Ring, Susan Margaret; (1992) An immunochemical investigation of the Wr(a) and Wr(b) blood group antigens. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the production of a monoclonal antibody to the low incidence blood group antigen Wra which is thought to be allelic to Wrb (a high frequency antigen known to be associated with glycophorin A and band 3). The main approach was to immunise mice with Wr(a+) red cells and select appropriate antibodies by screening them against a panel of red cells by haemagglutination. One anti-Wra antibody (BGU1-WR) was found from the 10 74 hybridomas screened.BGU1-WR belongs to immunoglobulin subclass IgG1 and has an affinity constant of 1.82x10 7. This antibody and a previously described anti-Wrb monoclonalantibody was used to investigate the nature of the antigens. Haemagglutination studies showed that both antigens are resistant to proteinase treatment, probably do not involve sialic acid and do not require intact disulphide bonds on native red cells. The site number of the Wra and Wrb antigens was determined by direct binding of 125I-labelled antibody to red cells. Wr(a+b+) cells were shown to have approximately 70,000 Wra and 150,000 to 350,000 Wrb sites per cell. The possible expression of these antigens on other cells was investigated using flow cytometry. The Wra antigen was not found on leucocytes from a Wr(a+) donor or on 19 cell lines of different cell and tissue origin of unknown Wra phenotype, including 2 erythroid-like lines. Wrb was only found on 2 myeloid-like cell lines. SDS-PAGE, immunoblotting and immunoprecipitation were used to investigate the molecular structure of the antigens. Neither antibody recognises the denatured, SDS- treated antigens under the wide variety of conditions used. Numerous immunoprecipitation experiments showed that anti-Wrb immunoprecipitates glycophorin A and band 3. Under identical conditions anti-Wra did not immunoprecipitate any detectable component of the red cell membrane. Thus the Wra antigen appears to differ from the Wrb antigen casting doubt on the antithetical relationship of these antigens.

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