Human monoclonal antibodies encoded by the V4-34 gene segment show cold agglutinin activity and variable multireactivity which correlates with the predicted charge of the heavy-chain variable region

We have characterized the reactivities of a panel of V4-34-encoded human IgM monoclonal antibodies (mAb) which bind the erythrocyte Rh D antigen, derived from an immunized individual. These were compared with the specificities of V4-34-encoded autoantibodies with I/i reactivity produced from patients with cold agglutinin disease (CAD), and other V4-34-encoded autoantibodies. The antibodies were evaluated for cold agglutinin activity using haemagglutination tests, immunofluorescence microscopy for reactivity with tissue components, and in solid phase radiobinding assays with purified antigens. We found that (i) cold agglutinin activity was a property of all the V4-34-encoded mAb (ii) the cold agglutinins from CAD patients were generally monospecific for I/i whereas most of the anti-D and the other V4-34-encoded mAb displayed multireactive properties, frequently binding to strongly acidic antigens (iii) computation of the net charge of the heavy-chain V regions showed that the multireactive mAb were generally more positively charged than the monospecific cold agglutinins, which could contribute to their multireactive phenotype. The involvement of charge interactions was further indicated by the effects of pH and ionic strength on the immunofluorescence staining patterns.