Diverse antigen specificity of erythrocyte‐reactive monoclonal autoantibodies from NZB mice

The specificities of a panel of erythrocyte‐reactive MoAbs derived from NZB mice with autoimmune haemolytic anaemia (AIHA) were determined by immunoprecipitation and immunoblotting. Of the eight antibodies, two (IgG1 MoAb 105‐2H and IgG2a MoAb 34‐3C) immunoprecipitated a 105‐kD component identified as the erythrocyte anion channel band 3. A similar band was also immunoprecipitated by the IgG2b MoAb 34‐2B when used at relatively high concentrations, but none of the remaining hybridoma antibodies precipitated any labelled erythrocyte components. In immunoblotting experiments only 34‐2B reacted with band 3, indicating that the epitope recognized by this MoAb is robust and differs from the determinant(s) recognized by 105‐2H and 34‐3C. The remaining MoAbs to react by immunoblotting were the IgM antibodies IE10 and 4C8, both of which bound to a doublet corresponding to band 4.1 from the internal erythrocyte membrane skeleton. Of the three MoAbs which gave negative results in immunoprecipitation and immunoblotting, the IgM antibodies 103‐7E and 106‐10E reacted poorly with intact erythrocytes by flow cytometry, but the IgG1 antibody 31‐9D bound well. ELISAs demonstrated that all four IgM MoAbs are polyreactive, since they bound to histones from a panel of nuclear antigens, and additionally 103‐7E reacted with phosphatidyl choline. It is concluded that band 3 is an important autoantigen in NZB AIHA. However, since 3/5 haemolytic MoAbs failed to precipitate this antigen, either these antibodies represent minor components of the total autoantibody response, or responses to diverse possibly non‐protein surface antigens also contribute to the pathogenesis of the disease.