Electrophoretic transfer blotting analysis of immune complexes in rheumatoid arthritis

Immunochemical analysis of immune complexes (IC) derived from synovial fluid and serum of patients with rheumatoid arthritis (RA) was undertaken. IC were isolated by differential polyethylene glycol precipitation and competitive binding to staphylococcal Protein A. Anti-IC antisera were raised in rabbits by immunization with purified IC. IC were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic transfer to nitrocellulose sheets, reaction with either anti-IC antiserum or monospecific antiserum against putative components, then developed with 125I-Protein A and autoradiography. Synovial fluid IC from both RA and control inflammatory joint diseases contained immunoglobulin light chains, and mu and gamma heavy chains. IC from RA synovial fluid also contained alpha chain, and Clq. Cross-over studies of IC and antisera revealed cross-reactive antigens in IC of RA synovial fluid and that remained unidentified were of the following relative molecular masses: 26,000; 39,000; 40,000; and 43,000. Studies using early and late serum samples to probe the blotted IC failed to disclose serological autoreactivity. This sensitive analytical technique provides a means for detailed description of the constituents of IC in inflammatory joint diseases.