Topological mapping of murine leukemia virus proteins by competition-binding assays with monoclonal antibodies

102

SUMMARYA panel of hybridoma antibodies that react with the surface peplomer glycoprotein (E2) of the murine coronavirus JHM were produced to characterize major antigenic domains associated with functions related to virulence. Three groups of hybridoma antibodies were differentiated by immunoprecipitation of lysates from JHM-infected cells. One group precipitated the virion structural proteins gpl70 and gp98 together with the intracellular form of E2, gpl50. A second group reacted with gp98 and gpl50, and a third group precipitated gpl50 only. Competition assays with biotinylated hybridoma antibodies allowed the definition of at least six different epitope groups. Only those antibodies which immunoprecipitated both gpl70 and gp98 neutralized infectivity, inhibited cell fusion and protected infected rats against acute disease. Another class of antibodies binding to gpl70 and gp98 also neutralized JHM virus, but did not inhibit fusion and did not protect against disease. Antibodies that immunoprecipitated gp 150 and gp98 revealed only weak neutralization and did not inhibit cell fusion or protect animals. Four epitopes were defined by antibodies that immunoprecipitated gp 150, but revealed no biological activity. These data indicate that the site responsible for cell fusion is associated with an epitope group carried by gpl70 and gp98. Neutralizing antibodies bind to this and another epitope. Furthermore, protection of JHM-infected rats against acute disease requires both inhibition of cell fusion and neutralization of virus.

Section: Discussionmentioning

confidence: 99%

Hybridoma Antibodies to the Murine Coronavirus JHM: Characterization of Epitopes on the Peplomer Protein (E2)

Dörries

Wege

1984

102

“…Complete competition (> 90~) was required for classification as a distinct epitope. The competition criteria were partially based upon the values obtained, and are similar to criteria used by other investigators (Stone & Nowinski, 1980;Iorio & Bratt, 1983). Although Stone & Nowinski suggest that partial competition between antibodies may result from differences in avidity, we did not find that this occurred.…”

Section: Binding Oj" Mabs To Vp70 and Competition Bindingmentioning

confidence: 99%

Analysis of the Respiratory Syncytial Virus Fusion Protein Using Monoclonal and Polyclonal Antibodies

Walsh

Côté

Fernie

et al. 1986

505 SUMMARYAt least four distinct epitopes are described on the respiratory syncytial virus fusion protein (VP70) using 13 monoclonal antibodies in solid-phase competitive binding studies. Two, and possibly three, fusion-inhibiting epitopes, one non-fusion-inhibiting neutralizing epitope, and one non-neutralizing epitope are described. All but the latter site demonstrated partial overlap, suggesting possible topographical proximity of these epitopes. Polyclonal rabbit sera to VP70, which neutralized virus but did not inhibit fusion of infected cells, blocked the binding of all fusion-inhibiting monoclonal antibodies to VP70 in the solid-phase assay but did not inhibit their effect in vitro. Western blot analysis of these monoclonal antibodies demonstrates that one fusioninhibiting epitope is localized on the 48K fragment of VP70 and is resistant to denaturation by heat, 2-mercaptoethanol and SDS.

“…It has been claimed previously that the results of competitive binding assays, if performed with two antibodies of different activities, must be interpreted carefully since the competitive reaction invariably favours the binding of the antibody with higher avidity (Stone & Nowinski, 1980;Massey & Schochetman, 1981). Prior to the competitive binding studies, therefore, the avidities of the anti-NP and anti-M MAbs were compared by ELISA, assuming that at antigen saturation the maximum amount of antibody bound to the wells is a direct reflection of the avidity of the antibody for its epitope (Frankel & Gerhard, 1979).…”

Section: Antigenic Analysis Of Np and M By Competitive Binding Assaysmentioning

confidence: 99%

Antigenic Characterization of the Nucleoprotein and Matrix Protein of Influenza C Virus with Monoclonal Antibodies

Sugawara

Nishimura

Hongō

et al. 1991

Monoclonal antibodies against the nucleoprotein (NP) and matrix (M) protein of influenza C virus were prepared and characterized. At least two non-overlapping or partially overlapping antigenic sites were delineated on each of the proteins by competitive binding assays. Western blot analysis showed that two antigenic sites on the M protein were highly resistant to conformational changes whereas two sites on NP were sensitive. No antigenic variation was seen in either the NP or the M protein when the reactivity of monoclonal antibodies with 23 different influenza C strain s isolated over a 41 year period was studied by radioimmunoprecipitation and enzyme-linked immunosorbent assay. Immunofluorescence analysis with the monoclonal antibodies revealed that both the NP and M proteins migrated to the cell nucleus during the replication cycle of influenza C virus.