Binding characteristics of three complement dependent assays for the detection of immune complexes in human serum

The fluid phase C1q binding, the solid phase C1q binding and the Raji cell assay are the most widely employed methods for the detection of complement fixing immune complexes in human disease. However, their binding characteristics for complexes formed in native serum have not been compared and studied in detail. For this purpose, Tetanustoxoid (TT): anti-TT complexes were formed closely to in vivo conditions by incubating sera of immunized people with TT. Then the molecular characteristics of those complexes, which could be detected by the 3 immune complex assays, were defined. The methods used are precipitation curve, gel chromatography and measuring the complexed antibody with a sensitive Elisa method. All 3 assays detected only complexes near the equivalence zone; the Raji cell assay being the most sensitive detecting 1.5 micrograms complexed antibody/ml serum followed by the solid phase (6 micrograms/ml) and the fluid phase C1q binding assay (50 micrograms/ml). As estimated by gel chromatography, both the C1q binding assays measured most sensitively complexes with a molecular weight distinctly over 2,000,000 daltons, whereas the Raji cell assay detected preferentially complexes in the range of 2,000,000 daltons. Smaller TT: anti-TT complexes with a molecular weight less than 600,000 daltons were not detectable by the 3 assays. In conclusion, the 3 assays, while differing in sensitivity, showed similar binding patterns with preference for high molecular complexes near equivalence. Subsequently, they might be insensitive to small complexes persisting in the circulation.