Isolation of soluble immune complexes by affinity chromatography using staphylogoccal protein A-Sepharose as substrate

Abstract

This report describes a technique for the general isolation of immune complexes, based on a combination of gel filtration and affinity chromatography. The first step is the preparation of a globulin-enriched fraction by precipitation with ammonium sulfate at 50% saturation, or of an immune-complex-enriched fraction by precipitation with 5% polyethylene glycol 6000. The enriched fraction is then subfractional by gel filtration in Ultrogel AcA 34. The immune complexes elute close to the void volume in the macroglobulin peak, separated from monomeric IgG molecules. This peak (sometimes subdivided into two fractions) is then submitted to affinity chromatography on a protein A-Sepharose column. Most immune complexes contain IgG molecules and therefore bind to the column. Almost no protein is bound when normal serum is fractionated according to this method, and no immunoglobulins are detectable in the acid-eluted fraction from the protein A-Sepharose column. In two patients with soluble immune complexes in their sera we eluted immunoglobulin-containing fractions from the column; in one, these fractions had high rheumatoid factor titers; and in the second, with a clinical diagnosis of systemic lupus erythematosus, a similar fraction contained RNA.