Advantages and limitations of staphylococcal protein A-Sepharose for isolating soluble immune complexes from goat, rabbit and human sera.
Staphylococcal protein A (SPA), bound to CNBr-activated Sepharose, was evaluated as a selective adsorbent for soluble immune complexes (ICs) prepared in antigen (Ag) excess. Goat antibody (Ab) to human serum albumin (HSA) and rabbit and human antisera to diphtheria toxoid (DT) were utilized for complex formation. Monomeric goat IgG did not bind SPA. However, HSA-goat anti-HSA complexes which were greater than 12S by sucrose density-gradient ultracentrifugation and had molar Ab:Ag ratios greater than 1.5 were adsorbed, and could subsequently be eluted with acidic phosphate-buffered saline, pH less than or equal to 3.8. Elution with 3.5 M MgCl2 enhanced recovery, but also resulted in hydrolysis of the bound Ab. Ninety per cent of the DT-anti-DT ICs prepared with rabbit Ab and 55% of those prepared with human Ab, in the presence of excess free Ag, bound to the SPA columns. However, only 42% of the DT-rabbit anti-DT complexes, and 32% of those prepared with human antisera were isolated in the acidic phosphate-buffered eluate, free of contaminating proteins. Recovery of ICs by SPA affinity chromatography was significantly decreased when the ICs were partially purified by PEG or ammonium sulphate precipitation before application to the SPA-Sepharose columns. These studies indicate that SPA can be used to isolate ICs prepared in far Ag excess and with Abs which, by themselves, do not bind to this absorbent. They also demonstrate that recovery of ICs from sera using this adsorbent is invariably incomplete.
D000645:Ammonium Sulfate; D000818:Animals; D000936:Antigen-Antibody Complex / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D002847:Chromatography, Agarose; D004168:Diphtheria Toxoid / Q000276:immunology; D006041:Goats; D006801:Humans; D007164:Immunosorbents; D008722:Methods; D011092:Polyethylene Glycols; D011817:Rabbits; D012685:Sepharose; D012709:Serum Albumin / Q000276:immunology; D013205:Staphylococcal Protein A
D000936:Antigen-Antibody Complex; D004168:Diphtheria Toxoid; D007164:Immunosorbents; D012709:Serum Albumin; D013205:Staphylococcal Protein A; D011092:Polyethylene Glycols; D012685:Sepharose; D000645:Ammonium Sulfate