An Antibody Specific for Component 8 of Myelin Basic Protein from Normal Brain Reacts Strongly with Component 8 from Multiple Sclerosis Brain

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies reveal antigenic differences in refractile bodies of avian Eimeria sporozoites

Monoclonal antibodies were developed against refractile body antigens of 4 species of avian Eimeria, E. meleagrimitis, E. adenoeides, E. acervulina, and E. tenella. Although antibodies from 8 different cell lines were used in this study, all produced similar fluorescent and gold-labeling patterns. By immunofluorescent antibody techniques, 5 of the 8 antibodies cross-reacted with all 4 of the Eimeria species that were examined; the other 3 antibodies reacted only with the species against which they were produced or with a limited number of species. In Western blot analyses using SDS-solubilized sporozoites as antigen, 4 of the cross-reactive antibodies recognized multiple bands; the predominant bands had molecular weights of approximately 23, 45, and 90 kilodaltons (kDa). Two of the antibodies with more limited reactivity recognized either a single band at 23 kDa (91C7), or bands at 23 and 45 kDa (4115); another reacted only with several bands greater than 100 kDa (4D10). The molecular weights of the antigens did not decrease markedly after digestion with N-glycanase F, indicating that if the refractile body antigens contained significant amounts of N-linked carbohydrate it was refractory to the enzyme. Collectively, the data indicate that antigens of the sporozoite refractile bodies differ among the Eimeria species. Some antigens are conserved, whereas others differ in distribution or frequency among the individual species.     

Characterization of Various Recombinant Antigens from <Emphasis Type="Italic">Echinococcus multilocularis</Emphasis> for Use in the Immunodiagnosis

Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser²³–Glu¹⁷⁶ of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen B1 did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.     

Recognition of human activated CD44 by tumor vasculature-targeted antibody

TES-23 monoclonal antibody (MAb), which targets rat CD44H on tumor vascular endothelial cells (TEC), dominantly reacted to human activated CD44 rather than human inactive CD44. TES-23 MAb reacted to HT-1080 fibrosarcoma cells almost comparably to anti-human CD44 MAb and moderately to HUVEC; however, it hardly reacted to PBMC. The binding of soluble hyaluronate to HT-1080 cells and HUVEC was clearly noted, but not to PBMC. In addition, stimulation with phorbol 12-myristate 13-acetate induced soluble hyaluronate binding of MOLT-4 human T lymphoma cells and relatively increased the reactivity of TES-23 MAb. Our results suggest that TES-23 MAb can potentially recognize human activated CD44 and hence might be potentially useful for the treatment of human solid tumors containing TEC that express activated CD44.     

Analysis of human C8 with monoclonal antibodies. Characterization of a monoclonal antibody that recognizes free C8 alpha-gamma subunit

The eighth component of human C is essential for the formation of the membranolytic C attack complex. C8 has a unique structure in that two covalently linked chains, C8 alpha and C8 gamma, are associated non-covalently with the third chain, C8 beta. In order to study the structure and assembly of the C8 molecule, a panel of mAb has been produced against the C component C8. Eight of these mAb had reactivity to the C8 alpha-gamma subunit, whereas four reacted with C8 beta. One of the C8 alpha-gamma mAb, C8A2, had specificity for an epitope on the C8 alpha-chain and exhibited no cross-reactivity to any of the other terminal C components, including C8 beta. C8A2 inhibited the hemolytic activity of the C8 alpha-gamma subunit but had no effect on the activity of fluid phase whole C8 or C8 within membrane-bound C5b-8. Functional experiments suggest that C8A2 inhibits C8 alpha-gamma activity by interfering with its interaction with the C8 beta-chain. In an enzyme immunoassay using the C8A2 mAb, free C8 alpha-gamma subunit could be detected in both homozygous and heterozygous C8 beta-deficient serum. However, only low level binding was observed when homozygous C5- and C7-deficient sera were tested. Thus the mAb, C8A2, recognizes an epitope expressed on the C8 alpha-gamma subunit but not on intact C8 and can detect free C8 alpha-gamma in the presence of native C8.     

Reaction of poliovirus RNAs with antibodies to double-stranded RNA demonstrated by an immunochemical binding assay.

An immunochemical binding assay was used to investigate the reactivity of radioactively labeled viral RNAs from poliovirus-infected cells with antibodies to the synthetic double-stranded RNA, poly(I)-poly(C). A RNase-free antibody-containing serum fraction was employed. Poliovirus replicative form reacted with the antibodies to poly(I)-poly(C) as well as or better than poly(I)-poly(C). Poliovirus replicative intermediate reacted with the antibodies to a greater extent than poliovirus single-stranded RNA, but both were less reactive than replicative form. The use of the immunochemical binding assay with sucrose-gradient fractions demonstrated that for both poliovirus single-stranded RNA and replicative form the peak of reactivity with the antibodies was coincident with the peak of radioactive material precipitated by trichloroacetic acid. The proportion of replicative intermediate that reacted with the antibody increased in sucrose-gradient fractions containing the more slowly sedimenting RI RNA.     

Characterization of rat gastric mucins using a monoclonal antibody,RGM23, recognizing surface mucous cell-type mucins

A novel anti-mucin monoclonal antibody (mAb), designated RGM23, was developed against mucin purified from rat gastric mucosa. RGM23 reacted with the mucin attached to the ELISA well. The reactivity was lost by trypsin treatment, but not by periodate oxidation, indicating that RGM23 recognizes the peptide moiety of the mucin molecule. Histochemical study showed that RGM23 stained the corpus and antral surface mucosa of rat stomach, but not their glandular mucosa, nor duodenal, small intestinal or large intestinal mucosa. The area stained with RGM23 was coincident with that stained with 45M1, a mAb reacting with MUC5AC mucin. Examination of the mucin subunits extracted from rat stomach by Sepharose CL-4B and Q-Sepharose chromatography and CsTFA equilibrium centrifugation showed that RGM23 reacted with the surface mucous cell-type mucins that were stained with periodate-Schiff (PAS) and reacted with mAb RGM21. The gastric gland-type mucin, which reacted with mAb HIK1083, did not react with RGM23. On Q-Sepharose chromatography, a part of the RGM21-reactive mucins was only faintly stained with PAS and did not react with RGM23. The results together indicated that RGM23 probably reacted with the rat MUC5AC (rMuc5AC) mucin present in the surface mucosa of the stomach, and that the surface mucosal cells in rat stomach may contain mucin bearing non-rMuc5AC core protein in addition to rMuc5AC mucins.     

Immunophenotypic difference of keratin expression in normal mammary glandular cells from five different species.

The immunohistochemical reactivity of human, monkey, shrew, rat and mouse normal mammary glands was examined using methacarn-fixed paraffin-embedded specimens and acetone-fixed frozen sections using the avidinbiotin-peroxidase method for cell phenotype comparison. Actin was visualized using anti-smooth muscle actin antibody and keratin expression was determined by employing 12 different monoclonal antibodies. All these antibodies cross-reacted specifically with the species examined. Basal (myoepithelial) cells from all species showed muscle-specific actin according to reactivity with HHF35 monoclonal antibody. Keratin expression showed significant phenotypic differences among species. In human and monkey, AEL-KS2, KL1, CK8.13, AE3 and 34BE12 stained luminal cells as well as basal cells. AE1, RPN1165, CK4.62, 35BE11, M20 and RPN1162 labeled only luminal cells whereas 312C8-1 preferentially bound to basal cells. In shrews, AEL-KS2, CK8.13 and AE3 reacted to both cell types, AE1 reacted only with luminal cells, and 35BE12 and 312C8-1 selectively stained basal cells. In rodents, AEL-KS2 reacted to both cell types, CK8.13, AE3, 34BE12 and 312C8-1 stained rat basal cells, and 34BE12 and 312C8-1 reacted to mouse basal cells. The data represents cytoskeletal differences among species.     

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

A novel cell surface antigen, 4C8, is expressed on human eosinophils

BACKGROUND: A novel monoclonal antibody, anti-4C8, reacted with human peripheral lymphocytes and monocytes but not with neutrophils. In this study, we investigated whether the 4C8 antigen is expressed on human peripheral eosinophils. METHODS: Expression of the 4C8 antigen on eosinophils was analyzed by flow cytometry and molecular analysis of the antigen was performed with eosinophils by Western blotting. RESULTS: Among human peripheral granulocytes, the 4C8 antigen was expressed on CD16-negative cells but not on CD16-positive cells. The 4C8 antigen also appeared to be expressed on eosinophils. To confirm the latter finding, eosinophils were purified from peripheral blood. On flow cytometric analysis, anti-4C8 antibody reacted with purified eosinophils. On Western blotting analysis, anti-4C8 reacted with a single band of 80 kDa in lysates from purified eosinophils. The correlation between the percentage of eosinophils determined by May-Giemsa staining and the percentage of 4C8-positive/CD16-negative cells among granulocytes was good (r = 0.91, P < 0.0001). CONCLUSIONS: Only a few cell surface antigens are available to distinguish human peripheral eosinophils from neutrophils. The novel cell surface antigen, 4C8, is a useful new marker of human eosinophils.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23α. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: identification of its antigen as a neutral glycolipid

 A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23α. These components had R _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway. Received: 24 April 1995 / Accepted: 11 July 1995     

Neurofilament Monoclonal Antibodies RT97 and 8D8 Recognize Different Modified Epitopes in Paired Helical Filament-τ in Alzheimer's Disease

Abstract: Neurofibrillary tangles in Alzheimer's disease have been previously found to be labeled by some neurofilament antibodies that also recognize τ proteins. We have studied the reactivity of two such monoclonal antibodies, RT97 and 8D8, and of an anti-ubiquitin serum with the abnormal paired helical filaments (PHF)-τ (A68) polypeptides known to be the main component of the PHFs constituting the neurofibrillary tangles. 8D8 recognized the three major PHF-τ polypeptides, but RT97 reacted only with the two larger PHF-τ species. PHF-τ polypeptides were labeled by 8D8 and RT97 much more strongly than normal human τ and this labeling was decreased after alkaline phosphatase treatment. Anti-ubiquitin and anti-phosphotyrosine antibodies did not label PHF-τ polypeptides. The immunoreactivity of proteolytic fragments of PHF-τ polypeptides was studied with RT97, 8D8, and a panel of τ antibodies. The epitope for 8D8 on PHF-τ was localized between amino acids 222 and 427 in the carboxyl half of τ. The RT97 epitope on PHF-τ was localized in the amino domain of τ, probably in the 29-amino-acid insertion (insert 1) found towards the amino terminus of some τ isoforms. These results show that the basis for the labeling of neurofibrillary tangles by antibodies 8D8 and RT97 to neurofilament is their ability to react with PHF-τ polypeptides by recognizing sites specifically modified on PHF-τ, including a site specific to some τ isoforms.     

Detection of Specific IgA Antibodies against a Novel Deamidated 8-Mer Gliadin Peptide in Blood Plasma Samples from Celiac Patients

We studied whether celiac disease (CD) patients produce antibodies against a novel gliadin peptide specifically generated in the duodenum of CD patients by a previously described pattern of CD-specific duodenal proteases. Fingerprinting and ion-trap mass spectrometry of CD-specific duodenal gliadin-degrading protease pattern revealed a new 8-mer gliadin-derived peptide. An ELISA against synthetic deamidated 8-mer peptides (DGP 8-mer) was used to study the presence of IgA anti-DGP 8-mer antibodies in plasma samples from 81 children (31 active CD patients (aCD), 17 CD patients on a gluten-free diet (GFD), 10 healthy controls (C) and 23 patients with other gastrointestinal pathology (GP)) and 101 adults (16 aCD, 12 GFD, 27 C and 46 GP-patients). Deamidation of the 8-mer peptide significantly increased the reactivity of the IgA antibodies from CD patients against the peptide. Significant IgA anti-DGP 8-mer antibodies levels were detected in 93.5% of aCD-, 11.8% of GFD- and 4.3% of GP-patients in children. In adults, antibodies were detected in 81.3% of aCD-patients and 8.3% of GFD-patients while were absent in 100% of C- and GP-patients. Duodenal CD-specific gliadin degrading proteases release an 8-mer gliadin peptide that once deamidated is an antigen for specific IgA antibodies in CD patients which may provide a new accurate diagnostic tool in CD.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

Effects of methanol and temperature on enzyme immunoassay with monoclonal antibodies specific to the insecticide etofenprox

We examined the effects of methanol and temperature on the reactivity of monoclonal antibodies specific to the insecticide etofenprox. When the antigen-antibody reaction was done at 4 degrees C in 10% methanol, the sensitivity in the enzyme immunoassay with each antibody was more than 10-fold higher than that measured at 37 degrees C. Although in 10% methanol one of the antibodies reacted equally with both etofenprox and the carbonate-derivative of etofenprox, in 50% methanol the antibody reacted with etofenprox, but not with the derivative.     

The preparation and characterization of monoclonal antibodies to human complement component C8 and their use in purification of C8 and C8 subunits.

1. Ten mouse monoclonal antibodies to human complement component C8 were prepared. It was found that six of these antibodies reacted with the alpha-subunit, two with the beta-subunit and two with the gamma-subunit, when assessed by immunoblotting after separation of C8 subunits by SDS/polyacrylamide-gel electrophoresis. 2. Epitope analysis of the ten monoclonal antibodies in a competitive binding assay showed that the six antibodies to the alpha-subunit could be classified in four overlapping epitope groups. The antibodies to the beta- and gamma-subunits bound to a single antigenic site on each, but also cross-reacted with the antigenic sites on the alpha-subunit. 3. Monoclonal anti-C8 immunoaffinity columns were used to purify C8 from fresh human plasma and to prepare C8-depleted serum. Immunoaffinity purified C8 was biologically active when assessed by using haemolysis assays of sheep and rabbit erythrocytes. 4. Salt elution was used to purify either alpha gamma- or beta-subunits when C8 was respectively bound to an anti-beta or anti-alpha immunoaffinity column. The purified subunits reconstituted C8-depleted serum when added together in a haemolysis assay.     

Epitopes on proliferating cell nuclear antigen recognized by human lupus autoantibody and murine monoclonal antibody

The immune epitopes of proliferating cell nuclear antigen (PCNA), also called cyclin, were analyzed by determining the reactivity between PCNA peptide fragments and anti-PCNA antibodies from lupus patients, murine monoclonal antibody (19A2), and rabbit anti-NH2-terminal peptide antibody. Limited digestion of PCNA/cyclin with Staphylococcus aureus V8 protease resulted in several peptide fragments. Five fragments of 30, 20, 15, 14, and 13 kDa were reactive with rabbit anti-NH2-terminal peptide antibody denoting that they contained the NH2-terminal peptide. The 30- and 20-kDa fragments reacted with 19A2 but the others did not. Lupus sera reacted with 17- and 15-kDa peptide fragments allowing their classification into three groups. Two of eight sera (type A) reacted only with the 17-kDa fragment. Two others (type B) reacted with both the 17- and 15-kDa fragments and the remaining four sera (type C) reacted only with the 15-kDa fragment. The sera reacting with the 15-kDa fragment also reacted with the 20-kDa fragment, but the sera reactive only with the 17-kDa fragment did not, indicating that the 17-kDa fragment was not a degradation product of 20-kDa fragments. The 19A2 epitope resided in the region between 15 and 20 kDa from the NH2 terminus, whereas there was at least one distinct epitope on each 15- and 17-kDa peptide, which were recognized by lupus autoantibodies.