Gupta R C, Tan E M
Clin Exp Immunol. 1981 Oct;46(1):9-19.
A method for the isolation of complement-fixing immune complexes from human serum and the separation of antigen from antibody is described. In order to isolate the complexes, we used soluble bovine conglutinin in a three-step procedure: (1) serum containing immune complexes is reacted with conglutinin in the presence of 10 mM calcium; (2) the conglutinin-bound immune complexes are precipitated by anti-conglutinin rabbit serum; (3) the precipitate is washed and the complexes are eluted from the precipitate by EDTA (pH 7.5) which chelates calcium and releases C3-associated immune complexes from conglutinin. To separate the antigen from the antibody, the isolated complexes are acid-dissociated (pH 3.0), and the antibody is absorbed to staphylococcal protein A conjugated to Sepharose leaving the antigen in solution. The antibody bound to Sepharose-protein A is recovered by elution with 3.5 M magnesium chloride. This procedure permitted the isolation of immune complexes from sera of hepatitis B surface antigen (HBsAg) positive chronic active hepatitis. In addition, immune complexes were isolated from sera of patients with rheumatoid arthritis, systemic lupus erythematosus and primary biliary cirrhosis. The isolated immune complexes contained IgG, IgM, C3 and albumin. Specific antibodies such as rheumatoid factors, anti-nuclear antibodies and antimitochondrial antibodies in varying titres have been found to be present in the isolated immune complexes. The conglutinin method has proven to be a useful technique for the isolation of immune complexes and for the identification of antibody and could be applied to the identification of the antigen in immune complexes.
本文描述了一种从人血清中分离补体结合免疫复合物以及从抗体中分离抗原的方法。为了分离复合物，我们采用了可溶性牛胶固素，分三步进行：（1）含有免疫复合物的血清在10 mM钙存在的情况下与胶固素反应；（2）与胶固素结合的免疫复合物用抗胶固素兔血清沉淀；（3）沉淀物经洗涤后，用EDTA（pH 7.5）从沉淀物中洗脱复合物，EDTA螯合钙并从胶固素中释放出与C3相关的免疫复合物。为了从抗体中分离抗原，将分离出的复合物进行酸解离（pH 3.0），抗体被吸附到与琼脂糖偶联的葡萄球菌蛋白A上，而抗原留在溶液中。与琼脂糖-蛋白A结合的抗体用3.5 M氯化镁洗脱回收。该方法可从乙肝表面抗原（HBsAg）阳性慢性活动性肝炎患者的血清中分离免疫复合物。此外，还从类风湿性关节炎、系统性红斑狼疮和原发性胆汁性肝硬化患者的血清中分离出免疫复合物。分离出的免疫复合物含有IgG、IgM、C3和白蛋白。已发现分离出的免疫复合物中存在不同滴度的特异性抗体，如类风湿因子、抗核抗体和抗线粒体抗体。胶固素方法已被证明是一种用于分离免疫复合物和鉴定抗体的有用技术，也可应用于免疫复合物中抗原的鉴定。
