprising: a) removing amine-containing molecules from said antibody or antigen- binding fragment; b) contacting said antibody or antigen-binding fragment with said immunogen, under conditions in which said antibody or antigen- binding fragment binds to said immunogen, thereby producing an antibody-immunogen complex or an antigen-binding fragment- immunogen complex; and c) conjugating said conjugating molecule to said antibody-immunogen complex or said antigen-binding fragment-immunogen complex through a primary amine linkage, thereby conjugating said antibody or antigen-binding fragment to said conjugating molecule, wherein said antibody or antigen-binding fragment retains an ability to bind to said immunogen for which said antibody or antigen-binding fragment has binding specificity.

16. The method of Claim 15 further comprising separating said conjugated antibody or conjugated antigen-binding fragment from said immunogen.

17. The method of Claim 15 wherein said conjugating molecule comprises one or more of a protein moiety, a peptide moiety, a peptidomimetic moiety, an organic moiety, a lipid moiety, a nucleic acid moiety and a carbohydrate moiety.

18. The method of Claim 15 wherein said conjugating molecule comprises a label selected from the group consisting of an affinity label, a spin label, an enzyme label, a fluorescent group, a chemiluminescent group, a radioactive label and a solvent soluble dye.

19. The method of Claim 15 wherein said conjugating molecule comprises an affinity label selected from the group consisting of: biotin and avidin.

20. The method of Claim 15 wherein said amine-containing molecules are removed using dialysis.

21. The method of Claim 15 wherein said immunogen is conjugated to a support matrix prior to contacting said antibody or antigen-binding fragment.

22. The method of Claim 21 wherein said support matrix is an affinity column.

23. The method of Claim 15 wherein said antibody or antigen-binding fragment has binding specificity for a heat shock protein or a poly-His tag.

24. The method of Claim 15 wherein said antibody or antigen-binding fragment has binding specificity for an epitope containing a modification selected from the group consisting of an acetylation moiety, a methylation moiety, a ribosylation moiety, a phosphorylation moiety and a ubiquitination moiety.

25. The method of Claim 15 wherein said conjugating of said conjugating molecule to said antibody-immunogen complex or antigen-binding fragment- immunogen complex is accomplished using a crosslinking agent.

26. A method of conjugating an antibody or antigen-binding fragment to a conjugating molecule through a primary amine linkage, wherein said antibody or antigen-binding fragment retains an ability to bind to an immunogen for which said antibody or antigen-binding fragment has binding specificity, comprising: a) removing amine-containing molecules from said antibody or antigen- binding fragment; b) contacting said antibody or antigen-binding fragment with said immunogen, wherein said immunogen is conjugated to a support matrix, under conditions in which said antibody or antigen-binding fragment binds to said immunogen, thereby producing an antibody- immunogen complex or an antigen-binding fragment-immunogen complex; and c) conjugating said conjugating molecule to said antibody-immunogen complex or said antigen-binding fragment-immunogen complex through a primary amine linkage, thereby conjugating said antibody or antigen-binding fragment to said conjugating molecule through a primary amine linkage, wherein said antibody or antigen-binding fragment retains an ability to bind to said immunogen for which said antibody or antigen-binding fragment has binding specificity.

27. The method of Claim 26 further comprising separating said conjugated antibody or conjugated antigen-binding fragment from said immunogen.

28. The method of Claim 26 wherein said conjugating molecule comprises one or more of a protein moiety, a peptide moiety, a peptidomimetic moiety, an organic moiety, a lipid moiety, a nucleic acid moiety and a carbohydrate moiety.

29. The method of Claim 26 wherein said conjugating molecule comprises a label selected from the group consisting of an affinity label, a spin label, an enzyme label, a fluorescent group, a chemiluminescent group, a radioactive label and a solvent soluble dye.

30. The method of Claim 26 wherein said conjugating molecule comprises an affinity label selected from the group consisting of: biotin and avidin.

31. The method of Claim 26 wherein said amine-containing molecules are removed using dialysis.

32. The method of Claim 26 wherein said support matrix is an affinity column.

33. The method of Claim 26 wherein said antibody or antigen-binding fragment has binding specificity for a heat shock protein or a poly-His tag.

34. The method of Claim 26 wherein said antibody or antigen-binding fragment has binding specificity for an epitope containing a modification selected from the group consisting of an acetylation moiety, a methylation moiety, a ribosylation moiety, a phosphorylation moiety and a ubiquitination moiety.

35. A conjugated antibody or conjugated antigen-binding fragment produced by the method of Claim 1.

36. A conjugated antibody or conjugated antigen-binding fragment produced by the method of Claim 15.

37. A conjugated antibody or conjugated antigen-binding fragment produced by the method of Claim 26.