PURIFICATION OF ANTIGEN-ANTIBODY COMPLEXES FROM SELECTED SPECIES BY STAPHYLOCOCCAL PROTEIN A
Abstract
The isolation of antigen-antibody complexes from serum and their subsequent analysis, particularly the identification of constituent antigens, may provide clues to the etiology of many diseases of unknown origin. This work examines the effectiveness of Staphylococcal protein A (SpA) as a reagent for the purification of immune complexes (IC) from the serum of species selected because of availability of sera containing elevated quantities of IC of unknown composition. A second goal of this work was to adapt two-dimensional gel electrophoresis, silver staining, and Western blotting to the analysis of isolated 1C. For SpA to be an effective reagent for the isolation of circulating IC, it should meet the requirements of binding preferentially to oligomeric, as opposed to monomeric, IgG, and allow for good recoveries of IC from serum. Hence, binding studies were done in polyvinyl chloride (PVC) plates, and Ka were determined by Scatchard analysis for hamster, mouse and human IgG, and for IC models prepared by alkaline aggregation of hamster or mouse IgG. The Ka were 4.5 x 10('6) M('-1), 7.5 x 10('6) M('-1), and 1.1 x 10('7) M('-1) for monomeric hamster, mouse, and human IgG, respectively. The Ka for the binding of SpA to hamster and mouse IgG aggregates increased as a function of size of the aggregate. The validity of using IgG aggregates as a model for IC was assessed by comparing the Ka of the binding of SpA to similar sized fractions of preformed antigen-antibody complexes. The enhancement of binding caused by chemical aggregation of IgG was less than the enhancement caused by antigen-induced polymerization of antibodies. Additional studies performed in PVC plates demonstrated that, on the average, monomeric mouse IgG inhibited the binding of mouse IC to SpA more than monomeric hamster IgG inhibited the binding of hamster IC. This possibly is because of the greater Ka of monomeric mouse IgG. Recovery studies were performed to evaluate the strength of the purification protocol used to isolate IC. A combination of sucrose density gradient ultracentrifugation and affinity chromatography on SpA-Sepharose gave a net recovery of 26.5% of a model IC (('125)I-EA hamster anti-EA) in hamster serum, and a 21.2% recovery of a model IC (('125)I-BSA mouse anti-BSA) in mouse serum. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI
Subject Area
Immunology
Recommended Citation
PER, STEVEN ROBERT, "PURIFICATION OF ANTIGEN-ANTIBODY COMPLEXES FROM SELECTED SPECIES BY STAPHYLOCOCCAL PROTEIN A" (1984). ProQuest ETD Collection - Thomas Jefferson University. AAI8421834.
https://jdc.jefferson.edu/dissertations/AAI8421834
