Monoclonal antibodies directed against AFAP-110 recognize species-specific and conserved epitopes.

The actin filament-associated protein, AFAP-110, is a Src SH2/SH3 binding partner that can modulate changes in actin filament structure. AFAP-110 contains a carboxy terminal motif that facilitates actin filament interactions, as well as amino terminal protein binding motifs, including an SH3 binding motif, two SH2 binding motifs, and two Pleckstrin homology domains. Two monoclonal antibodies (MAbs) were developed that recognized epitopes in either the amino terminus (MAb 4C3) or the carboxy terminus (anti-AFAP-110) of AFAP-110. Site-directed mutations that change key proline residues to alanine in the SH3 binding motif and an adjacent proline-rich motif abrogated MAb 4C3 binding. These same mutations have been shown to prevent SH3 interactions between AFAP-110 and Src527F. These data indicate that MAb 4C3 recognizes an epitope that is part of the SH3 binding motif. Interestingly, MAb 4C3 is not efficiently reactive with mammalian homologs of AFAP-110. Sequence analysis of a putative cDNA clone that encodes the amino terminus of the human AFAP-110 isoform predicted a one amino acid difference within this epitope, indicating a mechanism for species-specific binding by MAb 4C3. A second, MAb anti-AFAP-110, recognizes AFAP-110 across species and binds to an epitope within the carboxy terminus. This epitope includes the 5th heptad repeat of the carboxy terminal, leucine zipper motif (amino acids 592-598)--a motif that facilitates self-associations and may regulate the function of AFAP-110. These MAbs will be useful for analyzing the effects of AFAP-110 upon cell morphology and actin filament integrity. In addition, the avian-specific MAb 4C3 may be useful for studying the effects of avian AFAP-110 constructs expressed in mammalian cells, by providing an internal epitope tag.     

Monoclonal antibodies to HLA recognize monomorphic and polymorphic epitopes on BoLA

Fourteen monoclonal antibodies recognizing monomorphic and polymorphic epitopes on class I and class II antigens of the human MHC have been assayed on lymphocytes of a panel of 20–150 BoLA typed bovine animals from 12 different breeds. Some monomorphic antibodies cross-reacted and others did not. Two polymorphic monoclonal antibodies in man recognize a polymorphism in cows that follows allospecificities (BoLA-w3, w9) already described. Immunoprecipitation experiments with monomorphic anti-B2m and anti-HLA-DR monoclonal antibodies have shown that these cross-reactions concern BoLA antigens. They also revealed that Ia-like antigens in cattle present the same two chain features characterized in other species.     

Monoclonal antibodies to human C-reactive protein (CRP) that recognize epitopes in functional regions

Thirteen mouse hybridomas secreting monoclonal antibodies (MAbs) to human C-reactive protein (CRP) were generated and characterized. The relative avidity of the MAbs for CRP ranged from 0.005 to 8.6 micrograms/ml of purified Ig. Several of the MAbs recognized an epitope on CRP expressed only in the presence of physiological levels of Ca++. The epitope specificity of the 13 MAbs was examined by testing their cross-reactivity and allowed us to assign them to two groups. Group I MAbs recognized the Ca++-dependent phosphorylcholine (PC)-binding site of CRP since their reactivity was inhibited by PC. Group II MAbs recognized epitopes not affected by binding of PC by CRP. The results suggest that these MAbs may be suitable for assigning functional properties of CRP to specific peptide sequences.     

Protective monoclonal antibodies recognize heat-labile epitopes on surface proteins of spotted fever group rickettsiae.

Thirty-eight monoclonal antibodies that have not been reported previously were developed from mice immunized with Rickettsia rickettsii, R. conorii, and R. sibirica. Western immunoblotting showed that these monoclonal antibodies are directed against heat-sensitive epitopes which are located on two major surface polypeptides with molecular sizes ranging from 115 to 150 kilodaltons. The detection of the two bands did not depend on the presence of 2-mercaptoethanol. Both bands were destroyed by treatment with proteinase K. Monoclonal antibodies examined by immunofluorescence assay reacted with epitopes that are species specific, group reactive, or shared among a smaller subset of species of spotted fever group rickettsiae. Nine of the monoclonal antibodies were evaluated for their ability to neutralize rickettsial infection and thus protect animals against disease caused by homologous species of rickettsiae. Treatment of rickettsiae with monoclonal antibodies F3-12, F3-14, and F3-36 completely protected guinea pigs against illness caused by the homologous organism R. rickettsii. Monoclonal antibodies F9-5G11 and F15-5B12, derived from mice immunized with R. sibirica, conferred partial protection by delaying the onset and shortening the duration of fever in guinea pigs inoculated with R. sibirica. Monoclonal antibodies F2-15, F2-31, F2-53, and F3-12 protected mice from a lethal infection with R. conorii. Heat-labile epitopes of spotted fever group rickettsial surface proteins are important candidate antigens for development of vaccines to confer protective immunity.     

Monoclonal antibodies defining epitopes on human IgE.

Twelve monoclonal antibodies (mAb) were isolated that bound to six clusters of epitopes on the constant region of the epsilon chain of human IgE. Four of the mAb bound to the C epsilon 1 or early C epsilon 2 regions; three of these bound to the IgE myeloma protein PS and to serum IgE but not to the IgE myeloma protein ND. These mAb probably recognize an allotypic marker. Another mAb reacted with heat-denatured, but not native IgE. Four of the mAb failed to release histamine; the epitopes recognized by these mAb are in the C epsilon 1, C epsilon 2 and C epsilon 3-4 regions of IgE. Three of these non-histamine releasing mAb did not bind to IgE on the basophil surface. These mAb recognize epitopes in C epsilon 2 and C epsilon 3-4 that are not accessible when IgE is bound to its receptor. Four mAb inhibited IgE binding to basophils; two of these did not release histamine, and two others that bind to epitopes in the C epsilon 2-4 domain, released histamine and therefore blocked IgE binding by steric hindrance. Inhibition of IgE binding by different mAb suggest that the Fc epsilon RI and Fc epsilon RII bind to partly overlapping regions of the IgE molecule although the sites do not appear to be identical. A number of sites on C epsilon 1 and C epsilon 3-4 were accessible when IgE is bound to its basophil receptor. The data support the concept that only part of the Fc portion of IgE is hidden in the receptor and that portions of C epsilon 1-4 are accessible on the cell surface. These mAb should be useful in determining the domains of IgE that are critical for its biological activity.     

How antibodies recognize virus proteins

Numerous studies have addressed the nature of antibody-antigen interaction, but only recently have three-dimensional structures of complexes of antibodies with protein antigens been reported, one with lysozyme and one with the influenza virus antigen neuraminidase. Both structures show that there are epitopes involving about 16 amino acids on surface loops of the antigen. In the lysozyme complex the interaction of the components is rigid, but there is a degree of structural flexibility in the formation of the complex with neuraminidase. In this article, Peter Colman, Graeme Laver and their colleagues discuss some speculative implications of the results currently available.     

Monoclonal antibodies to sweet taste proteins. I. Analysis of antigenic epitopes on thaumatin by competitive inhibition assays.

Using a competitive inhibition binding immunoassay, we have examined some of the antigenic epitopes present on thaumatin, an intense sweet tasting protein from the African fruit katemfe. We have developed a library of monoclonal antibodies which react with different surface antigenic epitopes on thaumatin. Some of these monoclonal antibodies also cross-react with monellin, another unrelated sweet tasting protein. The competitive binding immunoassay examines the immunoreactivity of both solid-phase and liquid-phase monoclonal antibodies. At least six major antigenic epitopes on thaumatin were identified by our library of monoclonal antibodies. This type of competitive binding analysis may prove useful in the discovery of "sweet taste determinants" on plant proteins and in the development of tandem immunoassays for quantitation of sweet tasting proteins in plant extracts.     

Monoclonal antibodies to bluetongue virus define two neutralizing epitopes and a hemagglutinating epitope.

Monoclonal antibodies were used to characterize neutralizing epitopes on VP2 of bluetongue virus serotype 10 (BTV-10). Six neutralizing monoclonal antibodies that immune precipitated VP2 demonstrated two distinct patterns of reactivity in the competitive enzyme-linked immune absorbent assay (ELISA). These results suggest that there are at least two distinct domains of neutralization on VP2 of BTV. Monoclonal antibodies defining the two domains were serotype-restricted in plaque neutralization, immune precipitation or ELISA. One of the two neutralizing domains also demonstrated significant hemagglutinating activity. Both cattle and sheep infected with BTV-10 produce antibodies to the two neutralizing epitopes.     

Detection of species-specific and cross-reactive epitopes inSarcocystic cystozoites by monoclonal antibodies

A total of 24 hybridoma cell lines producing monoclonal antibodies (mAbs) toSarcocystis muris were established from 3 fusions performed on mice that had been immunized with total protein derived from cystozoites. In all, 19 mAbs were of the IgG₁ isotype, 3 were of the IgG_2a isotype, and 2 were of the IgG₃ isotype. The reactivity of the mAbs with homologous and heterologous antigens was examined by an indirect fluorescent antibody test, an enzyme-linked immunosorbent assay (ELISA), and a dot ELISA. MAbs were directed against various structures of the cystozoite as revealed by different immunofluorescence patterns. In all, 12 mAbs recognized only antigens ofS. muris cystozoites, whereas the other 12 mAbs also recognized various antigens of cystozoites ofS. gigantea, S. tenella, S. arieticanis, S. capracanis, S. miescheriana, orS. suihominis. No cross-reactions with endozoites ofToxoplasma gondii were observed.This publication is dedicated to Prof. Dr. J. Eckert on the occasion of his 60th birthday     

Monoclonal antibodies that recognize a specific surface antigen of Treponema denticola.

Spirochetes have been implicated as potential etiologic agents of periodontitis in humans. Murine monoclonal antibodies (MAbs) specific for a serogroup of Treponema denticola, an oral spirochete, were developed and characterized in this study. Antibodies secreted by clone IAA11 were judged to be the most useful, since they were able to detect 8 of 15 T. denticola strains. This MAb consisted of an immunoglobulin G3 heavy chain and a kappa light chain. MAb IAA11 was found to react with an epitope target located on the outer sheath of the cell wall. This MAb should be of diagnostic and scientific value in the study of T. denticola populations in human periodontitis.     

Monoclonal antibodies detecting discrete epitopes of human perforin

Perforin is a cytolytic protein of natural killer (NK) cells and cytotoxic T cells (CTL). Purified perforin has been shown to cause cell lysis and to form stable pores in the target cell membrane, but its relevance to cytolysis in vivo is not clear. The gene for human perforin has been cloned, but monoclonal antibodies (mabs) have not been available. In order to study further its role in cytotoxicity, we have generated mabs to different regions of human perforin. Four mabs were produced from mice immunized with hybrid proteins comprising E. coli TrpE protein at the N-terminus and different regions of human perforin at the C-terminus. These proteins were made using the pATH expression plasmids into which fragments of perforin cDNA were subcloned. Monoclonal antibody PA1 was made from a mouse immunized with a hybrid protein containing the C-terminal 240 amino acids (AA) of perforin, PE1 - the N-terminal 118 AA, and PB1 and PB2 - the central 199 AA. The three plasmid constructs contained non-overlapping cDNA segments which covered the entire sequence of perforin. All mabs reacted with the immunizing hybrid protein, but not with the other hybrid proteins, indicating that at least three epitopes are recognized by this set of mabs. All mabs immunoprecipitated a molecule of about 68 kd from lysates of metabolically-labelled cytolytic large granular lymphocytic leukemia cells, but not from control lysates of non-cytolytic promyelocytic U937 cells. These mabs should be of use in determining structure-function relationships for perforin.     

Monoclonal antibodies to murine CD40 define two distinct functional epitopes

Two rat IgG_2a antibodies which define distinct epitopes on murine CD40 have been generated. These antibodies specifically bind recombinant murine CD40 expressed on L cells, and the soluble extracellular domain of murine CD40 coated onto microtiter plates. Both antibodies bind B220⁺ but not B220 murine spleen cells, and immunoprecipitate a 45-kDa protein from the surface of purified murine splenic B cells. These antibodies exhibit separate functional properties, consistent with the notion that they define two distinct CD40 epitopes. One of the monoclonal antibodies (designated 1C10) directly induces a specific proliferative response from mature murine B cells, up-regulates several B cell surface antigens, and rescues immature B lymphoma cells from anti-IgM-induced growth arrest. The other monoclonal antibody (designated 4F11) exhibits none of these properties, but is capable of synergizing with suboptimal amounts of either anti-IgM antibodies or the 1C10 agonistic anti-CD40 antibody to produce an optimal proliferative response of purified small dense B cells. Furthermore, 4F11 antibody synergizes with suboptimal amounts of 1C10 antibody to rescue B lymphoma cells from anti-IgM-induced growth arrest. The 1C10 and 4F11 antibodies were unable to cross-block each other's binding to recombinant CD40 expressed in L cells, providing strong support for the notion that the antibodies recognize distinct epitopes on CD40. The potential implications of two functionally distinct CD40 epitopes are discussed.     

Inter-subtype cross-neutralizing antibodies recognize epitopes on cell-associated HIV-1 virions

HIV-1 infected individuals with cross-neutralizing antibodies against primary HIV-1 isolates belonging to Group M (envA-H) and O, are identified. To investigate the neutralization-kinetics of primary isolates with these antibodies, different neutralization assay conditions are compared. Each set is summarized as a/b/c where a is the time in hours for which antibody is incubated with virus, b is the time in hours allowed for virus to absorb to cells, c is the total culture period in days, from the cells' first exposure to virus, before antigen production (peripheral blood mononuclear cells) or number of fluorescent cells (GHOST) are measured. In HIV-infected individuals, neutralizing antibodies can be detected against a wide range of primary isolates (Group M; A-H and Group O) in PBMC-assays with short incubation phases (1/2/7 or 1/24/7). If cultures are extended (1/2/14 or 1/24/14), however, neutralization can be lost. In kinetic experiments, neutralization can even be seen without pre-incubation (a=0 hr). This study shows that neutralization of primary HIV isolates by cross-reactive antibodies can continue after the virus has bound to its target cell. This neutralization, however, is not an all or nothing loss in virus infectivity. Most often it leads only to a reduction in viral replication rates.     

Monoclonal antipeptide antibodies recognize epitopes upon VP4 and VP7 of simian rotavirus SA11 in infected MA104 cells

Summary To study morphogenetic events of rotavirus SA11-infected MA104 cells with strictly defined reagents we produced monoclonal antibodies against synthetic peptides from both outer capsid proteins VP4 (aa residues 228–241: QNTRNIVPVSIVSR) and VP7 (aa residues 319–326: SAAFYYRV) of simian rotavirus SA11. Two of the selected monoclonal antibodies proved to be reactive with determinants of SA11-infected MA104 rhesus monkey kidney cells, with purified SA11 as well as with the particular peptides used for immunization. The anti-VP4 antibody had a demonstrable neutralizing titer of 200 (50% focus reduction) whereas the anti-VP7 MuMAb revealed no detectable neutralizing activity. In peptide-inhibition experiments, the corresponding peptide inhibited its MuMAb whereas the noncorresponding peptide had no effect on antibody binding to intracellular viral antigen. Localization of VP7 was preceded by VP4 as shown by immunofluorescence microscopy.     

Monoclonal antibodies to distinct epitopes on human IgA and their use to IgA determination

Several monoclonal antibodies (MAbs) against human IgA have been obtained which specifically bind to human myeloma and polyclonal IgA. Three of these MAbs have been purified and studied further. They recognize both IgA subclasses and define three distinct epitopes on the IgA molecule. These MAbs were used to develop a solid-phase radioimmunoassay (RIA) in which one MAb was immobilized and the other two were labeled with 125I. The assay has a sensitivity in the nanogram range. A good correlation was found (r = 0.97, P less than 0.001) when the solid-phase RIA was compared with a commercially available immunodiffusion technique for the determination of IgA levels in serum samples.     

Monoclonal antibodies to human intermediate filament proteins. III. Analysis of tumors

A panel of monoclonal antibodies to human intermediate filament proteins was tested on an unselected series of 246 neoplasms. The antibody panel includes two different anti-cytokeratin antibodies, an anti-vimentin antibody, and an anti-neurofilament antibody (Gown and Vogel, Am J Pathol 114:309, 1984). The studies were done on Carnoy's or methacarn-fixed, paraffin-embedded tissue. When used as a panel, they can unequivocally distinguish carcinomas, melanomas, and lymphomas. All carcinomas react with at least one of the anti-cytokeratin antibodies, and carcinomas can be subtyped based upon the pattern of reactivity with the two anti-cytokeratin antibodies. Melanomas react only with the anti-vimentin antibody, and lymphomas react with none of the antibodies. Neural and neuroendocrine tumors can be identified with the anti-neurofilament antibody. A minority of neoplasms, including lymphomas, seminomas, and some sarcomas, do not react with any of the antibodies. These antibodies are reliable diagnostic reagents that are useful in distinguishing different categories of human tumors.     

Equine monoclonal antibodies recognize common epitopes on variants of equine infectious anaemia virus.

Equine-murine xenohybridoma cells were produced using SP2/0 murine myeloma cells and splenic lymph node cells obtained from horses infected with 10(6) TCID50 of single cloned variants of equine infectious anaemia virus (EIAV). The xenohybridomas secreted equine IgG monoclonal antibodies reactive with EIAV in enzyme immunoassays employing purified virus. Seven antibodies were studied in detail. They bound to viral glycoproteins (gp90 or gp45) in radioimmunoprecipitation assays, and reacted with homologous EIAV as well as five other cloned variants of EIAV. When evaluated against a single cloned variant of EIAV (EIAV-WSU5), two antibodies bound to different epitopes on gp90. The five remaining antibodies reacted with the same or overlapping epitopes on gp45. None of the antibodies exhibited viral neutralizing activity.     

Monoclonal antibodies to Staphylococcus aureus laminin-binding proteins cross-react with mammalian cells.

We and others have previously shown that some microorganisms, including bacteria, express on their surfaces receptors that specifically recognize extracellular matrix proteins, such as laminin, fibronectin, or both. The ability of microorganisms to adhere and to invade might depend on the existence of receptors which could, thus, be correlated with pathogenicity. In the present paper, we report the isolation of five stable cell lines that were producers of monoclonal antibodies to Staphylococcus aureus laminin receptors. One of these antibodies, which was of the immunoglobulin M isotype, blocked the binding of laminin to bacteria before and after fixation and recognized the putative 52-kilodalton laminin-binding protein in whole bacterial extracts. Also, purified receptor was isolated by immunoaffinity chromatography and shown to bind laminin. Furthermore, the same antibodies bound the 67-kilodalton putative receptor from mouse melanoma cells and gave positive immunofluorescence reactions against mammalian tumor cells. These data strongly suggest either the evolutionary conservation of at least some sequences in both procaryotic and eucaryotic laminin-binding proteins or convergent evolution and positive selection of epitopes cross-reacting with laminin. Some of these antibodies to the procaryotic protein could therefore become useful markers for the expression of laminin receptors by cancer cells.     

Anti-Hsp65 antibodies recognize M proteins of group A streptococci.

Group A streptococcal M protein and the mycobacterial heat shock protein, hsp65, are strong bacterial immunogens that have been linked to arthritis and autoimmunity. Recent evidence has shown that streptococcal arthritis and adjuvant arthritis may be related to epitopes shared between group A streptococci and hsp65. We investigated the possibility that immunological similarities were shared between streptococcal M protein and hsp65. Antibodies against the 65-kDa heat shock protein of Mycobacterium tuberculosis were tested for reactivity with group A streptococci and purified recombinant M proteins (rM5 and rM6). Rabbit polyclonal anti-hsp65 serum was highly reactive with M type 5 Streptococcus pyogenes and rM5 and rM6 proteins in an enzyme-linked immunosorbent assay (ELISA). A mouse anti-hsp65 monoclonal antibody (MAb), IIC8, reacted with streptococcal M types 5, 6, 19, 24, and 49 in an ELISA but showed no reactivity with an isogenic streptococcal mutant which did not express M protein. Anti-hsp65 MAb IIC8 recognized rM5 and rM6 proteins in the ELISA, and MAbs IIC8 and IIH9 reacted strongly with rM6 protein in Western immunoblots. The binding of M protein by anti-hsp65 MAbs was shown to be inhibited by both hsp65 and M protein. These data show that anti-hsp65 antibodies recognize streptococcal M proteins.     

Monoclonal antibodies and fusion proteins in medicine

Humanized antibodies and decoy receptors have been introduced in clinical practice to treat malignancies and systemic autoimmune disease because they ablate specific cells or disrupt pathogenic processes at distinct points. Reported clinical responses offer hope to treatment-resistant patients, particularly those with lymphomas and rheumatic diseases. Side effects from the use of biologic agents include lymphokine release syndrome, reactivation of tuberculosis, and immunosuppression. Further insights are needed regarding limitation of adverse effects, correct use in conjunction with existing drugs, and treatment of patients in whom resistance develops.