36] Quantitation of circulating immune complexes by combined PEG precipitation and immunoglobulin-specific radioimmunoassay (PICRIA)

Abstract

This chapter describes the quantitation of circulating immune complexes (ICs) by combined polyethylene glycol (PEG) precipitation and immunoglobulin-specific radioimmunoassay (PICRIA). PEG and other hydrophilic polymers induce the precipitation of proteins from neutral solutions largely in relation to the molecular weight of the protein. Conditions were established for minimal precipitation of the noncomplexed immunoglobulin and the precipitated complexes were solubilized, with acid, before the first step, in ensuing the nonequilibrium radioimmunoassay and the addition of antibody suitably diluted in a neutralizing buffer. Serial dilutions of the IgG standard and PEG precipitates from normal and patient sera were carried out in acid solution (pH 2.8) and in neutralized acid solution (pH 7.4), and then tested in κ-RIA. The immunoglobulin concentrations of 23 reference sera were measured in radial immunodiffusion with anti-γ, anti-κ, and anti-λ antisera, while the total protein content was estimated by the biuret method. It is found that the apparent loss of lgG determinants from the standard preparation after three days of incubation at pH 2.8 was 31% as judged from γ-RIA.