Critical aspects of immune complex assays employing polyethylene glycol

Treatment of artificial immune complexes (ICs) with 2.5% polyethylene glycol (PEG) — conditions under which C1q-binding activity is routinely measured in the fluid phase — produced marked changes in molecular size as determined by Sepharose 6B chromatography. The effect of PEG on the C1q-binding capacity of ICs, was therefore investigated using a solid phase (SP) system.

PEG enhanced the binding of aggregated human gammaglobulin (AHG) and artificial ICs to SP-C1q and, in reverse experiments, also increased the binding of C1q to SP-AHG. The degree of enhancement varied according to the Ag: Ab ratio employed; the binding of ICs formed in moderate Ab excess was only modestly enhanced but that of complexes formed at slight Ab excess, equivalence and Ag excess was markedly elevated. The profile of PEG-induced enhancement of binding paralleled that of similar ICs in the C1q fluid phase system, suggesting that C1q binding in the latter may be influenced by PEG. However, the C1q-binding activity of in vivo-formed ICs seemed to be relatively unaffected by PEG since enhanced binding was comparable in control and pathological sera.

The results indicate that PEG causes cross-linking and aggregation of ICs (and possibly other serum proteins) which may alter their biological activity and hence influence the results of IC assays that employ this agent.