Mapping and Regulation of the Tumor-associated Epitope Recognized by Monoclonal Antibody RS-11 (2000)

We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568–1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the α-helix 2B domain of keratin 18 in which two amino acids (Leu366 and Lys370) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha. We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568–1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the α-helix 2B domain of keratin 18 in which two amino acids (Leu366 and Lys370) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha. monoclonal antibody glutathione S-transferase transforming growth factor epidermal growth factor polymerase chain reaction SDS-polyacrylamide gel electrophoresis phosphate-buffered saline enzyme-linked immunosorbent assay synthetic peptide Many monoclonal antibodies (mAbs)1 have been raised against cancer cell preparations and have played a key role in the process of identification of antigens expressed uniquely on cancer cells. These mAbs help define specific differences between cancer and normal cells and elucidate cellular alterations associated with malignancy. Antigens overexpressed on cancer cells most commonly represent tumor-associated antigens. Many cDNAs that encode tumor-associated antigens have been cloned, including the folate-binding protein, a variable number of tandem repeats, and the transmembrane 4 superfamily proteins (1Coney L.R. Tomassetti A. Carayannopoulos L. Frasca V. Kamen B.A. Colnaghi M.I. Zurawski Jr., V.R. Cancer Res. 1991; 51: 6125-6132PubMed Google Scholar, 2Hareuveni M. Tsarfaty I. Zaretsky J. Kotkes P. Horev J. Zrihan S. Weiss M. Green S. Lathe R. Keydar I. Eur. J. Biochem. 1990; 189: 475-486Crossref PubMed Scopus (63) Google Scholar, 3Hotta H. Ross A.H. Huebner K. Isobe M. Wendeborn S. Chao M.V. Ricciardi R.P. Tsujimoto Y. Croce C.M. Koprowski H. Cancer Res. 1988; 48: 2955-2962PubMed Google Scholar, 4Szala S. Kasai Y. Steplewski Z. Rodeck U. Koprowski H. Linnenbach A.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6833-6837Crossref PubMed Scopus (134) Google Scholar, 5Marken J.S. Schieven G.L. Hellstrom I. Hellstrom K.E. Aruffo A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3503-3507Crossref PubMed Scopus (125) Google Scholar). The role of most of these antigens in the process of malignant transformation and tumor progression remains unclear. Intermediate filaments represent a superfamily of 10-nm fibers that are ubiquitous in multicellular eukaryotes. Each tissue type characteristically expresses specific intermediate filament proteins. All intermediate filament proteins possess a similar protein domain structure consisting of a central α-helical rod domain flanked by non-helical head and tail domains. The α-helical domain is conserved in size and secondary structure among all intermediate filament proteins, and the α-helical segments generally pair to form coiled coil dimers. Keratins, which are typically expressed in epithelial cells, assemble as obligate noncovalent heteropolymers from type I (acidic) and type II (basic or neutral) filaments. Keratin 8 and keratin 18 are the major components of intermediate filaments in simple or single layer epithelial tissue. They are the first intermediate filaments expressed in the embryo (6Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1281) Google Scholar, 7McLean W.H. Lane E.B. Curr. Opin. Cell Biol. 1995; 7: 118-125Crossref PubMed Scopus (219) Google Scholar). Of the more than 20 proteins in the keratin family, these two keratins are the ones that are most commonly overexpressed in association with malignant transformation. Experimental evidence suggests that keratin 8 and keratin 18 play a role in cell migration, invasion, apoptosis, signal transduction, and drug sensitivity (8Hendrix M.J. Seftor E.A. Chu Y.W. Seftor R.E. Nagle R.B. McDaniel K.M. Leong S.P. Yohem K.H. Leibovitz A.M. Meyskens Jr., F.L. J. Natl. Cancer Inst. 1992; 84: 165-174Crossref PubMed Scopus (161) Google Scholar, 9Chu Y.W. Runyan R.B. Oshima R.G. Hendrix M.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4261-4265Crossref PubMed Scopus (151) Google Scholar, 10Bauman P.A. Dalton W.S. Anderson J.M. Cress A.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5311-5314Crossref PubMed Scopus (73) Google Scholar, 11Parekh H.K. Simpkins H. Cancer Res. 1995; 55: 5203-5206PubMed Google Scholar). Many alterations in keratin 8 and keratin 18 expression have been identified in human tumors. Keratin 8 and keratin 18 expression are increased in mammary tumors (12Trask D.K. Band V. Zajchowski D.A. Yaswen P. Suh T. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2319-2323Crossref PubMed Scopus (169) Google Scholar), transitional cell carcinomas (13Schaafsma H.E. Ramaekers F.C. van Muijen G.N. Lane E.B. Leigh I.M. Robben H. Huijsmans A. Ooms E.C. Ruiter D.J. Am. J. Pathol. 1990; 136: 329-343PubMed Google Scholar, 14Schaafsma H.E. Ramaekers F.C. van Muijen G.N. Robben H. Lane E.B. Leigh I.M. Ooms E.C. Schalken J.A. van Moorselaar R.J. Ruiter D.J. Am. J. Pathol. 1991; 139: 1389-1400PubMed Google Scholar), and squamous cell carcinomas (15Schaafsma H.E. Van Der Velden L.A. Manni J.J. Peters H. Link M. Rutter D.J. Ramaekers F.C. J. Pathol. 1993; 170: 77-86Crossref PubMed Scopus (87) Google Scholar). Ubiquitin-conjugated keratin 8 fragments appear to accumulate in colorectal carcinomas (16Nishibori H. Matsuno Y. Iwaya M. Osada T. Kubomura N. Iwamatsu A. Kohno H. Sato S. Kitajima M. Hirohashi S. Cancer Res. 1996; 56: 2752-2757PubMed Google Scholar). Keratin 8 and 18 levels also correlate with tumor stage and prognosis (17Schaller G. Fuchs I. Pritze W. Ebert A. Herbst H. Pantel K. Weitzel H. Lengyel E. Clin. Cancer Res. 1996; 2: 1879-1885PubMed Google Scholar, 18Sundstrom B.E. Stigbrand T.I. Int. J. Biol. Markers. 1994; 9: 102-108Crossref PubMed Scopus (71) Google Scholar). We have previously described a rat mAb, RS-11, that we raised against a syngeneic rat bladder carcinoma (19Eto H. Saya H. Nakata M. Mizoguchi A. Kamidono S. Int. J. Cancer. 1989; 44: 454-459Crossref PubMed Scopus (2) Google Scholar). Immunocytochemical analyses have demonstrated that RS-11 reacts with rat, mouse, dog, and human bladder tumor cell lines as well as several non-bladder tumor cell lines. RS-11 reacts with a majority of low grade and well differentiated bladder tumors but reacts less commonly with advanced tumors. In contrast, RS-11 does not react with any normal rat tissues. Furthermore, RS-11 does not react with most normal human tissues examined immunohistochemically, and accordingly the antigen recognized by RS-11 appears to be a tumor-associated antigen common to several species. Immunofluorescent examination of malignant cells stained with RS-11 reveals a cytoplasmic, filamentous expression pattern (19Eto H. Saya H. Nakata M. Mizoguchi A. Kamidono S. Int. J. Cancer. 1989; 44: 454-459Crossref PubMed Scopus (2) Google Scholar). Biochemical analyses performed to further characterize the nature of the antigen suggest that the epitope is recognized on a cytoskeletal protein common to a wide variety of mammalian cells (19Eto H. Saya H. Nakata M. Mizoguchi A. Kamidono S. Int. J. Cancer. 1989; 44: 454-459Crossref PubMed Scopus (2) Google Scholar). In the present study we have cloned the RS-11 antigen from a cDNA library, and DNA sequence analysis indicates that the epitope is expressed on keratin 18. We have examined RS-11 interaction with a series of GST fusion proteins and synthetic peptides and have determined that the RS-11 epitope resides within a region of seven amino acids located on the end of the α-helix 2B domain of keratin 18. We have also demonstrated that whereas the RS-11 antigen is present in human primary cultured hepatocytes, the RS-11 epitope is not available for RS-11 immune recognition. The epitope becomes immunoreactive with RS-11 upon malignant transformation or exposure of the hepatocytes to TGFα or EGF. The KoTCC-1 human bladder carcinoma cell line has been previously described (19Eto H. Saya H. Nakata M. Mizoguchi A. Kamidono S. Int. J. Cancer. 1989; 44: 454-459Crossref PubMed Scopus (2) Google Scholar). T24 and HT1197 human bladder carcinoma and SK-HEP-1 human hepatocellular carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Anti-keratin 8 mAb (M20, Sigma), anti-keratin 18 mAb (CY90, Sigma), fluorescein isothiocyanate-conjugated anti-pan keratin mAb (C-11, Sigma), anti-proliferating cell nuclear antigen mAb (DC-10, Sigma), anti-GST mAb (Zymed Laboratories Inc., South San Francisco, CA), horseradish peroxidase-conjugated anti-rat IgM mouse mAb (Zymed Laboratories Inc.), biotinylated anti-rat κ and λ light chain antibody (Sigma), and avidin-conjugated Cy3 (Sigma) were used in this study. We used mAb RS-11 to screen 1 × 106 phage clones from a λgt11 phage expression library prepared from HeLa cDNA (CLONTECH, Palo Alto, CA). The immunoblotting conditions used for screening are described under “Immunoblot Analysis.” A single positive clone was identified, and after iterative screening to purify this positive plaque, the cDNA insert of the positive clone was PCR-amplified using specific oligonucleotide primers for the library insert (Forward, 5′-GACTCCTGGAGCCCG-3′; Reverse, 5′-GGTAGCGACCGGCGC-3′). The PCR fragment was directly sequenced with a 373S DNA sequencer (Applied Biosystems) using the same primers. Fragments of cDNA of the positive clone (ph/RS-11) were prepared by PCR amplification using primers with a unique 5′EcoRI site and another unique 3′ HindIII site (see Table I). PCR products were digested with EcoRI andHindIII and subcloned into the corresponding sites of the pGEX-2TH (kindly provided by Dr. Hiroshi Maruta), which is a modification of pGEX-2T and directs bacterial overexpression of foreign polypeptides fused to the carboxyl terminus of GST in the presence of isopropylthio-β-galactosidase. The correct sequences of all pGEX-2TH/RS-11 constructs were confirmed by DNA sequencing.Escherichia coli strain INVαF′ (Invitrogen, Carlsbad, CA) transformed with these constructs was cultured overnight and then diluted 10-fold and allowed to grow for 2 h. Protein production was then induced with 0.25 mmisopropylthio-β-galactosidase for 4 h.Table IOligonucleotides used for PCR amplification to create GST fusion protein constructsGST fusion constructKeratin/fragmentForward oligonucleotideReverse oligonucleotideFPΔ1K181-aK18, keratin 18; K8, keratin 8./178–3211-bAmino acid (aa) residues included in GST fusion protein.CGCGAATTCGTGGAGAACGACATCCATGGGTCAAAGCTTTCTCCAAGCTGGCCTTCAGATFPΔ2K18/178–338CGCGAATTCGTGGAGAACGACATCCATGGGGATAAGCTTGAGCTGCTCCATCTGTAFPΔ3K18/178–351CGCGAATTCGTGGAGAACGACATCCATGGGTCTAAGCTTGTCTGTGCCAGCTCTGAFPΔ4K18/178–364CGCGAATTCGTGGAGAACGACATCCATGGGTTCAAAGCTTCCTTCATACTCCTGGGCCFPΔ5K18/178–370CGCGAATTCGTGGAGAACGACATCCATGGGCTTAAGCTTGATGTTCAGCAGGGCCTCATAFPΔ6K18/178–375CGCGAATTCGTGGAGAACGACATCCATGGGTCTCAAGCTTCAGCTTGACCTTGATGTTCAFPΔ7K18/178–383CGCGAATTCGTGGAGAACGACATCCATGGGTTTGAAGCTTAGGCGGCGGTAGGTGGCGATFPΔ8K18/178–383 + 11 aaCGCGAATTCGTGGAGAACGACATCCATGGGTTCAAGCTTATTAAGCCAGCAGAAGCAGGGFPΔ9K18/178–383 + 19 aaCGCGAATTCGTGGAGAACGACATCCATGGGGAAAAGCTTAATGACACCAAAGTTCTGAGGFPΔ10K18/241–383 + 19 aaGATGAATTCAAATCTCAGGACCTCGCCAAGGAAAAGCTTAATGACACCAAAGTTCTGAGGFPΔ11K18/178–402CGCGAATTCGTGGAGAACGACATCCATGGGGATAAGCTTCATGGAGTTGCTGCTGTCCAAGST/Keratin 18 (full-length)K18/1–430CCGGAATTCATGAGCTTCACCACTCGCTTCTAAGCTTTTAATGCCTCAGAACTTTGGTGST/Keratin 8 (full-length)K8/1–483TGCCGGATCCATGTCCATCAGGGTGACGCGAATTCTCACTTGGGCAGGACGTCAGA1-a K18, keratin 18; K8, keratin 8.1-b Amino acid (aa) residues included in GST fusion protein. Open table in a new tab Full-length keratin 8 and keratin 18 cDNAs were prepared from T24 (human bladder cancer cell line) by reverse transcription-PCR amplification. The primers contain uniqueBamHI and EcoRI sites for keratin 8 and uniqueEcoRI and HindIII sites for keratin 18 (see Table I). PCR products were subcloned in the pGEX-2TH as described above. Bacteria cultured with isopropylthio-β-galactosidase were pelleted and suspended in lysis buffer (50 mm Tris-HCl (pH 8.0), 100 mm NaCl, 1 mm EDTA, and 5 mm β-mercaptoethanol) containing 1 mg/ml lysozyme at 4 °C for 15 min. The suspension was centrifuged, and the pellet was sonicated five times (30 s each time). After centrifugation, the pellet was washed once with a high salt buffer (1 m NaCl, 10 mm EDTA, 50 mm Tris-HCl (pH 8.0), 1% Nonidet P-40, 5 mm β-mercaptoethanol) and then with lysis buffer. The pellet was finally dissolved in 8 m urea solution containing 10 mm Tris-HCl (pH 8.0), 2 mm EDTA, 5 mm dithiothreitol and centrifuged at 10,000 ×g for 15 min. The supernatant was diluted 1:1 with 2× SDS sample buffer (4% SDS) and subjected to immunoblot analysis. Bacteria expressing truncated GST fusion proteins of the ph/RS-11 clone and partially purified recombinant keratin-GST fusion proteins were dissolved with 2× SDS sample buffer and separated by SDS-PAGE, and then the proteins were transferred onto a nitrocellulose membrane by electroblotting at 4 °C. The nitrocellulose sheets were incubated with PBS containing 5% nonfat milk for 2 h and then incubated with a 1:600 dilution of RS-11 ascites or 0.1–5.0 μg/ml mAbs for 1 h. After rinsing for 30 min with PBS containing 1% nonfat milk and 0.1% Tween 20, the filters were incubated with a 1:800 dilution of horseradish peroxidase-conjugated anti-rat IgM mouse mAb (Zymed Laboratories Inc.) or a 1:8000 dilution of horseradish peroxidase-conjugated anti-mouse immunoglobulins (Amersham Pharmacia Biotech). After rinsing for 30 min, specific proteins were detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech) and then exposed to RX film (Fuji, Tokyo, Japan). For immunoblot analyses of cultured cell protein lysates, cells were lysed with PBS (pH 7.4) containing 1% Triton X-100, 0.6 mKCl, 10 mm MgCl2, 1 mmphenylmethylsulfonyl fluoride, 1% mercaptoethanol and subjected to centrifugation. The insoluble residue was dissolved again with 2× SDS sample buffer (4% SDS) and subjected to SDS-PAGE, and immunoblot analysis was performed. A series of 20–23-residue peptides overlapping each other by 15–20 amino acids and covering the ph/RS-11 clone were synthesized (Sawady, Tokyo, Japan) (see Fig. 5). The peptides were reconstituted with dimethyl sulfoxide at a concentration of 5–10 mg/ml and diluted with 0.1 m NaHCO3 buffer (pH 9.6) at a concentration of 10–20 μg/ml for antigen coating to 96-well plates (PolySorp, Nunc, Naperville, IL). The plates were coated with synthetic peptides overnight at 4 °C. After blocking for 1 h with PBS containing 1% bovine serum albumin, the plates were incubated with a 1:1000 dilution of RS-11 ascites. After three rinses with PBS for 15 min, the plates were incubated with a 1:300 dilution of horseradish peroxidase-conjugated anti-rat IgM mouse mAb for 1 h. After three more rinses, enzyme substrate (citrate buffer (pH 5.0) containing 0.4 mg/ml o-phenylenediamine and 0.02% H2O2) was added to the wells, and the absorbance was determined at 492 nm on an ELISA reader. Human hepatocytes were isolated with perfusates I and II as described previously with minor modifications (20Li A.P. Roque M.A. Beck D.J. Kaminski D.L. J. Tissue Cult. Methods. 1992; 14: 139-146Crossref Scopus (102) Google Scholar). Briefly, fresh human liver specimens from the operating room were perfused with perfusate I followed by perfusate II at 40 °C. The digested specimens were gently agitated in perfusate II to release the dissociated hepatocytes, filtered through four layers of gauze, and centrifuged at 50 × g for 5 min. Dissociated hepatocytes were washed three times in perfusate II and resuspended in William's medium E containing 10% bovine serum albumin, insulin, transferrin, selenium, trace elements, dexamethasone, linoleic acid, linolenic acid, glucagon, nicotinamide, penicillin, streptomycin, and fungizone, and seeded on collagen-coated glass coverslips in 12-well plates at a density of 3 × 104 cells per well. 12-well plates were coated overnight with 5 μg/cm2 rat tail collagen (Collaborative Biomedical Products, Bedford, MA). Hepatocytes were incubated at 37 °C, and the medium was changed 2 h after plating, the following morning, and subsequently every 2 days. EGF (Sigma), TGFα (Intergen, Purchase, NY), and tyrosine kinase inhibitor AG-494 (Biomol, were added h after at of 20 20 and 20 Cells were subsequently with for 20 min at 37 °C and then with 0.1% Triton for 20 min at room After blocking for 1 h with PBS containing 1% bovine serum albumin, the cells were incubated with a dilution of RS-11 ascites for 1 h at room After three rinses with the cells were incubated with a dilution of biotinylated anti-rat κ and λ light chain After three rinses with the cells were incubated with a 1:800 dilution of avidin-conjugated Cy3 and a dilution of fluorescein isothiocyanate-conjugated anti-pan keratin We screened a λgt11 phage expression library prepared from HeLa cDNA and identified clone that reacts with RS-11. of were to this phage phage contains a pair cDNA to as which was PCR using oligonucleotide primers that the DNA sequence analysis of the PCR fragment revealed that ph/RS-11 is of sequences from human keratin 18. The first of ph/RS-11 are to a of the keratin 18 sequence (nucleotides is based on the sequence by R.G. G. PubMed Scopus Google and the of ph/RS-11 are to a sequence to a of keratin 18 cDNA (nucleotides other intermediate keratin 18 contains a non-helical head an α-helical rod and a non-helical tail The first of ph/RS-11 a sequence of keratin 18 of the α-helical The of ph/RS-11 the reverse sequence of the region and 3′ region of the keratin 18 of the reveals that expressed sequence contain sequences to the of also the that RS-11 reacts with an epitope expressed on an intermediate filament (19Eto H. Saya H. Nakata M. Mizoguchi A. Kamidono S. Int. J. Cancer. 1989; 44: 454-459Crossref PubMed Scopus (2) Google Scholar). These of cultured cells using RS-11, which a filamentous pattern similar to that using antibodies against keratin and other cytoskeletal proteins and filament protein. from also that the RS-11 antigen is in Triton These suggest that the RS-11 epitope is expressed on an intermediate filament and are with RS-11 immunoreactivity with a phage clone a cDNA insert that a of keratin 18. The unique DNA sequence of the ph/RS-11 insert raised the that the RS-11 epitope is by a tumor-associated end is to the keratin 18 sequence and 3′ end is to the 3′ end of the ph/RS-11 we examined tumor cells for containing the unique in ph/RS-11 which the keratin 18 is by the keratin the protein from this unique represent an antigen expressed in tumor cells. We performed PCR amplification on cDNA prepared from several cell lines using oligonucleotide primers that the unique that in We were to amplification of a correct size fragment not the λgt11 library from which we isolated ph/RS-11 was prepared from HeLa cells, we also PCR HeLa cell cDNA using these primers but again not an amplification the sensitivity of this we oligonucleotide primers to two of PCR amplification. this a amplification from several human tumor cell this also a similar amplification from normal human cDNA not We also used a oligonucleotide that is to the keratin 18 as a for a analysis of tumor cell line and not any not These suggest that ph/RS-11 does not represent a unique that is overexpressed in tumor cells but an of the λgt11 library that the RS-11 epitope is present on human keratin 18 and to more map the of the we prepared several GST fusion proteins in E. I and We PCR segments of ph/RS-11 using several of oligonucleotide primers and cloned the amplification into The correct sequence of each GST fusion protein was confirmed by DNA sequence analysis. protein were prepared from E. coli that been transformed with each GST fusion protein The were separated by in two gel was stained with and the other was used for immunoblotting with RS-11. GST fusion proteins were expressed in E. coli and as major These proteins were with anti-GST antibody by immunoblotting not with RS-11 demonstrated that RS-11 reacts with a protein by ph/RS-11 and expressed as a GST fusion protein. this system we examined RS-11 immunoreactivity with proteins consisting of GST fused to truncated of the protein. These suggest that amino acids are for the of the RS-11 epitope expressed as a GST fusion protein. RS-11 reacts with and 3 for and are not RS-11 also reacts with which is a GST fusion protein containing amino acids of keratin 18. fusion protein does not contain any of the amino acids by the ph/RS-11 and contains the same number of amino acids as the protein by RS-11 also with GST fusion proteins containing the of keratin evidence that the RS-11 epitope is expressed on keratin 18. RS-11 not react with or other GST fusion proteins that were used as and These that keratin 18 amino acids are to the of the RS-11 epitope expressed as of a GST fusion protein. the unique carboxyl terminus of the protein by ph/RS-11 does not appear to to the RS-11 RS-11 reacts with fusion proteins that not contain this peptide Furthermore, the antibody reacts with GST fused to either keratin 18 or the keratin of 18 fusion proteins with RS-11. protein prepared from E. coli transformed with 18 constructs were separated by and stained with GST fusion proteins were expressed and as major prepared coli expressing 18 fusion proteins were separated by SDS-PAGE, transferred to nitrocellulose and examined for immunoreactivity to RS-11 by further map the epitope recognized by RS-11 we constructed a of synthetic peptides with amino acid sequences of keratin 18 to be immunoreactive with RS-11 based on the GST fusion protein The amino acid sequence of this region is conserved among intermediate filaments peptides are of overlapping amino acid sequences in to amino acid residues 4 but not of these synthetic peptides residues We examined the immunoreactivity of RS-11 with each of the peptides in an RS-11 with two and The RS-11 immunoreactivity with with the of immunoreactivity with suggests that amino acids are to the of the RS-11 epitope in the of a synthetic the RS-11 immunoreactivity with with the of immunoreactivity with suggest that amino acids are to the of the RS-11 epitope in the of a synthetic described the epitope using the GST fusion proteins suggest that amino acids are for the of the RS-11 epitope in the of a fusion protein. is that the epitope is on the of amino acid that RS-11 reacts with GST fusion protein which does not contain amino acids residue the GST fusion protein and the synthetic peptide is to that the RS-11 epitope is located between amino acids and Amino acids be to this epitope in the of a synthetic whereas are not in the of the GST fusion protein. Of RS-11 not react with a synthetic peptide containing the unique carboxyl terminus by the of These further suggest that the of ph/RS-11 is an of the library and does not to the of the RS-11 epitope. RS-11 in the ELISA with and The amino acid sequence in this region is conserved among intermediate including keratin 8 4 We confirmed that RS-11 reacts with a fusion protein consisting of GST and keratin We examined RS-11 reactivity with synthetic peptides this domain of keratin keratin keratin filament protein, and 4 several differences in amino acids between these synthetic ELISA that RS-11 reacts well with each of 5). amino acids are completely conserved among these synthetic peptides and to the of the epitope recognized by RS-11. to residues of keratin but does not the tyrosine at residue which is conserved in most intermediate filaments. RS-11 with as as with which is to for its of the residue tyrosine at residue does not appear to to the of the RS-11 epitope. The pattern of RS-11 immunoreactivity with 18 fusion