Characterization of immune complexes from the pleural effusion of a breast cancer patient.

Abstract

AbstractPieural effusion from a patient with adenocarcinoma of the breast was precipitated by 50% ammonium sulfate and chromatographed on Sephadex G‐200. The resulting elution profile consisted of three protein peaks. Fractions were monitored by immunodiffusion and assayed for [125I]C1q binding activity (C1q B.A.). The first protein peak (eluted at the void volume) showed appreciable Clq B.A. and contained immunoglobulin G (IgG) and complement 3 (C3). These data suggested that antigen‐antibody complexes were present in this macromolecular peak. IgG and C3 were also present in the second peak of Sephadex G‐200 gel filtration while the third peak contained the albumin fraction. The known tumor‐associated antigens, carcinoembryonic antigen, alpha‐fetoprotein and ferritin, were not detected in any of the protein fractions as determined fay immunodiffusion analysis. The first Sephadex protein peak which contained high molecular weight IgG was chromatographed on Sepharose 6B. The second protein peak obtained by Sepharose filtration contained IgG and C3 and was further applied to a column of protein A‐Septtarose. Proteins which bound to the immobilized protein A were dissociated with 2.5 M KSCN and chromatographed on Sepharose CL‐6B under dissociating conditions. IgM rheumatoid factor, IgG and four putative antigens were obtained by these dissociation and chromatographic procedures. The isolated IgG exhibited a molecular weight of monomeric IgG and bound to saline extracts of malignant tissue at a greater amount titan to normal or benign breast extracts (p<0 05). The reactive breast tumor protein in these extracts was eluted in the molecular weight region of 67,000, as determined by gel filtration. The four putative pleural fluid antigens which dissociated from the IgG were tested for their ability to recombine with the isolated immunoglobulin. Results from radio‐immunoprecipitation and the [125I]C1q binding assay indicated that the putative antigens bound to the isolated IgG to form immune complexes.