Alteration in antibody reactivity with foot-and-mouth disease virus (FMDV) 146S antigen before and after binding to a solid phase or complexing with specific antibody

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.

Section: Discussionsupporting

confidence: 76%

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

Barnett

Rowlands

Parry

1993

“…None of these antibodies bound to virus directly adsorbed onto a solid phase, in contrast to their positive reactivity in an ELISA using antibody-captured virus. Given that virus particles may be distorted after adsorption onto a solid phase (McCullough et al, 1985), this suggests that these BMAbs recognize conformational-dependent discontinuous sites rather than linear epitopes. Eight of these antibodies, including three IgG " , neutralized homologous virus in vitro at various levels, and nearly all were effective at passively protecting suckling mice against virus challenge, indicating that neither in vitro neutralization nor protection is governed by the antibody's isotype.…”

Section: Discussionmentioning

confidence: 99%

Monoclonal antibodies, against O1 serotype foot-and-mouth disease virus, from a natural bovine host, recognize similar antigenic features to those defined by the mouse.

Barnett

Samuel

Püllen

et al. 1998

Eight neutralizing and two non-neutralizing antifoot-and-mouth disease virus (FMDV) bovine IgG1 and IgG2 monoclonal antibodies (BMAbs) recognize conformationally dependent epitopes. The majority of those shown to neutralize virus passively protected mice from virus challenge, regardless of isotype. Well-characterized anti-FMDV mouse MAbs, representing five independent neutralizing epitopes on O 1 serotype virus, were examined with each of the ten BMAbs in a competition-based ELISA. Five of the neutralizing BMAbs (C48, C65, C74, C83 and MH6) were shown to be directed against epitopes on, or in close proximity to, that previously defined as neutralizing antigenic site 2. Another neutralizing BMAb, MH5, bound to an epitope on, or in close proximity to antigenic site 3. Two neutralizing BMAbs (C2 and C96) simultaneously

“…However, binding of antigen to microtitre plates promotes substantial changes in conformation and antigenic character of proteins (Friguet et al, 1984;Halk, 1986;Muller et al, 1986). To minimize the influence of these factors on measurement of antibody specificity, liquid phase competition assays were performed (McCullough et al, 1985). These assays revealed a gradation in binding: top > middle, bottom ( Fig.…”

mentioning

confidence: 99%

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Kalmar

Eastwell

1989

SUMMARYRabbit antisera produced against two comoviruses (cowpea mosaic virus and cowpea severe mosaic virus) were used in plate-trapped ELISA and liquid phase competition ELISA. In the latter, the top component competed against bound unfractionated virus more effectively than did nucleoprotein components. One murine monoclonal antibody elicited to cowpea mosaic virus and three monoclonal antibodies to cowpea severe mosaic virus exhibited differential binding to top component, relative to either middle or bottom components in plate-trapped or antibody-trapped ELISAs. Differential binding to centrifugal components of virus by two of these monoclonal antibodies was maintained in liquid phase competition assays, These data suggest that the encapsidation of RNA alters the configuration of the virion surface in the vicinity of the antibody epitopes. Ribonuclease digestion of intact or denatured virus did not affect the binding of monoclonal antibodies.Like other comoviruses, cowpea mosaic virus (CPMV) and cowpea severe mosaic virus (CPSMV) are resolved into three major fractions by density gradient centrifugation (Bruening, 1969). The slowest sedimenting top component consists of empty capsids whereas the middle and bottom components are nucleocapsids containing one molecule of either RNA 2 (Mr 1"37 x 106) or RNA 1 (Mr 2.02 x 106), respectively. PAGE does not show differences between the protein composition of the three components (Geelen et al., 1972;Wu & Bruening, 1971) and rabbit antisera prepared against unfractionated virus reacts with all components in Ouchterlony double-diffusion assays (Bruening & Agrawal, 1967). This paper reports that liquid phase competition assays utilizing rabbit antisera reveal a difference between the binding of antibodies to empty capsids and to nucleoprotein virions. Antigenic differences between top component and middle or bottom components were confirmed by assays with monoclonal antibodies.CPSMV-DG and CPMV-SB isolates were originally obtained from G. Bruening (Department of Plant Pathology, University of California, Davis, Ca., U.S.A.), propagated in cowpeas (Vigna unguiculata Walp. cv. California Black Eye) and isolated as previously described (Bruening, 1969). Approximately 5 mg virus was suspended in 0-5 ml gradient buffer (50 mM-KH2PO, pH 7.0, 2 mM-disodium EDTA) and layered onto 11 ml of 39 ~ (w/w) caesium chloride in gradient buffer. Gradients were centrifuged for 36 h at 36000 r.p.m, and 4 °C in an SW 40Ti rotor (Beckman) and fractionated by side-puncture. Fractions were diluted at least 10-fold in gradient buffer and pelleted by centrifugation at 45000r.p.m. for 2h. The pellets were resuspended in gradient buffer. Concentrations of virus components were calculated using A260 at 1 mg/ml of 6.2, 10-0 and 8.1 for middle component, bottom component and unfractionated virus and A2so at 1 mg/ml of 1.28 for top component (Geelen et al., 1972).The preparation of rabbit antisera and of the panel of monoclonal antibodies produced by hybridomas have been described (Kalmar & Eastwell,...