Murine monoclonal antibodies that identify antigenically distinct subpopulations of human sperm

Murine monoclonal antibodies (MoAbs) were isolated to characterize antigenically distinct subpopulations of human sperm. Spleen cells from Balb/c mice immunized with freshly prepared human sperm, were fused with murine P3-NS1-Ag4-1 myeloma cells by somatic cell hybridization, and supernatants from the IgG-secreting hybridomas were screened by an enzyme immunoassay (EIA) for reactivity against fresh human sperm and a panel of human somatic cells. Two MoAbs, SP1D1 and SP7A7, reacted specifically with human sperm, whereas three others, SP2A9, SP3B3, and SP4F5, cross-reacted with a variety of human somatic cells. The binding of MoAbs were characterized by immunofluorescence, agglutination, and Staphylococcus aureus binding assays. We found that certain MoAbs bound to common antigens of the head and tail, or to tail alone, and had agglutinating activity. However, not all sperm were reactive to antibody, and the binding activity could only be demonstrated in subpopulations of sperm ranging from 5 to 50% of the total number.     

Conserved antigen expression in epithelial tumors recognized by monoclonal antibody 4A9 generated against canine mammary carcinoma cells

Hybridoma-derived murine monoclonal antibodies (MoAbs) were generated by fusing P3X63-Ag8.653 myeloma cells with splenic cells from BALB/c mouse which had been immunized with viable canine mammary adenocarcinoma cells, CMT-2. Fifteen MoAbs were shown to react with immunizing cells in indirect immunofluorescence (IFA) and enzyme-linked immunosorbent (ELISA) assays. The reactivity of one IgM MoAb, designated 4A9, was evaluated. The antigen recognized by 4A9 on CMT-2 cells appeared to be localized both in cell membrane and cytoplasm against fixed and unfixed preparations by IFA. The 4A9 MoAb was found to bind with four of five canine mammary carcinoma cell lines while no binding was detected with normal fibroblastic cell lines. In vivo tissue distribution of 4A9 antigen was evaluated by indirect immunoperoxidase (IP) assay against formalin-fixed, paraffin-embedded sections of normal and neoplastic tissues. 4A9 MoAb reacted strongly to moderately with 75% of mammary carcinomas, moderately to weakly with 57% of benign mammary tumors, and strongly with squamous cell and perianal gland carcinomas (100%), interstitial cell tumors (100%), transitional cell carcinomas (43%), lung adenocarcinomas (40%), colon carcinomas (33%), and pancreatic adenocarcinomas (20%). Moderate to weak staining was detected with granulosa cell tumors (25%) and apocrine gland adenocarcinomas (50%). Strong reactivity with perianal gland carcinomas contrasted to no reactivity with perianal gland adenomas. No immunostaining was detected with a large variety and number of normal adult and fetal tissues tested; negligible and very restricted staining was observed in a few adult and fetal tissues. Normal mammary gland was negative. Since the antigen is expressed on the cell surface and in the cytoplasm of most mammary carcinoma cells and a variety of other epithelial tumor cells, the 4A9 antibody may have potential application in diagnosis and management of canine mammary cancer and a variety of other epithelial tumors.     

Polyreactive Binding of Antibodies Generated by Polyclonal B Cell Activation. I. Polyreactivity Could be Caused by Differential Glycosylation of Immunoglobulins

The aim of this study was to gain knowledge of the pre-immune repertoire with reactivity directed to the carbohydrate antigen dextran B512 (Dx). Polyclonal activation of spleen cells in mice has been estimated to be a method of revealing the available repertoire. Hybridoma cell lines derived from lipopolysaccharide-(LPS)stimulated C57BL/6 spleen cells were screened for reactivity with Dx and with the non-related protein bovine serum albumin (BSA). Despite the lack of structural similarity between Dx and BSA we observed that nearly all of the Dx positive monoclonal antibodies (MoAbs) cross-reacted with BSA. The Dx and BSA cross-reactive MoAbs were also found to bind to several foreign-and auto-antigens, and therefore we concluded that these MoAbs fulfilled the criteria of polyreactivity. The Dx and BSA cross-reactive antibodies were produced in an apparently random fashion as judged by the use of κ and λ light chains and the use of V_H J558 subfamily genes. There are two possible explanations for this type of polyreactivity: (1) LPS induces a randomly generated polyreactive and a randomly generated specific immunoglobulin (Ig) repertoire; (2) polyreactivity can be the result of post-translational modifications. Since immunoglobulins are glycoproteins we considered the possibility that post-translational modifications such as glycosylation could be responsible for the generation of the polyreactive pool. Dextran-specific and Dx/BSA cross-reactive MoAbs showed different degrees of sensitivity to inhibition of glycosylation performed by treatment with tunicamycin (Tm), an inhibitor of the formation of N-glycosidic linkages. Other polyreactive, connective and specific MoAbs were also tested. The authors found that Tm treatment had a more profound effect in reducing the binding capacity of the polyreactive antibodies (Abs), suggesting that the polyreactive Abs may be more dependent than the specific Abs on the carbohydrate content of the molecule for binding to the antigens. The authors propose that lymphocytes may use differential glycosylation as a means to generate polyreactive or monospecific Abs.     

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies, II

We have previously described complement-independent killing of human B lymphocytes by two IgM MoAbs derived from the VH4-34 (VH4.21) gene. Analysis of 17 independently derived VH4-34-encoded MoAbs shows that B cell toxicity is not limited to the two described MoAbs, but is a general property shared by a subset of MoAbs derived from the VH4-34 gene. As observed by two independent microscopy techniques, giant membrane pores were formed on target B cells within 10–15 min of exposure to cytotoxic VH4-34-derived MoAbs. Toxicity by individual MoAb correlated directly to its B cell binding intensity measured by FACS, i.e. stronger the binding greater the killing. Sequence analysis showed that V_H region in germ-line or in near germ-line configuration was necessary but not sufficient for B cell binding. In addition, a particular sequence motif enriched in basic amino acids in the CDR3 may be required to supplement the reactivity mediated by the V_H region of the MoAb molecule. VH4-34-encoded antibodies that fulfil the above sequence requirements have cold agglutinin activity towards the i antigen of cord erythrocytes. In vivo, such anti-i/anti-B cell antibodies are rarely detected in healthy adults, but serum levels are dramatically elevated in selective pathological conditions, such as systemic lupus erythematosus and infectious mononucleosis. This strict regulation may be related to the novel and rapid mechanism of human B cell toxicity demonstrated by antibodies encoded by a single human V_H gene.     

Comparative analysis of monoclonal antibody-drug conjugate binding by flow cytometry

Flow cytometric methods for the evaluation of the cell surface binding properties of monoclonal antibody (MoAb)-drug/toxin conjugates defining tumor-associated antigens are presented. In these techniques, suspension cultures of solid human tumor cell lines are incubated with either varying dilutions of MoAb or MoAb-drug conjugates followed by FITC-conjugated anti-mouse immunoglobulin antibodies in an indirect assay or with FITC-conjugated MoAbs specific for the tumor target cell line in a competition assay. The amount of fluorescent probe bound is measured by flow cytometry and the mean fluorescence intensity determined. The relative binding capacity is quantified by linear regression of the mean fluorescence versus the concentration of primary antibody or antibody conjugate. The application of these techniques to several drug and toxin conjugates of MoAb KS1/4, which defines a human adenocarcinoma-associated antigen, demonstrates that these assays can be effectively utilized to monitor the effects of covalent chemical modification on a MoAb's antigen binding reactivity.     

Monoclonal antibodies against the nucleoprotein of mumps virus: their binding characteristics and cross-reactivity with other paramyxoviruses

Twelve monoclonal antibodies (MoAbs) directed against the nucleoprotein (NP) of mumps virus were analysed for their binding characteristics. Competitive binding in enzyme-linked immunosorbent assay divided them into eight groups. Two of the MoAbs recognized exclusively 66 kD polypeptide of NP, two recognized 66 kD and 60 kD, and one recognized 66 kD (and 60 kD to a lesser extent) in Western blot assays under either denaturing or partially denaturing conditions. Under partially denaturing condition, another five MoAbs reacted faintly but the remaining two did not react at all. Under denaturing condition, on the other hand, these seven MoAbs showed little reactivity with any polypeptide. Furthermore, denaturation resulted in formation of other polypeptides 55 kD, 50 kD, and 43 kD which all were detected by MoAbs reacting with 66 kD and/or 60 kD. Previously demonstrated antigenic cross-reactivity among the NPs of mumps virus and those of human parainfluenza viruses type 2 and type 4 in radioimmunoprecipitation assay using polyclonal antisera was confirmed by an anti-NP MoAb which showed little reactivity in denaturing Western blot assay.     

Antibodies Directed Against Monomorphic and Evolutionary Conserved Self Epitopes may be Generated in 'Knock-Out' Mice. Development of Monoclonal Antibodies Directed Against Monomorphic MHC Class I Determinants

Beta-2 microglobulin (β₂m) gene ‘knock-out’ mice (C1D) were primed wilh purified H-2K^b and H-2D^b molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2^b binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2^b, H-2^d, as well as H-2^k cells by FACS analysis. The MoAbs stained to two β₂m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous β₂m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous β₂m suggesting that xenogeneic β₂m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA-S, when cultured at 26°C, however, staining was reduced five-fold when RMA-S cells were cultured at 37°C. In total, these observations suggest that the MoAbs recognize conformational, presumably β₂m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one-way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epilopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes.     

Keratin and vimentin distribution patterns in the epithelial structures of the cannie anal region

The intermediate filament labeling pattern of the epithelial structures of the canine anal region was studied with different polypeptide specific keratin monoclonal antibodies (MoAbs) and with a monoclonal and polyclonal vimentin antibody. The epithelial structures in this region could be discriminated and characterized by differences in their keratin staining pattern. The basal cells in the different epithelial structures showed a similar staining pattern characterized by reactivity with MoAbs staining keratins 5, 8, 14, and 17. Columnar epithelial cells showed a completely different phenotype mostly characterized by reactivity with MoAbs staining keratins 7, 5, 8, 18, and 19. A restricted number of differentiated perianal gland cells showed perinuclear vimentin staining. Myoepithelial cells did not stain for vimentin, but, as other basal cells, were positive for MoAbs staining keratins 5, 8, 14, and 17.© Willey-Liss, Inc.     

Human monoclonal anti-phospholipid antibodies selectively bind to membrane phospholipid and beta2-glycoprotein I (beta2-GPI) on apoptotic cells

The ability of an anti-phospholipid (LJ1) and an anti-beta2-GPI (RSP-57) human MoAb to bind to apoptotic but not viable cells was demonstrated in this study. Both MoAbs were derived from patients with systemic lupus erythematosus and anti-phospholipid antibody syndrome. The parallel analysis of the specificity and affinity of four anti-phospholipid human MoAbs suggests that the binding of LJ1 MoAb to apoptotic cells is a specific property of this MoAb. RSP-57 MoAb recognizes apoptotic cells through beta2-GPI which becomes available for binding after the interaction with negatively charged phospholipids. This observation provides evidence that the binding of human anti-phospholipid antibodies to apoptotic cells occurs in both a beta2-GPI-dependent and independent way and involves a restricted group of epitopes. The finding that LJ1 and RSP-57 MoAbs bind apoptotic cells underlines the property of these MoAbs to act as cell membrane markers of apoptosis. Major pathological implications derive from the observation that LJ1 and RSP-57 MoAbs recognize epitopes expressed on 'early' apoptotic cells. The interference with the in vivo clearance and processing of apoptotic cells is a potential pathogenic mechanism of these antibodies.     

Monoclonal antibodies that bind to the My23 human myeloid cell surface molecule: epitope analysis and antigen modulation studies

It has previously been shown that the AML-2-23 monoclonal antibody (MoAb) reacts with a glycoprotein on differentiated myeloid cells. The antigen, My23, is released from these cells in culture and is also detectable in normal human plasma. We have now raised a panel of MoAbs against the soluble form of the antigen which reacts with monocytes and calcitriol-treated U937 and HL-60 myeloid cell lines, but not with lymphocytes or undifferentiated U937 and HL-60 cells. As with the AML-2-23 MoAb, the anti-My23 MoAbs immunoprecipitated a 50,000-55,000 protein from calcitriol treated HL-60 cells. Besides binding to cell surface My23, all eight MoAbs as well as the 63D3 MoAb reacted with crude and purified forms of soluble My23. A novel ELISA epitope analysis assay was developed to identify four distinct antigenic determinants on My23. Thus, the soluble and cell surface forms of My23 share several antigenic determinants and are biochemically very similar. Pooled anti-My23 caused selective patching, capping and clearing of My23 from the cell surface. My23 was not associated with cell surface, HLA-DR, HLA-A,B,C, beta 2-microglobulin or the Fc receptors for IgG or IgA. These anti-My23 MoAbs should be of importance in antigen functional studies and in clinical treatment protocols for patients with acute myelogenous leukemia. Furthermore, we have shown that a soluble myeloid differentiation antigen can serve as an effective immunogen for the preparation of MoAbs against important cell surface molecules thus obviating many problems inherent in the use of whole cells or detergent lysate-derived immunoprecipitates as immunogens.     

Keratin and vimentin distribution patterns in the epithelial structures of the canine anal region.

The intermediate filament labeling pattern of the epithelial structures of the canine anal region was studied with different polypeptide specific keratin monoclonal antibodies (MoAbs) and with a monoclonal and polyclonal vimentin antibody. The epithelial structures in this region could be discriminated and characterized by differences in their keratin staining pattern. The basal cells in the different epithelial structures showed a similar staining pattern characterized by reactivity with MoAbs staining keratins 5, 8, 14, and 17. Columnar epithelial cells showed a completely different phenotype mostly characterized by reactivity with MoAbs staining keratins 7, 5, 8, 18, and 19. A restricted number of differentiated perianal gland cells showed perinuclear vimentin staining. Myoepithelial cells did not stain for vimentin, but, as other basal cells, were positive for MoAbs staining keratins 5, 8, 14, and 17.     

The Expression of Carbohydrate Antigens in Activated T Cells and in Autoimmune Diseases

Cell surface carbohydrate antigens have been implicated in cell differentiation and maturation and may play a role in immunoregulation. The expression of carbohydrates in peripheral blood lymphocytes (PBL) was studied by double immunofluorescence flow cytometry, using MoAbs CTI and CT2 but only a small proportion of cells bound these MoAbs. MoAbs CTI, CT2 and the lectin vicia villosa (VV) which share specificity for Gal NAc were then used to examine lymphocytes from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Behcet's disease (BD) and IgA nephropathy. A significant increase in MoAbs CTI CT2 and W binding CD4 or CD8 cells was found only with lymphocytes from patients with SLE. However, MoAbs CT or VV binding lymphocytes from healthy subjects were significantly up-regulated by activation with a mitogen (PHA), cross-linked anti-CD3 MoAb or a common antigen (65kDa heat shock protein), suggesting that an increased proportion of T cells expressing these carbohydrates results from any of the three types of lymphocyte activating agents. Inhibitioti studies were then carried out to determine the relationship between the MoAbs CTI and CT2, VV and GalNAc. Indeed. VV binding to T cells was significantly inhibited by either MoAbs CTI or CT2, or GalNAc but not GlucNAc. suggesting that VV shares a common binding site with MoAb CT and that GalNAc may constitute one of the sugar receptors. Investigations of lymphocytes from adult peripheral blood in health and disease suggest that carbohydrate antigens may play a role in activation and immunoregulation.     

Human Polyreactive IgM Monoclonal Antibodies with Blocking Activity Against Self-Reactive IgG

Natural IgM antibodies have been found to be involved in the control of IgG reactivity in normal serum. The authors investigated the blocking activity of four human IgM monoclonal antibodies (BY-2, BY-7, BY-10 and IRM-7) derived from B-cells from blood samples of three renal dialysis patients, which had shown multispecific properties similar to those observed for natural polyreactive autoantibodies. To achieve this, competitive inhibition assays were performed with these MoAbs on the binding of IgG purified from a healthy control, three patients with SLE, and two patients with autoimmune thyroiditis, to histone, dsDNA, RNP and thyroglobulin. MoAbs inhibited binding of self-reactive IgG to histone and dsDNA, but not to thyroglobulin or RNP, of natural and active or inactive phase disease-associated autoreactive IgG. The inhibitory effect of the MoAbs was mediated by V-region dependent interactions with autoreactive IgG, as shown by the ability of these MoAbs to block the binding of F(ab')₂ fragments of autoreactive IgG to antigens (histone and dsDNA). The blocking of autoantibody activity was dose-dependent with maximal inhibition occurring at a specific molar ratio between the patient's IgG and a given MoAb. In contrast, MoAbs did not inhibit binding of IgG alloantibodies present in the sera of four polytransfused renal dialysis patients to target antigens on the surface of different cells. These results support the concept of a functional idiotypic network regulating autoimmune responses, and suggest that the IgM MoAbs under study may be natural polyreactive antibodies belonging to the physiological network of autoantibodies with highly connected V-regions, capable of binding and functionally neutralizing V-regions of natural and pathologic autoantibodies.     

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4-34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement-independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, ³H-thymidine uptake, and the 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20⁺ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm-6, OCI-LY8, Arent and SUP-B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing was greater at 4°C than 37°C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular processes and independent of complement, suggests a novel mechanism of cell death via membrane perturbations.     

Relevance of Antibody Valency in EGF Receptor Modulation

Binding characteristics of a monovalenl bispecific monoclonal antibody (bsMoAb), which recognizes both epidermal growth factor receptor (EGF-R) and drug doxorubicin (DXR) were compared with those of the parental bivalent MoAb directed against the EGF-R binding site. Scatchard analysis indicated that both MoAbs bound to EGF-R-overexpressing A431 cells with the same affinity. In tracer amounts, both MoAbs also displayed the same capacity to be internalized after binding to the cell surface. However, when the MoAbs were used at saturating concentrations, down-modulation of the receptor was greater with the bivalent MoAb. The bivalent MoAb also inhibited proliferation of A431 cells both in vitro and in vivo whereas the bsMoAb was inhibitory only in vivo. These data suggest that MoAb bivalency is required for EGF-R down-modulation and in vitro cell growth inhibition.     

Monoclonal antibodies that distinguish between class II antigens (HLA-DP, DQ, and DR) in 14 haplotypes

The specificity of three commonly used monoclonal antibodies (MoAbs) reacting with human class II histocompatibility antigens, was analyzed to determine whether these MoAbs would distinguish between HLA-DP, DQ, and DR in a large number of haplotypes. The reactivity of these MoAbs (L243, Anti-Leu 10, and B7/21) was compared by serial immunoprecipitation of class II antigens from 11 B-cell lines. The cell lines examined expressed a total of five DP, three DQ, and nine DR types, which together represent most of the well-defined class II specificities. This is the first demonstration that one of these antibodies. B7/21, binds to at least five DP specificities, and does not bind to DR or DQ molecules as defined by reactivity with the two other MoAbs. Within the scope of these experiments, the B7/21 antibody was shown to react with a monomorphic DP determinant. A variant clone of the B7/21 hybridoma was isolated that secretes IgG₁ antibody with the same specificity as the original IgG₃ antibody. The two other antibodies studied have been previously shown to react with DR molecules (L243) or DQ molecules (Anti-Leu 10). Here, their lack of cross-reaction with DP molecules is demonstrated. Thus, each of the three MoAbs reacts exclusively with a distinct class II molecule in all haplotypes studied, and therefore should be useful for comparing the independent expression and function of DP, DQ, and DR molecules.     

Monoclonal antibodies bind identically to both spores and hyphae of Aspergillus fumigatus

Immunoelectron microscopy (IEM) was used to determine the binding of six monoclonal antibodies (MoAbs) produced against Aspergillus fumigatus antigens present on or within the conidia and hyphae of the fungus. Antigen-antibody complexes were demonstrated in EM using labelled colloidal gold particles (15 nm). Three out of 6 MoAbs (C9, F12 and H10) reacted only with the cytoplasmic components of A. fumigatus while the remaining three (B12, F6G5 and D6E6) showed reactivity to both cytoplasm and cell wall of the conidia and hyphae. The results indicate that IEM is of considerable value in determining and selecting monoclonal antibodies having specific reactivity with diverse antigenic components.     

Lupus-derived autoantibodies with dual autoactivity: anti-DNA and anti-Fc. II. Fine specificity of anti-self autoreactivity.

The anti-immunoglobulin reactivity of two monoclonal, dual specific, autoantibodies, BV 17-45 and BV 04-01 was examined. The current study further defined the anti-immunoglobulin autoreactivity of these MoAbs to be Fc-specific. Both BV 17-45 and BV 04-01 bound their own Fc domains in addition to Fc regions of other MoAbs of similar isotype with varying levels of activity. The different anti-Fc reactivity patterns of BV 17-45 and BV 04-01 suggested that these MoAbs recognized distinct epitopes. Neither BV 17-45 nor BV 04-01 bound Fab fragments or single-chain antibody derivatives, which confirmed that the anti-immunoglobulin reactivity of these autoantibodies was Fc-specific. In addition, abrogation of anti-Fc reactivity was observed when affinity-labelled MoAbs were used as coating antigens in solid-phase ELISAs. These results implied that active-site ligand binding induced conformational changes which altered the Fc epitope(s) recognized by BV 17-45 and BV 04-01.     

Inhibition of influenza virus haemolytic and haemagglutination activities by monoclonal antibodies to haemagglutinin glycopolypeptides HA1 and HA2

Acid treatment of influenza virus enhanced haemagglutination inhibiting (HI) activity of some anti-HA1 monoclonal antibodies (MoAbs). These changes in the HI-activity could be either due to alteration in the mutual orientation of MoAb (e.g. IC8, IB8) binding epitope to receptor site or to an increase in the number of epitopes accessible to the corresponding MoAbs (e.g. IVA1). HI test with pH 5-virus revealed similar (although not identical) antigenic differences among related virus strains as the HI test with pH 7-virus. Anti-HA2 MoAbs were negative in the HI test with both pH 5- and pH 7-virus. Anti-HA1 MoAbs showed a HI activity with pH 5-treated BHA similar to that with pH 5-treated virus. Surprisingly one out of eight anti-HA2 MoAbs (IIF4) exhibited a relatively high HI activity to pH 5-BHA-mediated haemagglutination. Virus-induced red blood cell haemolysis was efficiently inhibited with several anti-HA1 MoAbs (e.g. IC8, IB8, and IIB4) while other anti-HA1 antibodies, including IVA1 and IVG6 with preferential reactivity with pH 5-treated antigens in RIA, gave no inhibition. As a rule, anti-HA2 MoAbs were poor haemolysis inhibitors.     

Common and different antigenic properties of the rabies virus glycoprotein of strains SAD-Vnukovo and Pitman-Moore.

Two fixed rabies virus strains, SAD-Vnukovo and Pitman-Moore (PM) were used as combined immunogens for the generation of hybridomas secreting specific monoclonal antibodies (MoAbs). The obtained hybridomas were primarily screened by an ELISA for production of MoAbs to antigen of SAD-Vnukovo strain. Six positive clones were established. A panel of MoAbs has been characterized according to reactivity in immunofluorescence, immunoblot, ELISA and neutralization tests. All MoAbs were positive in immunofluorescence when cells infected with the SAD-Vnukovo strain were used. By immunoblot, four MoAbs showed specificity for the viral glycoprotein of both SAD-Vnukovo and PM rabies strains. This pattern of reactivity indicated the existence of shared conformation-independent epitopes located on the related antigens. However, in ELISA, the tested MoAbs did not recognize viral glycoproteins of the PM strain. This indicates, that the different strain-specific conformations of the native glycoprotein determine the accessibility of the common linear determinants for respective antibodies. Only one antibody, with conformation-dependent glycoprotein specificity, was capable to neutralize the CVS strain of rabies virus.