Reisberg M A, Rossen R D
Clin Exp Immunol. 1981 Nov;46(2):443-52.
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.
结合在溴化氰活化琼脂糖上的葡萄球菌蛋白A（SPA），被评估为一种用于分离在抗原（Ag）过量情况下制备的可溶性免疫复合物（ICs）的选择性吸附剂。使用了抗人血清白蛋白（HSA）的山羊抗体（Ab）以及抗白喉类毒素（DT）的兔和人抗血清来形成复合物。单体山羊IgG不与SPA结合。然而，通过蔗糖密度梯度超速离心法测定大于12S且摩尔Ab:Ag比大于1.5的HSA - 山羊抗HSA复合物可被吸附，随后可用pH小于或等于3.8的酸性磷酸盐缓冲盐水洗脱。用3.5 M MgCl₂洗脱可提高回收率，但也会导致结合的抗体水解。在存在过量游离Ag的情况下，用兔抗体制备的DT - 抗DT ICs的90%以及用人抗体制备的55%与SPA柱结合。然而，在酸性磷酸盐缓冲洗脱液中分离得到的DT - 兔抗DT复合物仅为42%，用人抗血清制备的复合物为32%，且不含污染蛋白。当ICs在应用于SPA - 琼脂糖柱之前通过聚乙二醇（PEG）或硫酸铵沉淀进行部分纯化时，通过SPA亲和色谱法回收ICs的效率显著降低。这些研究表明，SPA可用于分离在远过量Ag情况下制备的ICs以及本身不与该吸附剂结合的抗体形成的ICs。它们还表明，使用这种吸附剂从血清中回收ICs总是不完全的。
