JournaloflmmunologicalMethods, 84 (1985) 125-134 125 Elsevier JIM03678 A Rapid and Reproducible Method for the Analysis of Immune Complexes Using Affinity Chromatography and Western Blotting A.J. Smith, T.E. Cawston and B.L. Hazleman Rheumatology Research Unit, Addenbrooke's Hospital, Hills Road, Cambridge, U.K. (Received 8 May 1985, accepted 10 July 1985) A new procedure which couples different analytical techniques in a format permitting the rapid analysis of immune complex components is described. Complexes obtained from sera by polyethylene glycol (PEG) precipitation were resuspended and then added, using a batch method, to antibody coupled to Sepharose beads. Antibody directed against either human Clq or human C3c were used in the present study. Bound immune complexes were washed and then eluted from the Sepharose by sodium dodecyl sulphate (SDS) treatment and simultaneously reduced with dithiothreitol. Individual components were separated by SDS gradient polyacrylamide gel electrophoresis and then transferred to nitrocellulose by Western blotting. Individual strips of nitrocellulose were investigated using specific antisera and a radiolabelled probe. Immune complexes (IC) isolated from the sera of 7 rheumatoid arthritis (RA) patients were analysed using this method and the results obtained for both affinity adsorbents compared. Key words: immune complexes - rheumatoid arthritis - affinity chromatography - Western blotting Introduction Circulating immune complexes (CIC) are known to be present in the sera of patients with autoimmune diseases but the clinical significance of their presence is not clear. Often separate assay methods give quite different results (Halla et al., 1979; McDougal et al., 1982; Moore et al., 1982), and large changes in immune complex levels can occur with no accompanying clinical change. Consequently, techniques have recently developed which allow CIC to be isolated in sufficient quantity to permit analysis of the individual components. The identifi- cation of these components of CIC, particularly the antigen, is attractive since it could clarify both the pathological and aetiological mechanisms of several auto-im- mune diseases. Abbreviations." PEG, polyethylene glycol; SDS, sodium dodecyl sulphate; IC, immune complexes; CIC, circulating immune complexes; RA. rheumatoid arthritis; lg, immunoglobulin; EDTA, ethylene- diamine tetra-acetic acid. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)