Advantages of sequential polyethylene glycol (PEG) precipitations to isolate and analyze circulating immune complexes

The present report describes a new approach utilizing sequential polyethylene glycol 6000 (PEG) precipitations to isolate and analyze circulating immune complexes (IC). Two experimental systems were studied. First, IC were formed by adding labeled or unlabeled bovine serum albumin (BSA) to anti-BSA antiserum. Sequential precipitations with PEG concentrations ranging from 2 to 6.2 indicated that immunoglobulin (Ig) molecules were precipitated in all experimental and control sera tested. In contrast, optimal precipitations of IC (i.e., highest BSA/Ig ratio) were a function both of PEG concentration and of BSA/anti-BSA ratio (i.e., of IC size). Satisfactory IC purification was achieved by precipitation with appropriate PEG concentration followed by incubation with STaphylococcus aureus, Cowan I. Second, in preliminary experiments, the same procedure was applied to human sera. The findings paralleled those of the BSA/anti-BSA system. While PEG precipitated Ig in all sera tested, a potential antigen was found only in the most IC-positive serum, and only at the higher of the two PEG concentrations tested. From our data, we conclude that PEG concentrations needed to precipitate IC cannot be predetermined, as they are a function of the size and probably the nature of the IC studied. This limitation can be overcome by the systematic sequential precipitations which increase the chances of identifying antigens and decrease the risks of confusing antigen-antibody complexes and Ig aggregates.