DETECTION AND CHARACTERIZATION OF CIRCULATING IMMUNE-COMPLEXES BY ULTRA-CENTRIFUGATION - TECHNICAL ASPECTS

Most techniques for detection of immune complexes (IC) in biological fluids do not fulfil criteria essential for IC definition, i.e. identification of the antigen and antibody after dissociation and demonstration of their immunological binding. We have fractionated plasmas by sucrose density gradient ultracentrifugation at pH 7.4 and 2.8 and studied the effects of acid pH on isolated and plasma immunoglobulins, on antibody titers, on IC formed in vitro and on aggregated immunoglobulins. There was no effect on the distribution of isolated radiolabeled IgG, IgA or IgM or on IgG and IgA of fresh serum samples. Heavy IgG appeared at pH 2.8 in samples stored at -20°C in excess of 1 month and also at pH 7.4 when stored in excess of 6 months. Heavy IgA was present in samples frozen for 1 week or more at both pHs. Anti-BSA and anti-DNA antibody titers and distribution were identical at both pHs. BSA : anti-BSA and DNA : anti-DNA IC formed in vitro were almost completely dissociated with recovery of antigen and antibody. IC obtained by fractionation at pH 7.4 could be refractionated at pH 2.8 with recovery of antibody and antigen. The distribution of aggregated IgG and IgA was unchanged at acid pH. This technique allows differentiation of complexed and aggregated Ig, identification of specific IC by comparing antibody titers at physiologic and acid pH, isolation of IC and the recovery of antigen and antibody from IC formed in vitro. © 1979.