MONOCLONAL ANTIBODIES SPECIFIC FOR THE HARVEY, KIRSTEN AND N RAS PROTEINS AND THEIR USE AS DIAGNOSTIC REAGENTS

Abstract: The subject of this invention is the induction, production, and characterization of monoclonal antibodies (Mabs) that react with the individual H, Ki2A, Ki2B, and N ras p21s. Mabs described in this invention are valuable tools for the detection, quantitation and classification of the individual ras p21s in normal, neoplastic, preneoplastic and dysplastic cells, as well as in bodily fluids, using a variety of techniques.

Description: TITLE Monoclonal Antibodies Specific for the Harvey, Kirsten and N Ras Proteins and Their Use as Diagnostic Reagents CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of co- pending patent application U.S.S.N. 07/185,194 filed on April 22, 1988. FIELD OF THE INVENTION This invention relates to murine monoclonal antibodies and immunoreactive fragments specific for the Harvey, Kirsten and N ras proteins and, more particularly, to their use as diagnostic reagents. BACKGROUND OF THE INVENTION Harvey, Kirsten and N ras proteins are immunologically related proteins and are collectively termed p21. They are products of the ras family of cellular genes which are found in a wide variety of nucleated mammalian cells. The ras genes appear to be frequent targets of genetic alterations that can lead normal cells along the pathway to malignancy. Ras oncogenes have been identified in a wide array of premalignant and malignant cells. The three members of the ras gene family, H, Ki, and N, are very highly conserved at the amino-terminal two-thirds of the molecule, but diverge somewhat over the remaining one-third of the molecule near the carboxyl-terminal end. Cellular Harvey (c-Ha) and cellular Kirsten (c-Ki) have been shown to be homologous to the Harvey πmrine sarcoma viral oncogene (v-Ha-ras) and the Kirsten murine sarcoma viral oncogene (v-Ki-ras) . (Weinberg et al., U.S. Patent 4,535,058; Harvey, Nature 104:1104 (1964); Kirsten et al., J. .C.I. 39:311 (1967) .) The third member of the ras family, N ras, was so designated because it was identified from a human neuroblastoma (Shimizu et al., PNAS (USA) 80:2112 (1983)). It has not yet been found to have a retroviral counterpart. The p21 proteins consist of about 188-189 amino acids having a molecular weight of about 21,000 daltons. Viral and cellular ras genes encode membrane bound proteins ( illingham et al., Cell 19:1005 ζl 9BO) ) which bind guanine nucleotides (Scolnick et al. r PNAS (USA) 76:5355 (1979); Papageorge et al., J. Virol. 44:509 (1982); and Finkel et al.. Cell-. 37:151 (1984)) and possess intrinsic GTPase activity (McGrath et al.. Nature 301:644 (1984); Sweet et al.. Nature 311:273 (1984); Gibbs et al., PNAS (USA) 81:5704 (1984); and Manne et al., PNAS 82:376 (1985)). Reports have demonstrated a partial homology with guanine nucleotide binding proteins, thus, leading to the hypothesis that ras p21 proteins may play a role in signal transduction at the cell membrane (Lochrie, M. A., et al.. Science 28:96-99 (1985); Hatley, J. B. et al.. Science 226:850-862 (1984)). Reports have also suggested that the 188-189 amino acid sequence of the H, Ki and N ras p21s have been greatly conserved throughout evolution. However, the most significant differences between the H, Ki, and N p21 proteins appear to be localized in a segment having 15-20 amino acids located at the carboxy end of the p21 protein (Taparowsky et al.. Nature 300:762 (1982)). Furthermore, McGrath ' et al.. Nature 304:501-506 (1983), and Shimizu et al., Nature 304:497-500 (1983), have shown that the Ki gene has the capacity to encode two distinct proteins referred to as Ki2A and Ki2B. Since this variable region exists among amino acids 160-180 at the C-terminal end of the ras p21, then it is theoretically possible to generate monoclonal antibodies that could specifically bind the individual H, Ki2A, Ki2B, and N ras p21s. Reports indicate that activation of a particular ras gene is frequently associated with a specific type of cancer, for example, the N ras gene in acute myelogenous leukemia (Bos et al.. Nature 315:726 (1985)) or the overexpression of the particular H-ras gene in breast cancer (Spandidos et al., Anticancer Res. 4:269 (1984)) . European Patent Application No. 86107244.5, published on December 3, 1986, discloses polypeptides having amino acid sequences derived from the variable regions of ras H , ras N , ras^ KA and ras^ KB protein families, immunogenic compositions wherein these polypeptides are covalently attached to immunogenic carriers, antibodies produced from such immunogens wherein these antibodies are specific for the ras oncogene from which the polypeptide sequence was derived and immunoassays employing these antibodies to distinguish among the individual p21 ras oncogene families. The peptide structures disclosed correspond to amino acids 171-189 and 170-189 of p21 H ras, 170- 186 of p21 N ras, 171-186 of p21 Ki4A ras, and 170-185 of p21 Ki4B ras. Although hybridoma technology was disclosed, it appears that the peptide antibodies generated were polyclonal antibodies. Tanaka et al., PNAS 82:3400 (1985), reported the generation of a series of rabbit sera to a variety of synthetic, peptides corresponding to various portions of the ras p21s. Tanaka et al. reported the production of rabbit sera to a peptide corresponding to amino acids 160-179 of the vr-Ha-ras . The anti-p21 sera was prepared by affinity purifying the rabbit sera and evaluating their specificity by biochemical assays. The specificity of these reagents, however, is questionable. Srivastava et al.. Molecular and Cellular Biology 5(11):3316 (1985), reported a series of rabbit polyclonal sera to synthetic peptides corresponding to various segments of the protein and in particular a segment corresponding to amino acid 161-176 of the H ras p21. The specificity of these reagents, O however, is questionable. Tahara et al. r Jpn. J. Cancer Res. (Gann) 77:517- 522 (1986), disclose that a sheep anti-p21 antibody was generated against a synthetic peptide corresponding to positions 160-179 of the v-Ha-p21. 5 The specificity of this reagent is also questionable. Bizub et al., Oncogene 1:131-142 (1987), raised antisera in mice, rats and rabbits to a variety of peptides in the H, Ki and N ras variable regions. Some of the polyclonal antibodies described in this Q report were affinity purified rabbit sera raised against peptides corresponding to amino acids 171-189 of the H p21 or to peptides corresponding to amino acids 171-186 of the Ki4B p21. According to this report, additional antibodies to the ras p21s are 5 available at the N.C.I- repository (Microbiological Assoc. Inc., Bethesda, Maryland). The antisera mentioned in the Bizub et al. report indicated that antibodies available at the N.C.I, were raised against a peptide structure correlating to the amino acid 0/ sequence 157-180 of the ras p21s. STATEMENT OF DEPOSIT The hybrido a cell lines which were found to secrete monoclonal antibodies reactive with the H ras p21s and the subject of this invention were deposited in the American Type Tissue Culture Collection (ATCC) under the Budapest Treaty on March 25, 1988. Hybridoma H-770-1.1.4 was designated ATCC No. HB 9673. Hybridoma H-784-4.7.7 was designated ATCC No. HB 9675. 5 Hybridoma H-873-3.5.3 was designated ATCC No. HB 9674. The hybridoma cell lines which were found to secrete monoclonal antibodies reactive with the N ras p21s and the subject of this invention were deposited in the American Type Tissue Culture Collection (ATCC) J O under the Budapest Treaty on March 25, 1988. Hybridoma N-821-1.1.9 was designated ATCC No. HB 9671. Hybridoma N-838-1.1.6 was designated ATCC No. HB 9672. The hybridoma cell lines which were found to secrete monoclonal antibodies reactive with Ki2A p21s 15 and the subject of this invention were deposited in the ATCC under the Budapest Treaty. Hybridoma K2A-1 was designated ATCC No. HB10072, date of deposit was March 30, 1989. Hybridoma K2A-2 was designated ATCC No. HB10065, date 0 of deposit was March 30, 1989. The hybridoma cell lines which were found to secrete monoclonal antibodies reactive with Ki2B p21s and the subject of this invention were deposited in the ATCC under the Budapest Treaty. 5 Hybridoma K2B-1 was designated ATCC No. HB10064, date of deposit was March 30, 1989. Hybridoma K2B-2 was designated ATCC No. HB10055, date of deposit was March 29, 1989. Hybridoma K2B-3 was designated ATCC No. HB10066, date 0 of deposit was March 30, 1989. Hybridoma K2B-4 was designated ATCC No. HB10085, date of deposit was March 31, 1989. Hybridoma K2B-5 was designated ATCC No. HB10056, ' date of deposit was March 29, 1989 Hybridoma K2B-6 was designated ATCC No. HB10067, date of deposit was March 30, 1989. Hybridoma K2B-7 was designated ATCC No. HB10091, date of deposit was March 31, 1989. SUMMARY OF THE INVENTION The production of monoclonal antibodies and i munoreactive fragments thereof specific for Harvey, Kirsten and N ras proteins has widespread diagnostic and therapeutic rami ications. Such monoclonal antibodies can be used to detect, quantify, and classify the three ras families of proteins in cells, tissues, and other bodily substances such as serum plasma, urine, effusions, and feces. Thus, these monoclonal antibodies and fragments can be used to diagnose, stage, monitor, and classify ras proteins in normal cells, malignant cells, premalignant cells as well as in a variety of bodily substances. Moreover, these monoclonal antibodies and fragments can be useful as therapeutic tools to deliver drugs, radionuclides, and the like to malignant areas to arrest development. Thus, the subject of this invention is the induction, production, and characterization of monoclonal antibodies (Mabs) that react with the individual H, Ki2A, Ki2B, and N ras p21s. Mabs described in this invention are valuable tools for the detection, quantitation and classification of the individual ras p21s in normal, neoplastic, preneoplastic and dysplastic cells, as well as in bodily fluids, using a variety of techniques. In the case of the anti-H specific Mabs, Balb/c x C57B1/6 mice were immunized on several occasions with a synthetic peptide coupled to carrier protein. Mabs specific for the H ras p21 were raised using a synthetic peptide corresponding to amino acids 163-180 of the H p21. Spleen cells from mice immunized with the H-specific peptide were fused with Sp2/0 mouse myeloma cells and two weeks later culture supernatants from the hybridomas were screened by ELISA. Mabs H-770-1.1.4, H-784-4.7.7, and H-873-3.5.3 were selected because of their reactivity with the H- specific peptide and because of their lack of reactivity with the N-specific peptide. These Mabs were also selected because of their reactivity and specificity for the H ras cellular p21s and because of their lack of reactivity with the other cellular p21s as determined by biochemical assays, such as, im unoblots, immunoprecipitation, immunohistochemistry and immunoassays. Ir. the case of the anti-N specific Mabs Balb/c x C57B1/5 mice were immunized on several occasions with a synthetic peptide coupled to carrier protein. Mabs specific for the N ras p21s were raised using a synthetic peptide corresponding to amino acids 163-180 of the N ras p21. Spleen cells from mice immunized with an N-specific peptide were fused with Sp2/0 mouse myeloma cells. Two weeks later the culture supernatants from the hybridomas were screened by ELISA. Mabs N-821-1.1.9 and N-838-1.1.6 were selected because of their reactivity with the N- specific peptide and because of their lack of reactivity with the H-specific peptide. These Mabs were also selected because of their specificity and reactivity for the N ras cellular p21s and because of their lack of reactivity with other cellular p21s as determined by biochemical assays such as, immunoblots, immunoprecipitation, immunohistochemistry and immunoassays . In the case of Mabs to the Ki2A and Ki2B Balb/c x C57B1/6 mice were immunized with synthetic peptides coupled to carrier proteins. Mabs specific for the Ki2A ras p21s were raised using synthetic peptides corresponding to amino acids 163-180. Spleen cells from mice immunized with the Ki2A-specific peptides were fused with Sρ2/0 mouse myeloma cells. After two weeks the culture supernatants from the hybridomas were screened by ELISA. Mabs were selected because of their reactivity with the Ki2A-specific peptides and because of their lack of reactivity with the H-specific, N-specific, and Ki2B-specific peptides. It is believed that these Mabs have specificity and reactivity for the Ki2A ras cellular p21s and lack reactivity with other cellular p21s as can be determined by immunoassays. Mabs specific for the Ki2B ras p21s were raised using a synthetic peptide corresponding to amino acids 164-175 of the Ki ras p21. Spleen cells from mice immunized with Ki2B-sρecific peptides were fused with Sρ2/0 mouse myeloma cells. After two weeks the culture supernatants from the hybridomas were screened by ELISA. Mabs were selected because of their reactivity with the Ki2B specific peptides and because of their lack of reactivity with the H-specific, N- specific, and Ki2A-specific peptides. It is believed these Mabs have specificity and reactivity for the K12B ras cellular p21s and lack reactivity with other cellular p21s as can be determined by immunoassays. DETAILED DESCRIPTION OF THE INVENTION It should be noted at the outset that the abbreviations H or Ha can be used interchangeably to designate the Harvey family of ras proteins. Similarly, K or Ki can be used interchangeably to designate the Kirsten family of ras proteins and the designations 2A and 2B are used interchangeably with 4A and 4B respectively. Immunizations In the case of Mab H-770-1.1.4, Balb/c x C57B1/6 mouse designated 4607 was immunized with the H- specific peptide coupled to carrier protein Keyhole Limpet Hemacyanin (KLH) via the glutaraldehyde method of A. Kagan et al.. Methods of Hormone Radioirnmunoassay, pp. 327-339 (2d Ed. ) (1979) . Unless stated otherwise, the Kagan method is the preferred method for all coupling/conjugation discussed herein. The H-specific peptide was composed of 18 amino acids corresponding to positions 163-180 of the ras H p21. The structure of the immunizing peptide was as follows: ^^Isoleucine-arginine-glutamine-histidine- lysine-leucine-arginine-lysine-leucine-asparagine- proline-proline-aspartic acid-glutamic acid-serine- glycine-proline-glycine^O. In the case of Mab H-784-4.7.7, Balb/c x C57B1/6 mouse designated 4615 was immunized with the H- specific peptide coupled to carrier protein KLH. In the case of Mab H-873-3.5.3, Balb/c x C57B1/6 mouse designated 4606 was immunized with the H- specific peptide coupled to carrier protein KLH. In the case of Mab N-821-1.1.9, Balb/c x C57B1/6 mouse designated 4486 was immunized with the N- specific peptide coupled to carrier protein Bovine Thyroglobulin (BTG) as discussed above. (0 In the case of Mab N-838-1.1.6, Balb/c x C57B1/6 mouse designated 4480 was immunized with the N- specific peptide coupled to BTG. The N-specific peptide was composed of 18 amino acids corresponding to positions 163-180 of the N ras p21. The structure of the N ras peptide was as follows: l^Isoleucine- arginine—glutamine-tyrosine-arginine-methionine-lysine- lysine-leucine-asparagine-serine-serine-aspartic acid- aspartic acid-glycine-threonine-glutamic acid- glycine 180 . The immunizing N and H-specific peptides were coupled to carrier proteins KLH and BTG respectively to enhance i munogenicity of the peptide. The first inoculation consisted of the peptide-carrier conjugate mixed with Complete Freunds Adjuvant. Total protein inoculated was 500 micrograms. Subsequent inoculations were given at two-week intervals. Three days before fusion mice were given an intraperitoneal inoculation of the appropriate immunogen. In the case of Mabs to the Ki2A p21, Balb/c x C57B1/6 mice designated 5399 and 5191 were immunized with, a peptide corresponding to amino acids 163-180. A peptide corresponding to amino acids 170- 184 of the Ki2A p21 protein can also be used as an immunogen. Once again, peptides were coupled to carrier proteins prior to inoculations. The structures of the Ki2A peptides were as follows: The peptide corresponding to amino acids 163-180 was composed of 18 amino acids and had the following structure: ^-"^Isoleucine-arginine- glutamine-tyrosine-arginine-leucine-lysine-lysine- isoleucine-serine-serine-glutamic acid-glutamic acid- lysine-threonine-proline-glycine-cysteine!80 φ It The peptide corresponding to amino acids 170-184 is composed of 15 amino acids and has the following structure: ^ 7 ^Lysine-isoleucine-serine- lysine-glutamic acid-glutamic acid-lysine-threonine- proline-gl cine-cysteine-valine-lysine-isoleucine- lysine 184 . In the case of Mabs to the Ki2B p2l, Balb/c x C57B1/6 mice designated 5606, 5602 and 6061 were immunized with peptides corresponding to amino acids 164-175. Peptides corresponding to amino acids 163-180 and 168-183 of the Ki2B p21 protein can also be used as an immunogen. Peptides were again coupled to carrier proteins prior to inoculation. The peptide corresponding to amino acid positions 163-180 of the Ki2B ras p21 is composed of 18 amino acids and had the following structure: ^^Isoleucine- arginine-lysine-histidine-lysine- glutamic acid-lysine-methionine-serine-lysine-aspartic acid-glycine-lysine-lysine-lysine-lysine-lysine- lysine 180 . The peptide corresponding to amino acid positions 164-175 of the Ki2B ras p21 is composed of 12 amino acids and had the following structure: ^Arginine- lysine-histidine-lysine-glutamic acid- lysine-methionine-serine-lysine-aspartic acid-glycine- lysine 17 ^. The peptide corresponding to amino acid positions 168-183 of the Ki2B ras p21 is composed of 16 amino acids and has the following structure: ^-^^Glutamic acid-lysine-methionine-serine-lysine- aspart ic acid-glycine-lysine-lysine-lysine-lysine- lysine-lysine-serine-lysine-threonine 8 . The Ki2A and Ki2B-specific peptides were coupled to carrier proteins KLH or BTG to enhance immunogenicity of the peptide. The first inoculation it consisted of the peptide carrier conjugate mixed with Complete Freunds Adjuvant. Total protein inoculated was 500 micrograms. Subsequent inoculations were given at two-week intervals. Three days before fusion mice were given an intraperitoneal inoculation of the appropriate immunogen. Hybridoma Methodology Three days after an intraperitoneal boost the spleens of the appropriate immune mice were removed and fused with the non-secretor myeloma cell Sp2/0. Spleen cell suspensions were prepared in serumless DMEM-high glucose medium and mixed with myeloma cells at a ratio of 4:1. This cell mixture was centrifuged at 1200 x g for 10 minutes at room temperature. After removal of the supernatant, the cells were resuspended by gently tapping the tube. The fiαsion procedure was initiated by adding 1.0 ml of 45% w/v polyethylene glycol 3350 (^aker) at 37°C over a 30-second period. The cells were occasionally mixed with a pipette tip for 90 seconds and 5 ml of serumless DMEM-high glucose medium was added over a 3-minute period. This was followed by the addition of 14 ml of DMEM-high glucose supplemented with 10% fetal calf serum, L- glutamine, hypoxanthine, aminopterin and thymidine (referred to as HAT medium) . The HAT medium was added over a 1-minute period. Appropriate volumes of HAT medium were added to cells and then the cells were centrifuged at 800 x g for 7 minutes at room temperature. Supernatants were aspirated and the.cell pellet disrupted with 10 ml of HAT medium. Peritoneal cells from Balb/c x C57B1/6 were added and the final volume adjusted so that two hundred thousand spleen cells were dispensed to each well. Approximately 14 days later, tissue culture supernatants from wells containing hybridoma colonies were tested by ELISA for the desired reactivity with peptides conjugated to carrier proteins. Screening Procedures and ELISA Protocol For screening purposes, the H-specific peptide described above was conjugated to the BTG carrier protein while the N-specific peptide was conjugated to the KLH carrier protein. The rationale for coupling peptides to different carrier proteins for immunization and screening was to avoid selecting antibodies reactive with the carrier protein. The same rationale applied to the selection of carrier to conjugate to the Ki2A and Ki2B specific peptides. Prior to screening hybridoma supernatants, 500 mg of the peptide-carrier conjugate was dispensed to 96 well microtiter plates for overnight incubation at 37°C. After incubation, plates were washed and unbound sites on the plates were blocked with bovine serum albumin (BSA) . At the time of screening hybridoma supernatants, 50 microliters of fluid was added to wells containing the appropriate peptide-carrier conjugate. Hybridoma supernatants were allowed to incubate overnight at 4°C. Supernatants were removed the next day and wells washed with the BSA solution. Each well subsequently received 50 microliters of goat anti-mouse IgG antibody conjugated to horseradish peroxidase (GAMHRP) diluted in BSA phosphate buffered saline (PBS) . Wells were incubated for 60 minutes at 37°C. GAMHRP was removed after incubation and wells were washed three times with PBS-BSA mixtures. The presence of bound GAMHRP was determined by adding 50 microliters of the substrate o-phenylenediamine (OPD) in phosphate buffer containing 0.15% hydrogen peroxide. HRP, in combination with its substrate OPD, results in a yellow colored product. Development of the yellow product was allowed to occur at room temperature for 15 minutes. The enzymatic reaction was terminated by the addition of 50 microliters of 4.5% M sulfuric acid. Measurement of the resultant reaction product was accomplished by determining optical density at 488 nm on a Nunc Plate Reader (Nunc, Inc., Newbury Park, CA) . Presence of the yellow color in the wells indicated that antibodies of interest were present in the hybridoma supernatants. The more antibody present in the culture fluid, the higher the optical density. Using the above-described assay, Mabs H-770- 1.1.4, H-784-4.7.7, and H-873-3.5.3 were found to be reactive with the H-specific peptide coupled to carrier protein and not reactive with the N, Ki2A and Ki2B specific peptides also coupled to carrier protein. Mabs N-821-1.1.9 and N-838-1.1.6 were found to be reactive with the N ras specific peptide coupled to carrier protein and not reactive with the H, Ki2A and Ki2B peptides coupled to carrier protein. Mabs Ki2A-l and Ki2A-2 were found to be reactive with the Ki2A specific peptide and not reactive with the H, N and Ki2B specific peptides also coupled to carrier protei . Mabs Ki2B-l, Ki2B-2, Ki2B-3, Ki2B-4, Ki2-5, Ki2B- 6 and Ki2B-7 were found to be reactive with the Ki2B specific peptide (amino acids 164-175) and not reactive with the H, N, and Ki2A specific peptides also coupled to carrier proteins. Specificity of Mabs for Peptides of Interest In the next series of experiments, the anti-N and anti-H Mabs were tested for specificity with free peptides (i.e., peptides not coupled to carrier proteins) to ensure that the Mabs were specific for the peptides of interest and not reactive with the bond attaching the carrier protein to the peptide. Table 1 summarizes ELISA results of testing the anti-H and anti-N Mabs against the H, N, Ki2A and Ki2B specific peptides. Table 1 Ki2A Ki2B Peptide Peptide H- N- (AminoAcids (AminoAcids Hybridoma Pept Lde Peptide 163-180) 163-180) H-770-1.1.4 + - H-7B4-4.7.7 + - H-873-3.5.3 + - N-821-1.1.9 - + N-838-1.1.6 — + These results show that Mabs raised against the H peptide were specific for that peptide and not with the N peptide, Ki2A peptide or with the Ki2B peptide (amino acids 163-180) . Results also demonstrated that the Mabs raised against the N peptide were specific for that peptide and not reactive or cross-reactive with the H peptide, the Ki2A peptide or with the Ki2B peptide (amino acids 163-180) . Similarly, Mabs for Ki2A and Ki2B are tested for specificity with peptides not coupled to carrier proteins. It is believed that Ki2A and Ki2B Mabs are specific for the peptides of interest and not reactive with the bond attaching the carrier protein to the peptide. Reactivity and Specificity of the Anti-Peptide Mabs for Cellular ras Proteins Immunoprecipitation and western blotting were used to determine whether the Mabs raised against the H and N ras peptides reacted with and were specific for the cellular H and N ras p21s. Experiments were performed using the following four cell lines: 1. Cell line designated 3T3-Hras (also designated PSV-13) overexpressed the H ras p21 protein. 2. Cell line designated 3T3-Nras overexpressed the N ras p21 protein. 3. Cell line (KNRK) designated 3T3-Ki2A expressed the viral form of the Ki p21 protein. 4. Cell line (SW480) designated 3T3-Ki2B expressed the cellular form of the Ki ras protein. Nonradioactive extracts of the above-mentioned cells were incubated with an anti-H Mab, anti-N Mab, or an anti-p21 Mab designated Ras 10 for 1 hour. The Ras 10 anti-p21 Mab reacted with normal and oncogenic forms of the ras p21 proteins, as discussed in co- pending patent application having Serial Number 071,175, filed on July 8, 1987. The anti-p21 monoclonal antibodies described therein were generated by using a recombinant Ha-ras p-21 protein having an arginine amino acid as position 12 instead of glycine which is present in the normal Ha-ras p21 protein. Hybridomas secreting these anti-p21 monoclonal antibodies were designated ras 8, ras 10, and ras 11, all of which were deposited in the American Type Tissue Culture Collection (ATCC) under the Budapest Treaty. Hybridoma ras 8 was designated HB-9428, hybridoma ras 10 was designated HB-9426, and hybridoma ras 11 was designated HB-9427. After incubation, a complex of rabbit anti-mouse Ig was added to protein A Sepharose for 30 minutes at 4°C to capture the anti-H, anti-N, and anti-p21 Mabs. After the 1 hour incubation, the samples were centrifuged and the resulting pellets washed. After the final wash, 50 microliters of a sodium dodecylsulfate reducing buffer was added to the pellet and heated for 5 minutes at 100°C. This material was then applied to a 12.5% polyacrylamide gel. Cellular proteins were separated according to molecular weight by running an electric current through the gel. After this electrophoresis procedure, the proteins were electrophoretically transferred to nitrocellulose membranes which had been blocked with PBS containing 5% BSA. The membranes were incubated for 1 hour with either an anti-p21 Mab ras 10 or mouse serum which served as a negative control. After incubation with Ras 10 or a negative control antibody, membranes were washed three times with PBS-NP-40. Membranes were then incubated with an anti-mouse immunoglobulin coupled to HRP for 1 hour to detect the mouse Mabs. Membranes were then washed three times with PBS-NP-40 and incubated with 4-chloro-l-naphthol substrate to complete the reaction. Experiments demonstrated that the anti-H Mabs were able -to immunoprecipitate or capture the cellular H ras p21 from the 3T3-Hras cell line but did not react with cellular p21s from the N ras family, Ki2A family, or Ki2B family. Anti-N ras Mabs specifically immunoprecipitated or captured the cellular N ras p21 but did not react with the other cellular H, Ki2A, and Ki2B p21 protein families. The broadly reactive anti-p21 Mab Ras 10 reacted with p21 proteins from all the cells, whereas the negative control antibody did not react with any of the cellular ras p21s. These results are summarized below in Table 2. Table 2 Cell Line Hybridoma 3T3Hras 3T3Nras 3T3Ki2Aras 3T3Ki2Bras H-770-1. 1 .4 + H-784-4.7 .7 + H-873-3.5 .3 + N-821-1. 1. S + N-838-1.1.6 + Ras 10 • * ■ + Negative Control - (mouse serum) In a separate series of experiments the anti-H and anti-N Mabs were evaluated for their ability to detect the cellular H and N ras p21s without prior immunoprecipitation. To do this, cell extracts were applied directly to the 12.5% gel and electrophoresed to separate proteins. Proteins were transferred to nitrocellulose membranes and reacted with either the anti-H, anti-N or the anti-p21 Mab Ras 10. The mouse antibodies were detected as described above. The results demonstrated that the anti-H Mabs reacted with only the cellular H ras p21s, whereas the anti-N Mabs reacted with only the cellular R ras p21s. It is not clear whether the Kirsten Mabs react in an immunoblot format. The ability to react in a particular analytical format is believed to be a function of how the epitope is presented. Thus, two antibodies may recognize the same epitope and, yet, react differently in, for example, an immunoblot format. Epitope specificity can be determined using conventional techniques such as a competition immunoassay.

Claims: CLAIMS 1. Monoclonal antibodies reactive with an epitope on H ras proteins and not reactive with an epitope on N ras proteins, Ki2A ras proteins, or Ki2B ras proteins. 2. Monoclonal antibodies reactive with an epitope on N ras proteins and not reactive with an epitope on H ras proteins, Ki2A ras proteins, or Ki2B ras proteins. 3. Monoclonal antibodies reactive with an epitope on Ki2A ras proteins and not reactive with an epitope on N ras proteins, Ki2B ras proteins, or H ras proteins. 4. Monoclonal antibodies reactive with an epitope on Ki2B ras proteins and not reactive with an epitope on N ras proteins, Ki2A ras proteins, or H ras proteins. 5. Monoclonal antibodies reactive with an epitope on H ras proteins and not reactive with an epitope on N ras proteins, Ki2A ras proteins, or Ki2B ras proteins, the epitope of the H ras protein is also bound by a reference antibody having ATCC accession number HB9673, HB9675 or HB9674 as determined by immunoassay. 6. Monoclonal antibodies reactive with an epitope on N ras proteins and not reactive with an epitope on H ras proteins, Ki2A ras proteins, or Ki2B ras proteins, the epitope of the N ras proteins is also bound by a reference antibody having ATCC accession number HB9671 or HB9672 as determined by immunoassa . 7. Monoclonal antibodies reactive with an epitope on Ki2A ras proteins and not reactive with an epitope on N ras proteins, Ki2B ras proteins, or H ras proteins, the epitope of the Ki2A ras proteins is also ZP bound by a reference antibody having ATCC accession number HB10072 or HB10065, as determined by immunoassay. 8. Monoclonal antibodies reactive with an 5 epitope on Ki2B ras proteins and not reactive with an epitope on N ras proteins, Ki2A ras proteins, or H ras proteins, the epitope of the Ki2B ras proteins is also bound by a reference antibody having ATCC Accession Number HB10064, HB10055, HB10066, HB10085, HB10056, 10 HB10067, or HB10091, as determined by immunoassay. 9. The hybridoma cell line deposited under ATCC Accession Number HB9673, HB9674 or HB9675. 10. The hybridoma cell line deposited under ATCC Accession Number HB9671 or HB9672. 1.5 11. The hybridoma cell line deposited under ATCC Accession Number HB10072 or HB10065. 12. The hybridoma cell line deposited under ATCC Accession Number HB10064, HB10055, HB10066, HB10085, HB10056, HB10067 or HB10091. 20 * 13. An immunoreactive fragment of the antibody of claim 1 or 5. 1 . An immunoreactive fragment of the antibody of claim 2 or 6. 15. An immunoreactive fragment of the antibody 2 * 5 of claim 3 or 7. 16. An immunoreactive fragment of the antibody of claim 4 or 8. 17. A method for detecting the presence of Harvey Ras proteins in normal cells, malignant cells, 3TCt premalignant cells and bodily substances comprising contacting the cells or bodily substances with a monoclonal antibody of claim 1 and determining whether antibody binding has occurred. 18. A method for detecting the presence of N 35 Ras proteins in normal cells, malignant cells. . premalignant cells and bodily substances comprising contacting the cells or bodily substances with a monoclonal antibody of claim 2 and determining whether antibody binding has occurred. 19. A method for detecting the presence of Kirsten 2A Ras proteins in normal cells, malignant cells, premalignant cells and bodily substances comprising contacting the cells or bodily substances with a monoclonal antibody of claim 3 and determining whether antibody binding lias occurred. 20. A method for detecting the presence of Kirsten 2B Ras proteins in normal cells, malignant cells, premalignant cells and bodily substances comprising contacting the cells or bodily substances with a monoclonal antibody of claim 4 and determining whether antibody binding has occurred.

Inventors: CARNEY WALTER PATRICK, CARNEY, WALTER, PATRICK
Applicants: DU PONT, E.I. DU PONT DE NEMOURS AND COMPANY
Application year: 1989
Main classification: C07K15
IPC codes: C07K16, C12N15