IgM anti-erythrocyte autoantibodies specific for buried and neo-antigens using cellular-ELISA assays

The aim was to develop a cellular-ELISA assay to detect natural autoantibodies specific for bromelain-treated mouse red blood cells (BrMRBC). High, unexpected IgM titres against normal mouse red blood cells (NMRBC) were detected in day 7-14 sera of CBA mice treated with E. coli lipopolysaccharide (LPS). These "autoantibodies" bound to normal mouse red blood cells in the presence or absence of commonly used c-ELISA adhering agents. Such high reactivity to NMRBC was never detected using complement dependent haemolytic assays in earlier work in this system. The question whether these IgM alpha-NMRBC molecules were binding nonspecifically (via Fc) or specifically (via Fab) was answered indirectly by comparing the binding titres of LPS-stimulated serum and several purified IgM antibody preparations (alpha-PC, alpha-KLH, MOPC 104E) on the same antigen coated plates. The observed binding ratios (titre on antigen X: titre on NMRBC) varied widely between different antibody sources, indicative of specific binding. In addition no significant unequivocal binding against NMRBC could be detected in vivo (LPS-stimulated mice) nor could bound IgM antibody be detected in a suspension-c-ELISA assay (high binding titres to BrMRBC could be detected in the latter test system). In conventional c-ELISA assays, modification of normal erythrocyte by adhesion to plastic microtitre plates appears to expose or create "neoantigens" on NMRBC which are not encountered in suspension-type c-ELISA, nor in lytic or agglutination assays where the erythrocyte targets are in suspension at physiological pH and isotonicity.