Monoclonal antibodies directed against skeletal muscle actin

The development of a monoclonal antibody directed against rabbit skeletal muscle monomeric actin is described. The production of the monoclonal antibody followed a standard hybridoma technique, the antibody being purified by affinity chromatography. It was found to be of the IgM class. Antibody specificity for rabbit skeletal actin was demonstrated by radioimmunoassay. The antibody failed to bind to actin in Western Blot experiments, presumably due to modification of the antigenic determinant on actin during the Western Blot procedure. The antibody was also shown to bind to two other isotypes of actin, i.e. actin from squid mantle muscle and bovine myocardium.     

Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes.

Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.     

Ro60 peptides induce antibodies to similar epitopes shared among lupus-related autoantigens

The coexistence of autoantibodies to ribonucleoproteins (RNP) in sera of patients with systemic lupus erythematosus has been attributed to intermolecular determinant spreading among physically associated proteins. Recently, we showed that murine Ab responses to rRo60 or Ro60 peptides were diversified unexpectedly to small nuclear RNP. In this investigation, the mechanisms for this autoantibody diversification were examined. Intramolecular determinant spreading was demonstrated in mice immunized with human or mouse Ro60316-335. Immune sera depleted of anti-peptide Ab immunoprecipitated Ro60-associated mY1 and mY3 RNA and remained reactive to a determinant on Ro60128-285. Absorption with the immunogen depleted the immune sera completely of anti-Golgi complex Ab (inducible only with human Ro60316-335) and anti-La Ab, and reduced substantially Ab to SmD and 70-kDa U1RNP. Mouse rRo60 completely inhibited the immune sera reactivity to La, SmD, and 70-kDa U1RNP. However, La, SmD, and 70-kDa U1RNP preferentially inhibited the antiserum reactivities to these Ags, respectively. Affinity-purified anti-La Ab were reactive with Ro60, La, SmD, and 70-kDa U1RNP. These results provide evidence that a population of the induced autoantibodies recognized determinants shared by these autoantigens. Lack of sequence homology between Ro60316-335 and La, SmD, or 70-kDa U1RNP suggests that these determinants are conformational. Interestingly, similar cross-reactive autoantibodies were found in NZB/NZW F1 sera. Thus, a single molecular mimic may generate Ab to multiple RNP Ags. Furthermore, cross-reactive determinants shared between antigenic systems that are not associated physically (Ro/La RNP and small nuclear RNP) may be important in the generation of autoantibody diversity in systemic lupus erythematosus.     

Monoclonal antibodies to different protein-related epitopes of human articular cartilage proteoglycans.

Monoclonal antibodies produced against chondroitinase-treated human adult cartilage proteoglycans were selected for their ability to recognize epitopes on native proteoglycans. Binding analyses revealed that four of these monoclonal antibodies (BCD-4, BCD-7, EFG-4 and KPC-190) each recognized a different epitope on the same proteoglycan molecule which represents a subpopulation of a high buoyant density (D1) fraction of human articular cartilage proteoglycans (10, 30, 50 and 60% in fetal-newborn, 1.5 years old, 15 years old and 52-56 years old cartilages, respectively). Analysis of epitope specificities revealed that BCD-7 and EFG-4 monoclonal antibodies recognized epitopes on proteoglycan monomer which are associated with the protein structure in that they are sensitive to cleavage by Pronase, papain and alkali treatment and do not include keratan sulphate, chondroitin sulphate or oligosaccharides. The BCD-4 and KPC-190 epitopes also proved to be sensitive to Pronase or papain digestion or to alkali treatment, but keratanase or endo-beta-galactosidase also reduced the immunoreactivity of these epitopes. These observations indicate that the BCD-4 and KPC-190 epitopes represent peptides substituted with keratan sulphate or keratan sulphate-like structures. The BCD-4 epitope is, however, absent from a keratan sulphate-rich fragment of human adult proteoglycan, while the other three epitopes were detected in this fragment. None of these four epitopes were detected in the link proteins of human cartilage, in the hyaluronic acid-binding region of human newborn cartilage proteoglycan, in Swarm rat chondrosarcoma proteoglycan, in chicken limb bud proteoglycan monomer and in the small dermatan sulphate-proteoglycan of bovine costal cartilage. EFG-4 and KPC-190 epitopes were not detected in human fetal cartilage proteoglycans, although fetal molecules contained trace amounts of epitopes reactive with BCD-4 and BCD-7 antibodies.     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.     

Monoclonal antibodies detecting different epitopes on the Forssman glycolipid hapten

Two hybridomas, derived by fusing mouse myeloma cells with spleen cells from a rat immunized with mouse mammary tumors, have been shown to produce antibodies that recognize cell surface antigens on mesenchymal cells in a variety of tissues. Evidence presented in this report suggests that these antibodies detect overlapping epitopes on the Forssman glycolipid hapten (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). One antibody (33B12) reacts with the terminal sugar sequence GalNAc alpha 1-3GalNAc and is specific for Forssman. The other antibody (117C9) recognizes the internal sugar sequence GalNAc beta 1-3Gal. The terminal sugar sequence GalNAc beta 1-3Gal in globoside, as well as the internal sugar sequence GalNAc beta 1-4Gal in asialo-GM1, is not recognized as an antigenic determinant by 117C9. Nevertheless, the 117C9 antibody does not react exclusively with the Forssman antigen. In a lipid extract fractionated by Folch partition of mouse mammary tumors, the antibody also detects other glycolipids.     

Monoclonal antibodies identify multiple epitopes on maize leaf nitrate reductase

Nine hybridoma cell lines secreting antibodies against the maize leaf nitrate reductase have been distinguished by reciprocal competition for binding to the antigenic site. Inhibition of enzymatic activities, and western blots of native enzyme and denatured subunits revealed different behaviors of individual antibodies towards the antigen. Two classes of monoclonal antibodies are inhibitory of NADH and methyl viologen nitrate reductase activities, but only one affects also NADH cytochrome c reductase activity. The associated epitopes are sensitive to antigen conformation. Among the 4 other classes, one is specific for the native conformation of the molecule, another binds more strongly to the denatured antigen, and two recognize equally well the two forms.     

Specificity studies on anti-histone H1 antibodies obtained by different immunization methods

Antibodies were elicited against chromatographically purified histone H1 subfractions or against their complexes with RNA and their specificity studied by enzyme-linked immunosorbent assay. The results show that complexing of the pure protein with RNA does not lead to any significant increase in the antibody titer and results in obtaining antibodies predominantly against the common antigenic determinants present in the H1 histone class. On the other hand, using pure histone fractions for immunization gives rise to antibody populations reacting mainly with the subfraction-specific determinants on the histone molecule. In view of these results the literature data should be interpreted with caution.     

Monoclonal antibodies obtained against G-protein epitopes: comparison of immunoreactions seen in the brain, retina and striated muscular tissue]

Monoclonal antibodies have been obtained against a purified fraction of brain G proteins containing the Gi alpha, G0 alpha, G beta, and G gamma subunits. After characterization, two monoclonal antibodies have been used to detect the cellular distribution of the two epitopes in neural, retinal and muscular tissues: ELISA, cross-dot and Western blot demonstrated that F.IV.5 is an anti-G beta antibody specific for the 36 kDa beta-subunit. ELISA, cross-dot and immunocytochemical distribution of the epitopes recognized by F.VII.9 suggested that this antibody recognizes epitopes which are also detected with polyclonal anti-G0 alpha antibodies. With both monoclonal antibodies, we confirmed that G proteins demonstrated a sub-membranous distribution as well as extensively cytoplasmic, axoplasmic or sarcoplasmic distributions in different cell types.     

Monoclonal antibodies to distinctive epitopes on the alpha and beta subunits of the fibronectin receptor

Monoclonal antibodies (MAbs) have been developed that can recognize epitopes that are unique to either the alpha or beta subunit of the fibronectin receptor (FnR). MAbs 11B4 and 7A8 immunoblot the alpha subunit of FnR either in purified form from Chinese hamster ovary (CHO) cells or in nonionic detergent extracts of cells of human and rodent origin electrophoresed under reducing or nonreducing conditions. The MAbs seem to be more reactive to the subunit when it has been electrophoresed under reducing conditions, suggesting that the epitope may be partially masked by the conformation conferred by disulfide bonding. A second set of MAbs, 7E2 and 7F9, is directed to an epitope on the beta subunit that is conformationally dependent upon disulfide bonding, as reduction of the subunit leads to loss of reactivity with both MAbs. Further, 7E2/7F9 immunoblots of nonionic detergent extracts of CHO cells, run under nonreducing conditions, reveal the presence of a third band (90-kDa), immunologically related to the beta subunit, which is not surface-labeled with 125I in intact cells and which does not copurify with the alpha and beta subunits isolated by immunoaffinity purification of FnR using the MAb PB1. The 90-kDa component is not found associated with a plasma membrane fraction prepared by crude cell fractionation, but is abundant in a low-speed pellet containing nuclei and intracellular membranes. This finding suggests that the 90-kDa component is a precursor to the beta subunit. Finally, the epitope of 7E2/7F9 is unique to CHO cells, as cross-reactivity to other cell types cannot be demonstrated by either immunoblotting or immunoprecipitation.     

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity

Background

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity.

Methods and Principal Findings

Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity.

Conclusions

The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.     

A direct interaction between actin and vimentin filaments mediated by the tail domain of vimentin

The assembly and organization of the three major eukaryotic cytoskeleton proteins, actin, microtubules, and intermediate filaments, are highly interdependent. Through evolution, cells have developed specialized multifunctional proteins that mediate the cross-linking of these cytoskeleton filament networks. Here we test the hypothesis that two of these filamentous proteins, F-actin and vimentin filament, can interact directly, i.e. in the absence of auxiliary proteins. Through quantitative rheological studies, we find that a mixture of vimentin/actin filament network features a significantly higher stiffness than that of networks containing only actin filaments or only vimentin filaments. Maximum inter-filament interaction occurs at a vimentin/actin molar ratio of 3 to 1. Mixed networks of actin and tailless vimentin filaments show low mechanical stiffness and much weaker inter-filament interactions. Together with the fact that cells featuring prominent vimentin and actin networks are much stiffer than their counterparts lacking an organized actin or vimentin network, these results suggest that actin and vimentin filaments can interact directly through the tail domain of vimentin and that these inter-filament interactions may contribute to the overall mechanical integrity of cells and mediate cytoskeletal cross-talk.     

Monoclonal antibody to fibulin-1 generated by genetic immunization

Fibulin-1 (Fbln-1) is an extracellular matrix (ECM) and plasma glycoprotein. Considering the growing evidence indicating that Fbln-1 plays a role in cancer we sought to develop monospecific antibodies to better facilitate further studies of the function of Fbln-1 in breast cancer. Using a plasmid expression vector encoding full-length human Fbln-1D as an immunogen and CpG oligodeoxyribonucleotides as adjuvant a monoclonal antibody (MAb) against Fbln-1 was produced. This MAb, designated MEM-2 was of IgM isotype and reacted with bacterially expressed Fbln-1. Furthermore, MEM-2 reacted with Fbln-1 expressed in the ECM released by cultured human breast carcinoma SKBR-3 cells in ELISA, and also with Fbln-1 present in SKBR-3 cell extract in immunoprecipitation and Western blotting. MEM-2 also reacted with Fbln-1 in human breast carcinoma specimens. These findings illustrate the utility of genetic immunization as a means of generating monoclonal antibodies to tumor-related ECM proteins. MEM-2 represents a useful new tool for the study of Fbln-1 in breast cancer.     

Monoclonal antibodies to protein kinase C gamma. Functional relationship between epitopes and cofactor binding sites

Four monoclonal antibodies (mAbs), derived from high-responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype gamma 1, kappa and 15G4 is of isotype gamma 2b, kappa. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific for the bovine gamma isoform of protein kinase C and cross-react with protein kinase C gamma from a variety of animal species. Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase C gamma cofactor-dependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor-independent catalytic activity of protein kinase C gamma. Competition analysis between these mAbs and phosphatidylserine, 12-O-tetradecanoylphorbol 13-acetate and Ca2+ showed that 36G9 and 5A2 block cofactor binding to protein kinase C gamma. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase C gamma.     

Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins

Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.     

Monoclonal antibodies to different antigens of skin epithelium obtained by immunizing mice with streptococcus group A antigens

Monoclonal antibodies (MCA) were obtained by immunization of BALB/c mice with streptococcal group A protein antigens of the cellular wall, or with whole microbial cells. In immunofluorescence test, MCA react with different skin epithelial structures (basal, suprabasal or all the epidermal layers). The majority of MCA belong to autoantibodies. The same MCA revealed no cross-reactions with streptococcal antigens in immunoenzyme and inhibition tests. MCA reacting with epithelial cells are, apparently, obtained as a result of polyclonal activation of the autoreactive clones by streptococcal antigens.     

Monoclonal autoantibodies to different epithelial structures of the thymus gland obtained by the immunization with streptococcal group A antigens

By the indirect immunofluorescence method it was shown to which epithelial thymus structures monoclonal antibodies (mAT) reacting with the different epidermal structures are directed. These mAT related to the autoantibodies were obtained earlier, as a result of lymphoid cells polyclonal activation, by the immunization of BALB/c mice with streptococcal group A nonspecific protein antigens of the cell wall. It was shown that mAT A6/1, reacting with the basal layer of the skin epithelium are directed to the epithelium of the cortical and medullar thymus zones, which is regarded as the so called endocrinal epithelium. These mAT, by the study with immunoblotting method, react with the protein of mV SOkD, B5/1 mAT to the skin epithelium, on the thymus sections react with the single cells around the Hassel bodies.     

Monoclonal antibodies to newcastle disease virus: delineation of four epitopes on the HN glycoprotein

Eighteen independent hybridomas producing monoclonal antibodies to Newcastle disease virus have been prepared by fusion of SP2 cells with spleen lymphocytes from a BALB/c mouse immunized with intact UV-inactivated Newcastle disease virus strain Australia-Victoria. They have been divided into three groups on the basis of radioimmunoprecipitation, infected cell surface and cytoplasmic fluorescence, and isotype. The anti-HN group is made up of nine antibodies which give surface fluorescence on infected cells and immunoprecipitate the HN glycoprotein. These antibodies bind to HN in nitrocellulose transfers of sodium dodecyl sulfate gels, but only if it has been neither reduced nor boiled. To varying degrees, all of these HN antibodies neutralize infectivity. These results suggest that they recognize exposed determinants of a conformational nature on the native HN molecule. They have been used in competition antibody-binding radioimmunoassays and additive neutralization assays, and on the basis of these studies the epitopes they recognize have been subdivided into four domains, two of which are overlapping, on the HN glycoprotein. The relatively weaker neutralizing activity observed with some of these antibodies cannot be explained by lower avidities for their epitopes because there is not an inverse correlation between estimated binding constant and neutralizing activity. The four antibodies in the second group all give a predominantly cytoplasmic fluorescence pattern, immunoprecipitate the nucleocapsid protein, and bind to nucleocapsid protein in nitrocellulose transfers of reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gels. All five of the antibodies in the third group are of the immunoglobulin M class, unlike the others which are all immunoglobulin G antibodies. Members of this group show variable fluorescence patterns, but none is able to immunoprecipitate or bind to a specific viral antigen transferred to nitrocellulose paper from sodium dodecyl sulfate-polyacrylamide gels.