Antigen detection in immune complexes by a modified Staphylococci binding assay and Western blot analysis

The combination of a modified Staphylococci binding assay for immune complexes and Western blot analysis is described for the isolation and detection of antigen in immune complexes from human sera. The strategy of the procedure is to preclear immune complexes from other serum components by sequential polyethylene glycol precipitation and incubation with insoluble protein A under conditions in which immune complexes are preferentially bound. Immune complexes are eluted from protein A in sodium dodecyl sulfate buffer and dissociated by acrylamide electrophoresis. Resolved proteins are then transblotted to nitrocellulose, and antigen is detected with specific antibody. Immune complexes were prepared in vitro with an antigen (keyhole limpet hemocyanin) that focuses well on electrophoresis and for which a potent immunologic probe (antibody) was available. In this system, antigen could be detected when immune complexes were present in sera in concentrations as low as 20 micrograms aggregated-IgG eq/ml, regardless of antigen-antibody ratio. We demonstrate the detection of horse globulin in immune complexes from a patient with acute serum sickness and hepatitis B virus antigen in complexes from a patient with vasculitis.