De Beer F C, Pepys M B
J Immunol Methods. 1982;50(1):17-31. doi: 10.1016/0022-1759(82)90300-3.
Procedures are described for the isolation in high yield of consistent, highly purified preparations of human C-reactive protein (CRP) and serum amyloid P component (SAP). CRP was obtained from malignant ascitic and pleural fluids by calcium-dependent affinity chromatography on pneumococcal C-polysaccharide covalently coupled to cyanogen bromide-activated Sepharose. It was then gel filtered on Ultrogel AcA44 (acrylamide-agarose beads) in the presence of calcium ions, combining molecular sieve chromatography with removal of contaminating SAP by its affinity of agarose. Residual trace contaminants were removed by immunoabsorption with anti-normal human serum and anti-SAP antibodies insolubilised on Sepharose and/or by absorption with Sepharose-Con A to remove glycoproteins and Blue-Sepharose to remove albumin. After a final gel filtration step on Sephacryl S-300 35-45% of the initial CRP was recovered in pure form according to biochemical and immunochemical criteria. SAP was isolated from normal serum by calcium-dependent affinity chromatography on unsubstituted Sepharose beads, followed by solid-phase immunoabsorption of contaminants and finally gel filtration on Sephacryl S-300. At least 50% of the SAP in the starting material was recovered in pure form according to biochemical and immunochemical criteria. Ready availability of such preparations facilitates biochemical, biophysical and particularly biological studies of these plasma proteins.
本文描述了从人C反应蛋白（CRP）和血清淀粉样蛋白P成分（SAP）中高产率分离出一致性高、纯度高的制剂的方法。CRP是通过将共价偶联到溴化氰活化的琼脂糖珠上的肺炎球菌C多糖进行钙依赖性亲和色谱，从恶性腹水和胸水中获得的。然后在钙离子存在的情况下，在Ultrogel AcA44（丙烯酰胺-琼脂糖珠）上进行凝胶过滤，将分子筛色谱与通过琼脂糖亲和力去除污染性SAP相结合。通过用固定在琼脂糖珠上的抗正常人血清和抗SAP抗体进行免疫吸附和/或用琼脂糖-Con A吸附去除糖蛋白以及用蓝色琼脂糖吸附去除白蛋白，去除残留的痕量污染物。在Sephacryl S-300上进行最后一步凝胶过滤后，根据生化和免疫化学标准，以纯形式回收了初始CRP的35%-45%。通过在未取代的琼脂糖珠上进行钙依赖性亲和色谱，从正常血清中分离出SAP，随后对污染物进行固相免疫吸附，最后在Sephacryl S-300上进行凝胶过滤。根据生化和免疫化学标准，起始材料中至少50%的SAP以纯形式回收。这些制剂的现成可用性有助于对这些血浆蛋白进行生化、生物物理特别是生物学研究。
