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UBC Theses and Dissertations
Characterization of monoclonal antibodies to rubella virus structural proteins Brush, Bradley Alan
Monoclonal antibodies (MAbs), due to their intrinsic specificity to single determinants, are capable of defining the antigenic and structural properties of viruses. A panel of twenty-five monoclonal antibodies, the result of fusions between splenocytes from Balb/c mice immunized with RA 27/3 vaccine strain rubella virus and myeloma cell line NS1X63Ag8.653, were characterized by radioimmunoprecipitation of rubella virus structural proteins, IgG sub-classification, haemagglutination inhibition (HI) and virus neutralization (Nt) of rubella virus (RV). The antigen specificities of thirteen of twenty-five MAbs were identified by immunoprecipitation of [³⁵S]-methionine labelled virus. All showed reactivity to the E1 envelope glycoprotein of either M33 wild type, or RA 27/3 vaccine strain virus, or both. The remainder precipitated multiple viral proteins or none at all. These trials required numerous alterations in the composition of buffers to achieve unambiguous results. IgG subclassification was performed on hybridoma cell culture supernatants or purified ascites fluid by the Ouchterlony double immunodiffusion technique, with all immunoglobulins typed as IgG₁, IgG[sub 2a] or IgG[sub 2b], but with no cases of IgG₃ or IgM secretors. Inhibition of rubella virus agglutination of chick erythrocytes by nine of the monoclonal antibodies gave haemagglutination inhibition titres of greater than 8 with the highest titre at 16384. The panel of monoclonal antibodies was tested for its capacity to neutralize both M33 and RA 27/3 strains of rubella virus, with and without the presence of complement. To detect rubella plaque formation exclusively, an immunocytochemical method of detecting rubella virus proteins on cell surfaces was developed. Plaque reduction experiments showed that ten of the MAbs inhibited the cytopathic effect of rubella virus to some extent. Five MAbs neutralized M33 RV without complement present, one showing a four-fold increase in titre upon its addition, and one MAb, negative in the absence of complement displayed a positive titre in its presence. Three MAbs showed neutralizing activity toward RA 27/3 with no observed effect upon the addition of complement; two MAbs showed a shift from negativity to neutralizing titres. Of the ten MAbs that were active, four reacted with both rubella strains. The capacity of the monoclonal antibodies to neutralize rubella virus was found to be independent of their ability to inhibit the haemagglutinin of the virus. Therefore the epitopes for virus neutralization and haemagglutination appear to be different.
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