Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure ofEscherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structurein situ.     

Both ends ofEscherichia coli ribosomal protein S13 are immunochemically accessiblein situ

To investigate the structure ofEscherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopesin situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13in situ.     

Epitopes ofEscherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

Both ends of Escherichia coli ribosomal protein S13 are immunochemically accessible in situ.

To investigate the structure ofEscherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopesin situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13in situ.     

Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

Monoclonal antibodies to three separate domains on 124 kilodalton phytochrome from Avena

Forty-six monoclonal antibodies have been prepared against 124 kilodalton phytochrome from Avena sativa cv Garry. Clones grown in mice have yielded ascites fluids with antibodies which bind to three distinct regions of the molecule, as visualized by immunoblot analysis of proteolytically produced peptides of the protein. One antibody group (type 1) recognizes an antigenic domain(s) that lies within 6 kilodaltons of the amino terminus of the molecule, a region critical to correct protein-chromophore interaction. The second group (type 2) binds to an antigenic site(s) present within the chromophore-containing half of the molecule that is adjacent to the domain recognized by the type 1 antibodies. The third group (type 3) recognizes an antigenic site(s) that resides in the nonchromophoric, carboxy terminal end of the molecule between 88 and 97 kilodaltons from the amino terminus. One of the type 1 antibodies cross-reacts with apparently undegraded 120 kilodalton phytochrome from zucchini, and therefore may be useful for identifying conserved domains which are essential to the regulatory role of the photoreceptor.     

Structural characterization of a cross-reactive idiotype shared by monoclonal antibodies specific for the human CD4 molecule.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.     

Monoclonal antibody studies of the antigenic determinants of human plasma retinol-binding protein

A battery of monoclonal antibodies (MoAbs) against human retinol-binding protein (RBP) was produced to obtain useful probes for the study of the antigenic determinants of RBP. The 12 antibodies all reacted with human RBP by immunoblotting. Based on antibody cross-competition radioimmunoassays, four distinct and different groups of antibodies were identified: group I, 1A4 and 2F4; group II, 1G10, 5C5, 6F4, and 7G3; group III, 5H6, 6C7, 10G5, and 14E3; and group IV, 5H9 and 13A1. Information about the epitopes of RBP recognized by these MoAbs was obtained by testing the reactivity of each antibody with human, rabbit, and rat RBPs by immunoblotting. Group I and group IV antibodies reacted to a similar extent with human, rabbit, and rat RBPs. Group II antibodies reacted strongly with human and rabbit RBPs, but reacted very weakly with rat RBP. Group III antibodies reacted strongly with human RBP, but did not react with rabbit or rat RBP. Thus, the epitopes for group I and group IV antibodies appear to be regions of the RBP molecule that are conserved across the three species, whereas group III antibodies recognized only human RBP. In a preliminary study, the reactivity of each antibody with purified cyanogen bromide fragments of RBP was tested by slot immunoblotting. None of the MoAbs reacted with any of the cyanogen bromide fragments. This study shows that MoAbs specific for at least four different regions of the RBP molecule can be produced; hence, RBP contains at least four major antigenic domains.     

Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.     

Identification of immunodominant regions of transforming growth factor alpha. Implications of structure and function

Human transforming growth factor alpha (TGF alpha) is a 50-residue mitogenic peptide with a compact structure restrained by three disulfide bonds. Sequential and overlapping synthetic peptides were made to identify epitopes of TGF alpha using a panel of murine monoclonal antibodies and rabbit polyclonal antibodies. Antibodies were raised against human TGF alpha from different preparations obtained from either chemical synthesis or recombinant DNA techniques. Two related methodologies were used in these experiments. In the first method, probes were synthesized as peptides immobilized on polyethylene pins by the method of Geysen et al. (Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3998-4002). Three sets of sequentially overlapping tetrapeptides, hexapeptides, and octapeptides covering the entire length of the human TGF alpha sequence were synthesized. In the second method, a set of overlapping 8-residue synthetic peptides, freely soluble in solution, were used as probes. By both methods, the nonneutralizing monoclonal antibodies, i.e. those that did not inhibit TGF alpha in mitogenic assays, recognized two immunodominant regions represented by the NH2-terminal segment (residues 1-9) and the most prominent beta-sheet of the molecule (residues 22-31). The NH2 terminus and the beta-sheet-(22-31) are in the same face of the molecule as determined by the solution structure. These two immunodominant regions were also recognized by the polyclonal antibodies as well as regions in the COOH terminus as minor epitopes. However, none of the neutralizing monoclonal antibodies recognized any synthetic peptides. Thus, our results suggest that the receptor-binding surface of TGF alpha does not involve the face represented by the NH2-terminal fragment and the major beta-sheet of residues 22-31, but rather, that the opposite face represented by two loops formed by residues 12-20 and 34-43 may be involved in TGF alpha binding to its receptor.     

Monoclonal antibodies as probes for determining the microheterogeneity of the link proteins of cartilage proteoglycan

Monoclonal antibodies were raised against Swarm rat chondrosarcoma link protein 2. Two of the resultant hybridomas (9/30/6-A-1 and 9/30/8-A-4) were used in structural analyses of the link proteins. The 9/30/6-A-1 monoclonal antibody recognized an epitope which was only present on rat chondrosarcoma link protein 2. This epitope was absent in rat chondrosarcoma link protein 3 obtained after trypsin or clostripain treatment of rat chondrosarcoma proteoglycan aggregate, indicating that proteolytic digestion either removed or modified the epitope. Contrasting this, the 9/30/8-A-4 monoclonal antibody recognized an epitope present in link protein(s) 1, 2, or 3 isolated from cartilage of several animal species (rat, bovine, human, and chicken). Rat chondrosarcoma link protein 2 was digested with Staphylococcus aureus V8 protease, and the resulting peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunolocation analyses. The 9/30/6-A-1 and 9/30/8-A-4 monoclonal antibodies recognized epitopes in two different halves of the link protein molecule. The 9/30/8-A-4 monoclonal antibody was used to identify proteolytic cleavage peptides common to the individual link proteins (1, 2, or 3) purified from cartilage proteoglycans of several animal species. Digestion of rat chondrosarcoma link protein 2 with endoglycosidase H or alpha-mannosidase increased its electrophoretic mobility to that of link protein 3 and removed or altered the determinant recognized by the 9/30/6-A-1 monoclonal antibody, indicating that a high-mannose oligosaccharide chain was part of the antigenic determinant. The 9/30/8-A-4 monoclonal recognition of epitope was unaffected by endo- or exoglycosidase treatment. Endo- and exoglycosidase treatment of bovine nasal cartilage link proteins also altered their electrophoretic mobility, indicating that high-mannose oligosaccharide structures on the various link proteins (1, 2, or 3) accounted for the microheterogeneity observed in sodium dodecyl sulfate-polyacrylamide gels.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

A major part of the polypeptide chain of tobacco mosaic virus protein is antigenic

Eighteen synthetic peptides representing virtually the entire length of the polypeptide chain of tobacco mosaic virus coat protein (TMVP) have been analyzed for their ability to bind in an enzyme immunoassay to 30 monoclonal antibodies raised against the dissociated viral subunits. Only five of the monoclonal antibodies were able to bind a number of peptides while the other 25 antibodies recognized only the complete molecule and seemed to be specific for conformational features that are absent in the peptide fragments. The 18 peptides were also tested for their ability to bind to several antisera to TMVP. Virtually the entire sequence of TMVP possessed antigenic activity. Four new epitopes were identified in the vicinity of residues 19–32, 90–95, 115–134 and 134–146. These results bring to 11 the number of continuous epitopes that have been identified in the TMVP molecule and show that the entire surface of the molecule is antigenic. When peptides of TMVP of a length of 6–8 residues were tested for antigenic activity previously a correlation was found between the location of short continuous epitopes and mobile segments of the protein. In the present study, in which longer peptides as well as monoclonal antibodies were used to probe the antigenicity of TMVP, additional conformation-dependent epitopes were shown to be present. Our results illustrate the operational nature of any definition of antigenicity and caution against the use of any single criterion for distinguishing between antigenic and non-antigenic regions of a protein.     

Location of a blocking epitope on outer-membrane protein III of Neisseria gonorrhoeae by synthetic peptide analysis

A series of overlapping peptides spanning the deduced amino acid sequence of outer-membrane protein PIII of Neisseria gonorrhoeae have been synthesized on solid-phase supports. The peptides were used in an attempt to locate the epitopes recognized by anti-PIII monoclonal antibodies with defined biological properties. Four bactericidal and two nonbactericidal antibodies were reacted with the synthetic peptides. None of the bactericidal antibodies reacted with the linear peptides. However, the two nonbactericidal antibodies were found to react within the disulphide loop thought to be exposed on the bacterial surface. Monoclonal antibody SM51 recognized a decapeptide corresponding to amino acid residues 24-33, while monoclonal antibody SM50 recognized an octapeptide contained within the decapeptide. The difference in the ability of the two antibodies to block the bactericidal effect of antibodies directed against other surface antigens therefore appears to be related to a difference in their ability to activate complement rather than to the location of the epitope recognized.     

Immunochemical mapping of a specific domain on human choriogonadotropin using anti-protein and anti-peptide monoclonal antibodies

In an attempt to localize topographic domains specific to native human chorionic gonadotropin (hCG), we studied the discontinuous antigenic regions recognized by a monoclonal anti-hCG antibody designated as C8 which binds only to hCG and does not cross-react with either the free hCG-alpha and hCG-beta subunits or other glycoprotein hormones. Using two-site monoclonal immunoradiometric assays (M-IRMAs), we found that C8 antibody and an anti-peptide antibody (FB12) directed to residues 110-116 of hCG-beta did not bind simultaneously to hCG. This observation suggested that C8 binds to residues of hCG-beta included either in the antibody-binding region of FB12 or in close proximity to amino acids 110-116. To further delineate the regions of hCG-beta recognized by C8, we carried out hapten inhibition experiments with synthetic peptides corresponding to various regions of hCG-beta. The peptide corresponding to residues 109-122 and subpeptides (111-122 or 112-122) inhibited the binding of 125I-hCG to C8, whereas weak inhibition was observed with subpeptide 113-122. By studying the binding of C8 to the 1-112 disulfide-bonded part of hCG-beta (hCG-beta core) recombined with hCG-alpha, we were able to confirm that C8 binds to a region including or near to Asp112. M-IRMAs showed that C8 does not bind to the recombinant molecule lacking residues 113-145 of hCG. Taken together, these results indicate that a limited number of residues located on hCG-beta near to Asp112, and most likely the sequence Asp111-Asp112-Pro113, are included in the discontinuous antigenic region recognized by C8. We then attempted to localize residues of hCG-alpha that constitute another part of the determinant which bound to C8. Six synthetic peptides corresponding to various regions of hCG-alpha did not inhibit binding of 125I-hCG to C8. In contrast, M-IRMAs demonstrated that C8 is capable of binding recombinant products composed of the hCG-beta subunit and the alpha subunits from human, equine, and porcine species. These results indicate that C8 recognizes a region of the alpha subunit highly conserved in these three species. Finally, we determined that the discontinuous regions recognized by C8 are partially accessible on the CG/LH-receptor complex.     

Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus.

The amino acid sequences recognized by monoclonal antibodies (MAbs) specific for the antigenic site IV of the spike protein S of transmissible gastroenteritis virus were analyzed by PEPSCAN. All MAbs of group IV recognized peptides from the S region consisting of residues 378 to 390. In addition, the neutralizing MAbs (subgroup IV-A) also bound to peptides from the region consisting of residues 1173 to 1184 and to several other peptides with a related amino acid composition. The contribution of the individual residues of both sequences to the binding of a MAb was determined by varying the length of the peptide and by a consecutive deletion or replacement of parental residues by the 19 other amino acids. The sequence consisting of residues 326 to 558, tested as part of a cro-beta-galactosidase hybrid protein, was antigenic, but the sequence consisting of residues 1150 to 1239 was not. Furthermore, antibodies raised in rabbits against the peptide SDSSFFSYGEIPFGN (residues 377 to 391), but not those raised against the peptide VRASRQLAKDKVNEC (residues 1171 to 1185), recognized the virus and had neutralizing activity. We infer that the epitope of the neutralizing MAbs is composite and consists of the linear sequence SFFSYGEI (residues 380 to 387) with contributions of A, D, K, N, Q, or V residues from other parts of the S molecule. The complex epitope was simulated by synthesizing peptides in which the sequences consisting of residues 380 to 387 and 1176 to 1184 were combined. MAbs of subgroup IV-A recognized the combination peptides two to six times better than the individual sequences. These results may offer prospects for the development of an experimental vaccine.     

Monoclonal antibodies to gonococcal outer membrane protein I: location of a conserved epitope on protein IB

Hybrid cell lines have been derived which produce monoclonal antibodies reacting with outer membrane protein I from Neisseria gonorrhoeae strain P9. The antibodies obtained showed variable reactivity with other strains but one antibody recognized an epitope present on all of the strains tested which expressed the protease sensitive protein IB. Purified IgG labelled with 125I was used in competitive radioimmunoassays with unlabelled antibody to investigate the spacial distribution of the epitopes recognized. Each pair of antibodies showed some degree of inhibition. The relative magnitude of inhibition suggested that the conserved epitope lies within a variable region containing other epitopes which determine the antigenic specificity of the protein. Western blotting of peptides derived by proteolytic digestion of protein IB revealed that the conserved epitope is located close to the chymotrypsin cleavage site within a 7000 Mr surface exposed region of the molecule.     

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas’ disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2β protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.     

Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody

A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.     

Antigenic determinants on human choriogonadotropin alpha-subunit. I. Characterization of topographic sites recognized by monoclonal antibodies

Immunochemical studies were designed to localize antigenic regions recognized by two monoclonal antibodies directed against the alpha-subunit of human choriogonadotropin (hCG-alpha) and to provide information on the three-dimensional structure of hCG and its alpha-subunit. Monoclonal antibody HT13 bound to a region accessible on both hCG and the free alpha-subunit, whereas monoclonal antibody AHT20 recognized a site localized only on the free alpha-subunit. By studying the cross-reactivity of these antibodies to homologous proteins, we found that antibody HT13 did not bind to equine or ovine lutropin, whereas AHT20 was capable of binding to both subunits. This observation suggests that AHT20 recognized a structurally related antigenic determinant on alpha-subunits of different species. To delineate the portions of hCG-alpha contributing to the antigenic determinants of AHT20 and HT13, we performed competitive inhibition assays using reduced and carboxymethylated hCG-alpha, deglycosylated hCG-alpha, hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core 1), or disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-alpha (hCG-alpha core 2). Reduced and carboxymethylated hCG-alpha did not inhibit the binding of 125I-labeled hCG-alpha to both antibodies, whereas deglycosylated hCG-alpha was as active as hCG-alpha, suggesting that antigenic determinants of both antibodies are mainly discontinuous and do not reside on the oligosacharide part of the alpha-subunit. hCG-alpha core 1 had the same capacity as intact hCG-alpha to inhibit the binding of 125I-hCG-alpha to both antibodies, indicating that the 5 COOH-terminal residues of hCG-alpha do not participate in the antigenic determinants. hCG-alpha core 1 was as potent as hCG-alpha in inhibition experiments performed with HT13, whereas, in striking contrast, hCG-alpha core 2 did not compete with 125I-hCG-alpha for binding to AHT20, suggesting that the peptides released after proteolysis of the alpha-subunit by trypsin participate in the epitope of AHT20 and are not included in the antigenic determinant of HT13. In an attempt to elucidate the amino acid residues constituting the antigenic sites of HT13 and AHT20, hapten inhibition experiments were carried out using as competitive inhibitors five different synthetic peptides spanning the primary structure of hCG-alpha. None of these peptides inhibited the binding of 125I-hCG-alpha to HT13. In contrast, two peptides analogous to regions 23-43 and 33-59 of hCG-alpha exhibited significant potency in competing with 125I-hCG-alpha for binding to AHT20.(ABSTRACT TRUNCATED AT 400 WORDS)