Tumoricidal response following perfusion over immobilized protein A: identification of immunoglobulin oligomers in serum after perfusion and their partial characterization.

Previously, we showed that perfusion of plasma from hosts bearing breast adenocarcinoma over immobilized staphylococcal protein A resulted in objective tumor regressions. In the present study, sera perfused in vitro over immobilized staphylococcal protein A were analyzed by physicochemical and immunochemical methods to characterize newly formed products. Sera from normal and breast adenocarcinoma-bearing dogs showed increased levels of C1q-binding IgG after perfusion over a strain of staphylococcus that is protein A rich (Cowan I), but not protein A deficient (Woods 46). C1q binding levels were also increased in normal and tumor-bearing canine or human sera which were perfused over purified protein A immobilized in collodion charcoal (PACC), and this increase was localized in sucrose density gradient fractions ranging from 7S to 19S. Polyacrylamide gel electrophoresis analysis of the high-molecular-weight fraction in postperfusion canine sera, isolated by G-200 fractionation and immunoaffinity chromatography, showed predominantly heavy and light immunoglobulin chains of canine IgG. Furthermore, protein A was released from PACC after perfusion with serum or solutions containing IgG or albumin from humans, dogs, and chickens. After serum perfusion over PACC, protein A was identified in the effluent by additional studies as follows: (a) polyacrylamide gel electrophoresis analysis showed that eluted 125I-protein A comigrated with the protein A marker; (b) postperfusion C1q-binding complexes, isolated by gel filtration under dissociating conditions and affinity chromatography on IgG-Sepharose showed a single precipitin band with normal human (protein A reactive) but not chicken (protein A unreactive) serum. Protein A released from PACC which appeared in postperfusion sera was associated with immunoglobulins in macromolecular complexes since (a) eluted 125I-protein A was largely (NH4)2SO4 and polyethylene glycol precipitable, whereas free protein A was not, and it sedimented in sucrose density gradient fractions distributed beyond the 7S marker, compared to free protein A which localized below 7S; (b) radiolabeled protein A eluting from PACC after serum perfusion showed 8-fold greater binding to C1q-coated tubes compared to free protein A; and (c) increased C1q-binding IgG in postperfusion sucrose density gradient fractions corresponded to the appearance of protein A in parallel gradient fractions.(ABSTRACT TRUNCATED AT 400 WORDS)