Epitope mapping of homologous and cross-reactive antigens by monoclonal antibodies to streptococcal cell membrane (mAb to SCM).

An approach to epitope mapping of a series of anti-streptococcal cell membrane (SCM) mAbs is described. Evaluations by enzyme-linked immunosorbent assay (ELISA) of one control mAb HB-35 and 13 different anti-SCM mAbs were made on homologous SCM antigen and human basement membrane antigens isolated from glomeruli (GBM) and lung (LBM). These anti-SCM mAbs were previously shown to be cross-reactive in a variety of systems with both GBM and LBM. The binding capacities were measured for all 14 mAbs on ELISA plates sensitized with SCM antigen or the cross-reactive GBM or LBM antigens, at 5 micrograms/ml or approximately 20 pM/well. From the 50% binding capacity dilution the pM of mAb bound/pM antigen-well was calculated which translated into an estimate of the ratio-number of epitopes bound. Observations with the homologous and cross-reactive antigens showed multiple reactive epitope ratios to eight mAbs whereas the other five yielded a ratio value of one or two on the tested antigens. Plates blocked with a specific dilution of one mAb evaluated the binding by a second mAb providing both binding and specificity data. One mAb (I-F-3) blocked all the other anti-SCM mAbs on all three antigen plates. An additive effect was noted by three mAbs, I-G-8, II-C-4 and II-D-8 with most of the other mAbs. The order of placement, however, made distinctive differences; II-F-4 showed an additive or enhancement effect on I-B-5 but no reciprocal effect was seen. A similar effect was made with I-G-8 and II-C-4 or I-F-7. Possible interpretations are that each mAb is binding different epitopes each fully exposed, and the order of placement of the mAbs makes no difference. Where an enhanced effect was observed it is suggested that the binding of the first mAb changed the conformation of the antigen, thereby opening and exposing additional epitope(s) to the second antibody. Or, in contrast, where the second mAb was blocked by the first, a fixing of the protein conformation is suggested thereby occluding the other epitope, as seen with I-F-3 and II-C-4. These epitope mapping procedures confirmed that all 13 anti-SCM mAbs were binding at different epitopes. The nature of basement membrane collagens and how this relates to post-streptococcal sequelae will be discussed.