mAb Group Research Articles

New hapten-protein conjugation method using N-( m-aminobenzoyloxy) succinimide as a two-level heterobifunctional agent: thyrotropin-releasing hormone as a model peptide without free amino or carboxyl groups

The use of a two-level heterobifunctional agent N-( m-aminobenzoyloxy)succinimide ( m-ABS) allowed us to develop a new method for preparing hapten-protein conjugates. This was demonstrated by a conjugation between thyrotropin-releasing hormone (TRH) and bovine or human serum albumin (BSA or HSA). The conjugation is based on the principle that the succinimidyl ester group of m-ABS immediately acts on an ε-amino group of lysine residues of carrier protein BSA (or HSA) and a m-aminobenzoyl group incorporated into the protein is then activated by diazotization to a functional m-diazobenzoyl group ( m-DB) acting on a histidyl group of TRH. The TRH-BSA containing about 3.5 mol of TRH per BSA molecule, elicited the production of TRH antibody in rabbits. A new type of enzyme-linked immunosorbent assay (ELISA) for TRH was developed using the antiserum, the solid-phase antigen TRH-HSA and the commercially available horseradish peroxidase-labeled goat anti-rabbit IgG/Fab′ as a marker, revealing that the ELISA was monospecific to the hormone and measured as low as 50 pg of the hormone reproducibly. Also, using the antiserum by the indirect immunoperoxidase method the distribution of immunoreactive TRH in the rat brain was demonstrated in neurons of the paraventricular nucleus and neuronal processes of the median eminence. These results strongly suggested that the use of m-ABS provided a simple and efficient new method for preparing immunogens not only for the previously reported haptens with a primary amino group(s) (J. Immunol. Methods 134 (1990) 227), but also for haptens with an imidazole, phenolic, or indole group(s) in the molecule.

Group-selective immunoassay

A new solid-phase immunoassay technique has been applied for anti-digoxin monoclonal antibodies (Mabs) detection. At the first assay step, anti-digoxin Mabs (IgG1, K aff = 9.2 × 10 9 M −1) were bound to a specially prepared immunosorbent, the microtiter plates coated with digoxin-human serum albumin conjugate (Dig-HSA), in which free amino groups were protected by a glutaraldehyde cross-linking modification. The modification did not essentially influence the antibody-binding capacity of the immunosorbent. After antigen-antibody reaction, free amino groups were located only on the anti-digoxin Mabs, bound to chemically modified immunosorbent. At the second assay step, free amino groups of anti-digoxin Mabs were biotinylated by N-hydroxysuccinimide-biotin ester. Then the biotin residues were detected by the streptavidin-peroxidase conjugate. The method does not require second labeled antibodies and may be used for anti-hapten hybridoma screening.

Monoclonal antibodies against the heptose region of enterobacterial lipopolysaccharides

Murine monoclonal antibodies (MAbs) against the rough mutant lipopolysaccharides (LPS) of Salmonella minnesota strain R4 and R7 (chemotypes Rd2P - and Rd1P-, respectively) were obtained after immunization of BALB/c and NZB mice, respectively, with heat-killed bacteria. A total of 5 MAbs was obtained which were serologically characterized by hemagglutination, EIA and Western blot using LPS, de-O-acylated LPS and dephosphorylated LPS. In addition, the completely deacylated carbohydrate backbone, the deacylated, dephosphorylated and reduced carbohydrate backbone, and synthetic partial structures were covalently linked to bovine serum albumin resulting in artificial glycoconjugate antigens. Both groups of MAbs (anti-R4 and anti-R7) were highly specific for the Rd2P- and Rd1P- chemotypes, respectively, since they did not react with LPS of the Ra, Rb, Rc or Re chemotype. The R4 antibodies required for binding the core region [composed of 3-deoxy-α-D- manno-octulosonic acid (Kdo) and L-glycero-α-D- manno-heptopyranose (Hep)] and the phosphorylated glucosamine backbone of the lipid A moiety. The R7 antibodies bound to some extent to the core oligosaccharide Hep(1→3)Hep(1→5)Kdo(2→6), however, better binding was observed when the glucosamine backbone of lipid A was also present. In this case however, phosphoryl groups were not required. These antibodies can detect enterobacteria with an LPS of the Rd-chemotype, and may be useful to characterize also other naturally occurring rough mutants or genetically manipulated strains used to clone genes of the rfa locus.

Identification of a combinatorial epitope expressed by the integrin alpha 4 beta 1 heterodimer involved in the regulation of cell adhesion.

The alpha 4 integrin subunit can associate with either the beta 1- or beta 7-integrin subunit to form two unique adhesion receptors alpha 4 beta 1 and alpha 4 beta 7. We developed a monoclonal antibody (mAb 19H8) that immunoprecipitated alpha 4 beta 1, induced homotypic leukocyte aggregation, and blocked the binding of cells to a synthetic peptide corresponding to the CS-1 peptide region of fibronectin. Aggregation cross-blocking analysis suggested that mAb 19H8 belonged to the group of mAbs that react with the B2 epitope of the alpha 4 subunit (alpha 4.B2 epitope); however, unlike the alpha 4.B2-specific mAb L25, mAb 19H8 did not immunoprecipitate alpha 4 beta 7. In addition, mAb 19H8 did not bind to beta 1-positive cells unless transfected with alpha 4 cDNA. These results indicated that mAb 19H8 was not specific for an individual alpha 4, beta 1, or beta 7 subunit but reacted with an epitope formed from the association of alpha 4 with beta 1. Separating the alpha 4 from the beta 1 subunit, by removing divalent cations or by treatment with high pH, disrupted mAb 19H8 binding. In contrast, the alpha 4-specific mAb L25 and the beta 1-specific mAb 18D3 could react with their respective subunits without subunit association. Therefore, mAb 19H8 defined a novel regulatory epitope expressed by the integrin alpha 4 beta 1.

Antigenic characterization of serogroup 'A' of infectious pancreatic necrosis virus with three panels of monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against three serotypes of infectious pancreatic necrosis virus (IPNV): A1 (LWVRT 60-1, U.S.A.), A2 (d'Honnincthun, France) and A9 (Jasper, Canada). Each panel of MAbs (identified as LW, HF and JA) was analysed by ELISA with the 10 proposed serotypes of IPNV and their specificity defined by immunoprecipitation and Western immunoblotting analysis. A first group of MAbs, directed against the outer capsid protein VP2, reacted with linear or conformational epitopes. A second group of MAbs, directed against the internal protein VP3, reacted with linear epitopes. There was no relationship between the neutralizing property of anti-VP2 MAb and the configuration of the epitope that it recognized. The MAbs were used for antigenic characterization of serogroup A. Each panel of MAbs showed a characteristic pattern of reactivity. The European HF series was predominantly cross-reactive and detected conserved epitopes among the 10 serotypes for both VP2 and VP3. The North American LW and JA series identified a group of conserved epitopes on VP3 and new specific epitopes on VP2 and VP3. The higher variability observed for VP2 in comparison with VP3 is one example of how external pressures may promote natural selection of those epitopes required for virus survival. Our results are consistent with an ancestral relationship of the European to the North American strains, the latter having developed new antigenic determinants upon evolution in their new geographical location.

Characterization of monoclonal antibodies raised against a synthetic peptide capable of inducing a neutralizing response to human rhinovirus type 2

Synthetic peptides incorporating the derived amino acid sequence of VP2 residues 156 to 170 of human rhinovirus type 2 (HRV2) have previously been shown to elicit antibodies that neutralize virus infectivity. The proportion of virus-reactive antibodies present in polyclonal antisera to these peptides is, however, very low. Moreover, neutralization titrations of such antisera correlate poorly with other assays of either anti-virus or anti-peptide activity, suggesting the presence of antibodies with different specificities. To investigate these findings further, we produced a panel of monoclonal antibodies (MAbs) to VP2 peptides of residues 156 to 170 and characterized their reactions with a range of antigens in ELISA, precipitation and neutralization titrations. All the MAbs obtained recognized the homologous peptide, but could be divided into four main reaction groups according to their specificity for viral antigens. Antibodies in the first group recognized and neutralized native virus, apparently by preventing attachment to cells. A second group of MAbs bound to intact particles with similar affinities to the first group, but failed to neutralize infectivity. Antibodies in the third group recognized virus only after capsid distortions incurred by heating or by previous reaction with polyclonal antibodies. The fourth group comprised MAbs that were mainly peptide-specific. Some possible applications of anti-peptide MAbs to improving the design of peptide immunogens are considered.

Leukocyte CD18 monoclonal antibody worsens endotoxemia and cardiovascular injury in canines with septic shock

We investigated the effects of a murine monoclonal antibody directed against the canine leukocyte CD11/18 adhesion complex (MAb R15.7) in a canine model of septic shock. Awake 2-yr-old purpose-bred beagles were studied 7 days before and 1, 2, 4, and 10 days after intraperitoneal placement of an Escherichia coli-infected fibrin clot. Starting 12 h before clot placement, animals received 0.5-1 mg/kg iv every 12 h (4 doses total) of either MAb R15.7 (MAb group, n = 8) or, as controls, murine serum protein (n = 8). After infected clot placement, all animals received antibiotic (ceftriaxne, 100 mg.kg-1.day-1 for 4 days). Two of eight control animals and four of eight MAb animals died (P = 0.4). During the first 8 h after clot placement, MAb animals, compared with control animals, had greater (P < 0.06) increases in serum endotoxin levels and higher (P < 0.05) neutrophil counts. Day 1 after clot placement, MAb animals, compared with control animals, had decreased (P < 0.05) central venous pressure and arterial pH and increased (P < 0.05) arterial lactate. Day 2 after clot placement, MAb animals, compared with control animals, had decreased (P < 0.05) cardiac index and mean arterial pressure. In summary, MAb R15.7, although associated with increased neutrophil counts, worsened serum endotoxemia, acidosis, and cardiovascular function in this canine model of septic shock. These data suggest that in septic shock, antibody directed against this leukocyte membrane protein complex may be harmful, possibly via impairment of normal leukocyte function.

Structural requirement of CRK SH2 region for binding to phosphotyrosine-containing proteins. Evidence from reactivity to monoclonal antibodies.

The SH2 region of signal-transducing proteins mediates binding to phosphotyrosine-containing proteins. We analyzed the structure-function relationship of the SH2 region of the human CRK protein by using a series of monoclonal antibodies (mAbs). Seventeen mAbs against the CRK SH2 region were classified into 5 groups according to the reactivity with mutant CRK proteins expressed in COS7 cells and in Escherichia coli and by epitope scanning with synthetic nonapeptides. Two groups of mAbs (groups A and B) were reactive only with intact SH2. Mutation(s) in either the amino-terminal B box or the carboxyl-terminal C box, which are the two subdomains of SH2, abolished the reactivity of the CRK mutants to the mAbs of groups A and B. Group A mAbs competed the binding of the CRK SH2 region to the phosphotyrosine-containing proteins. Moreover, the spectrum of the CRK mutants which were recognized by group A mAbs coincided with that of the CRK mutants which could bind phosphotyrosine-containing proteins, suggesting that group A mAbs were directed against the site of binding to phosphotyrosine-containing proteins. Group C mAbs, directed against the region between the B and C boxes, were reactive with both wild-type and mutant CRK proteins and did not affect the capacity of SH2 to bind phosphotyrosine-containing proteins. Contrary to group A and B mAbs, mAbs belonging to groups D and E, which were mapped onto the C box, did not bind well to the native CRK proteins, but bound to CRK mutants with mutation(s) in either the B or the C box. These results suggest that the B and C boxes, which are separated by a hinge region, coordinately form the functional SH2 domain that binds to the phosphotyrosine-containing proteins and to the group A mAbs.

Monoclonal IgG and IgM autoantibodies obtained after polyclonal activation, show reactivities similar to those of polyclonal natural autoantibodies

In this study, we described the properties of two groups of polyreactive IgG monoclonal antibodies (mAb) derived from splenocytes of a Plasmodium chabaudi-infected BALB/c mouse. These IgG mAb reacted with self antigens, but not with the parasite. Depending upon the antigen under study, they showed low (10 −5 M) to high (10 −8 M) affinities. When examined by immunoblotting using mouse organ extracts as the antigen source, each IgG mAb had a different reactivity profile. The IgG2b, but not the IgG2a mAb group, reacted with F(ab') 2 fragments of IgG from normal mouse sera, as well as with F(ab') 2 fragments of other mAb from the same fusion. Their anti-F(ab') 2 activity was inhibited by various antigens, suggesting that the binding sites for these antigens and F(ab') 2 fragments were close or overlapping. We also characterized two monoclonal IgM that were strongly and almost exclusively reactive with polyclonal F(ab') 2 fragments of normal IgG, and that inhibited the binding of normal polyclonal IgG to self antigens. We previously described such properties for polyclonal IgM from normal mouse sera. The binding of the F(ab') 2-reactive IgG mAb to self antigens was also inhibited by normal polyclonal IgM. These results indicate that the IgG and IgM obtained after polyclonal stimulation, exhibit characteristics similar to those of autoantibodies present in normal mouse sera. Furthermore, they confirm our previous studies showing the polyreactivity of polyclonal IgG. Finally, they also show that such IgG mAb exhibit properties similar to those of natural IgM, namely polyreactivity, affinity and connectivity.

Epitope mapping of human immunoglobulin-specific murine monoclonal antibodies with domain-switched, deleted and point-mutated chimeric antibodies

27 engineered chimeric antibodies possessing human γ, ε, μ or α constant regions and V region specificity for nitrophenyl or dansyl were used to study the isotype specificity of 29 murine monoclonal antibodies (MAbs) specific for human immunoglobulins (IgG1–4, IgE, IgM, IgA or secretory piece). The isotype-restricted immunoreactivity observed with wild-type chimeric antibodies parallel the pattern of each MAb's reactivity with purified human myeloma proteins. 16 mutant IgG anti-dansyl chimeric antibodies with genetically engineered domain switches, deletions or point-mutations were used as antigens to further characterize the epitopes recognized by the human IgG subclass-specific MAbs. The binding of three human IgG1-specific MAbs (HP6069, HP6070 and HP6091) was mapped to similar epitopes on the C H2 domain of human IgG1. Of the two anti-human IgG2 MAbs tested, HP6002 reacted man IgG3 MAbs (HP6047, HP6050) reacted with different regions of the IgG3 hinge. The anti-human IgG4 MAbs (HP6023, HP6025) bound to a similar epitope on the carboxyl terminus of C H2 or the C H3 of human IgG4. The three exclusion antibodies (HP6019, HP6030 and HP6058) bound to different epitopes in the C H2 domain of three of four IgG subclasses. The domain mapping was confirmed by competitive inhibition experiments. These results were used to select a group of IgG-reactive MAbs for construction of a poly-monoclonal anti-IgG capture and detection reagent that uniformly bound all four subclasses of human IgG. This study provides support for the use of engineered chimeric human chimeric antibodies as replacements for increasingly rare, purified human paraproteins in the specificity analysis of immunochemical reagents used in clinical and research laboratories for the detection and quantitation of human antibodies. Moreover, these studies demonstrate how the MAbs can serve as effective probes for examining conformational differences among the four human IgG subclasses.

Two Immunodominant Regions Revealed by Monoclonal Antibodies on the Main Structural Protein p24 of Bovine Leukemia Virus

Eleven different monoclonal antibodies (Mabs) directed against the main structural protein p24 of bovine leukemia virus (BLV) were prepared. All Mabs reacted with p24 in Western blot and in radioimmunoprecipitation. Competition antibody binding assays with the prepared Mabs distinguished three independent groups of Mabs. Two immunodominant regions (IDRs) of p24 BLV were defined by these Mabs. The Mabs were induced preferentially against two immunodominant regions on the native form of p24 BLV (BLVp24 IDR-1 and BLVp24 IDR-2). Mab of the third group was directed against a different immunogenic epitope of p24 BLV. A model of the IDRs based on the differences in the fine epitope specificity of Mabs defining these immunodominant regions is proposed.

Protective murine monoclonal antibodies to Cryptococcus neoformans.

Several murine monoclonal antibodies (MAbs) specific for the capsular glucuronoxylomannan of Cryptococcus neoformans were studied for their capacity to confer protection when passively administered to lethally infected mice. The MAb group studied recognized at least three distinct epitopes and included immunoglobulin M (IgM), IgG3, IgG1, and IgA isotypes. The protection model used A/J and BALB/c mice infected intraperitoneally with 10(8) cryptococci. The MAbs were administered either immediately preceding or, in one experiment, 24 to 48 h prior to infection. Protective efficacy was assessed by the ability of passively administered MAbs to prolong the survival of lethally infected mice. Three IgM MAbs, each of which recognized a distinct epitope, were able to prolong survival of lethally infected mice to different extents. A set of IgM, IgG3, IgG1 and IgA MAbs which utilize the same immunoglobulin gene elements and were derived from the same B-cell clone exhibited significant class differences in protective efficacy with IgA, IgG1 > IgM > IgG3. The results confirm that protective MAbs against C. neoformans capsular polysaccharide exist and strongly suggest that both epitope specificity and isotype are important determinants of protective efficacy.

Enhanced association of plasminogen/plasmin with lesional epidermis of bullous pemphigoid.

The distribution of plasminogen/plasmin, the central proteolytic component of the plasminogen activator/plasmin system was analysed in lesional skin of bullous pemphigoid by using monoclonal antibodies (MAbs) specific for distinct epitopes of the plasminogen/plasmin molecule. Four groups of MAbs were used: (i) MAbs HD-PG 1 and HD-PG 2, specific for epitopes associated with the lysine-binding sites I (kringle domain 1 + 2 + 3) and II (kringle domain 4) of plasminogen/plasmin, (ii) MAbs HD-PG 6 and HD-PG 7, specific for the lysine binding site I only, (iii) MAbs HD-PG 12 (formerly designated P 2) and HD-PG 18, specific for non-kringle domains of glu- and lys-plasminogen, and (iv) MAb HD-PG 13 which recognizes glu-plasminogen, only. The basal cell layers of normal skin consistently reacted with MAb HD-PG 12, whereas only faint staining was seen with the other MAbs in the same biopsies. In contrast, all anti-plasminogen/plasmin MAbs strongly stained lower and intermediate epidermal cell layers of fully developed bullous pemphigoid lesions.

Epitope analysis of an immunodominant domain on the P1 protein of Haemophilus influenzae type b using synthetic peptides and anti-idiotypic antibodies

Synthetic peptides, anti-idiotypic antibodies (anti-Id) and human and murine monoclonal antibodies (mAbs) were used to further define a major antigenic domain on the outer membrane P1 protein (OMP) of Haemophilus influenzae type b (Hib). Synthetic peptides were elaborated from the known primary sequences of the P1 protein of prototype Hib strains MinnA (OMP subtype 1H) and 8358 (OMP subtype 6U). By peptide mapping, antibodies are categorized into three groups: A, B and C. A first epitope on the P1 from strain MinnA was identified by the reactivity of one set of murine anti-P1 mAbs with the two overlapping peptides 11H and 13H, corresponding to amino acid residues 384–412 and 400–437, respectively. On the basis of their reactivity with both peptides, these mAbs were designated as group A. Anti-Id obtained from mice immunized with two group A mAbs reacted specifically with all group A mAbs. A second epitope on the same P1 protein was identified by the reactivity of the peptide 13H with another distinct set of murine anti-P1 mAbs assigned to group B. This group of mAbs did not recognize the peptide 11H. Murine anti-Id which were prepared against one group B mAb inhibited the attachment of this mAb to outer membrane preparations, whereas the binding of the other group B mAbs was not affected, suggesting that these mAbs represent a heterologous group of mAbs. The epitope(s) recognized by two human anti-P1 mAbs was (were) distinct from the ones recognized by murine mAbs since no reactivity with the peptides was observed. Similarly, the binding of the two human mAbs to the P1 antigen was not inhibited by anti-Id raised against group A or B mAbs. Interestingly, an epitope on a different P1 protein recovered from strain 8358 was identified by the reactivity of group C murine mAbs with the peptide 13U, which occupies the same position on the P1 protein as 13H but differs from the latter by 10 amino acid residues. Our studies demonstrated the presence of several distinct surface-exposed B-cell epitopes within the antigenic domain which was defined previously on the P1 protein of Hib MinnA. Furthermore, we showed the immunodominance of this region on two different P1 proteins. None of the mAbs, however, had a bacteriolytic or protective activity against Hib strains. We suggest that the surface-exposed immunodominant region on the OMP P1 of Hib do not induce protective antibodies against Hib infection.

Cell surface display of rat invariant gamma chain: detection by monoclonal antibodies directed against a C-terminal gamma chain segment.

A series of 14 monoclonal antibodies (mAb) directed against the C-terminal part of the rat invariant gamma chain (amino acid 142-216) was generated using distinct fusion proteins that contain this gamma segment for immunization and hybridoma screening. Additional fusion protein were prepared carrying discrete regions of the gamma chain. Employing these reagents confirmed that the obtained mAb do indeed recognize the C-terminal portion of the invariant chain, as demonstrated by Western blot analysis. All mAb established recognize epitopes present on the native gamma chain, as revealed by immunoprecipitation analysis using nonionic detergent extracts of metabolically labeled Lewis rat splenocytes combined with two-dimensional gel electrophoresis. However, while the majority of the gamma chain-specific mAb precipitated gamma chain-containing polypeptide chain complexes in which immature, sialic acid-deficient and mature, terminally sialylated forms of the gamma chain were predominantly represented, a fraction of the antibodies preferentially precipitated the immature gamma forms. Cell surface binding of these two groups of mAb correlated with the immunoprecipitation data in that the former group of antibodies did bind to intact Lewis rat spleen cells, while essentially no binding was observed with the antibodies of the latter group. Double-fluorescence staining with the class II-specific fluorescein isothiocyanate-conjugated mAb OX3 and OX6, respectively, as well as a representative gamma chain-specific mAb visualized with phycoerythrin-coupled secondary antibody shows coexpression of class II determinants and the invariant chain at the cell surface.

Five novel antigens illustrate shared phenotype between mouse thymic stromal cells, thymocytes, and peripheral lymphocytes

Previously we characterized by immunohistology a group of rat anti-mouse thymic stromal mAbs (MTS 12, 32, 33, 35, and 37), which recognized novel plasma membrane determinants on both thymic stromal cells (TSCs) and thymocytes. The present study investigates in more detail this incidence of shared phenotype by an extensive flow cytometric analysis of MTS mAb reactivity on TSCs, thymocyte subsets, peripheral lymphocytes, and bone marrow cells. Examination of freshly isolated or cultured heterogeneous TSCs and TSC clones confirmed that the mAb identified plasma membrane molecules on distinct subsets of these cells. All but MTS 12 reacted with epithelial cells. Triple-labelling illustrated that MTS 32, 33, and 37 were also reactive with more than 90% of total thymocytes, but varied in their distribution on the four major CD4 and CD8 defined subsets. MTS 12, staining with thymic vascular endothelium by immunohistology, labelled more than 95% of each subset. MTS 35 reactivity in each subset correlated strongly with only the immature populations. Examination of peripheral lymphocytes by triple- and double-labelling unexpectedly showed that MTS 33, 35, and 37 did not recognize peripheral T cells but labelled all B cells. MTS 32 was negative for B cells, but positive for all CD8+ T cells, yet only a subset of CD4+ T cells. Further, MTS 33, 35, and 37 were present on a significant percentage of bone marrow cells. MTS 12 reacted with virtually all peripheral T and B cells, and about 50% bone marrow leukocytes. Collectively these results reveal the same novel epitopes on different thymic cell types and subsets thereof, highlighting specific similarities between cells of apparently diverse lineages. These findings may be of importance in the delineation of intercellular communications within the thymus and emphasize the integrated nature of the microenvironment in this organ.

Structure‐function studies on human C4b‐binding protein using monoclonal antibodies

Human C4b-binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K-dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160-kDa chymotryptic central core fragment were prepared to study the structure-function relationships of C4BP. By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By rotary shadowing, spider-like images of C4BP-antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody-C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1:1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme-linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide-linked beta chain of C4BP were identified that inhibited the binding of protein S to C4BP. In a binding assay using 125I-labeled C4BP and solid-phase C4b, the inhibitory effect of one group of anti-C4BP mAb on the binding of C4BP to C4b was demonstrated.

The eortc melanoma group exchange program: Evaluation of a multicenter monoclonal antibody study

In the framework of the European Organisation for Research and Treatment of Cancer (EORTC), the Immunology and Pathology Subgroups of the Malignant Melanoma Cooperative Group undertook a large multicenter monoclonal antibody (MAb) study. Fourteen laboratories from 7 European countries tested a panel of 23 MAbs for immunohistological staining reactivity for malignant and non-malignant lesions involving the melanocytic lineage. A standardized immunoperoxidase procedure was used and the results were evaluated using a standard protocol and data evaluation form developed in collaboration with the EORTC Data Center. According to this analysis, the antibodies in the panel could be classified into 3 main groups. The first group of MAbs includes those antibodies which stained the majority (greater than 80%) of all primary tumors, irrespective of their Breslow thickness and the majority of metastatic lesions. In addition, these MAbs stained a high percentage of cells within a given lesion. Several antibodies of Group I were likewise reactive with the majority of naevoblasts and with normal melanocytes. The second group of MAbs included antibodies reacting only with a limited number of primary melanomas and metastatic lesions. Antibodies of Group II reacted only weakly, if at all, with normal melanocytes or naevocytes. The percentage of cells within a malignant lesion stained by these MAbs was always rather low. The MAb group III detected surface structures whose expression appeared to be related to tumor progression; they did not react or reacted only weakly with naevi, and they all reacted with a small number of early primary melanomas (less than 0.75 mm). The number of lesions stained increased with increasing Breslow thickness. Our study suggests that the application of a panel of well defined MAbs might be of diagnostic and prognostic value in evaluating malignant melanoma.

Identification of a neutralization epitope on the envelope gp46 antigen of human T cell leukemia virus type I and induction of neutralizing antibody by peptide immunization.

We have generated a number of mAb against various epitopes on the external envelope glycoprotein, gp46, of human T cell leukemia virus type I (HTLV-I) from a WKA rat immunized with a recombinant vaccinia virus containing the HTLV-I env gene. Among these mAb, one group of mAb, represented by a mAb designated LAT-27, could neutralize the infectivity of HTLV-I, as determined by a HTLV-I-mediated cell fusion inhibition assay. LAT-27 also interfered with transformation of normal T lymphocytes by HTLV-I in vitro. An antibody-binding assay using overlapping synthetic oligopeptides showed that LAT-27 bound specifically to 10-mer peptides that contained the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser-Asn-Leu). Antibodies from HTLV-I+ humans interfered with the binding of LAT-27 to gp46 Ag. Sera from rabbits immunized with a LAT-27-reactive peptide, 190-199, conjugated with OVA, but not sera from OVA-immunized rabbits, reacted with gp46 Ag and neutralized infectivity of HTLV-I. These results show that the HTLV-I neutralization epitope recognized by LAT-27 locates to the gp46 amino acids 191-196, and that immunization with a peptide containing the LAT-27 epitope can elicit an HTLV-I neutralizing antibody response.

Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates

Monoclonal antibodies (MAbs) directed against two phenotypically distinct ovine lentivirus (OvLV) strains were generated by fusion of BALB/c SP2/0-Ag 14 myeloma cells with spleen cells from mice immunized with purified OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag-encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MA p17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competition ELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis-encephalitis virus (CAEV). Analysis of the data indicated that these epitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on immunoblots. An anti-MA p17 MAb recognized the same OvLV strains on immunoblots but failed to recognize CAEV. MAbs which recognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MA p17) are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats.