Cumber A J, Henry R V, Parnell G D, Wawrzynczak E J
Section of Medicine, Institute of Cancer Research, Sutton, U.K.
J Immunol Methods. 1990 Dec 31;135(1-2):15-24. doi: 10.1016/0022-1759(90)90251-p.
Comparable amounts of the ribosome-inactivating proteins (RIPs) ricin A chain, gelonin and momordin were allowed to bind to Blue Sepharose CL-6B (immobilised Cibacron Blue F3GA) in phosphate buffer, pH 7.5, and were then eluted quantitatively with buffer containing 0.5 M NaCl. Differences in the elution profiles indicated that the RIPs possessed different affinities for the Cibacron Blue F3GA dye. Conjugation of the RIPs to the monoclonal antibody Fib75 resulted in decreased affinity for Blue Sepharose. Under conditions allowing the complete separation of the Fib75-ricin A chain immunotoxin from unconjugated Fib75, the Fib75 immunotoxins made with gelonin and momordin failed to bind completely to the Blue Sepharose column. The Fib75-gelonin and Fib75-momordin fractions that eluted from the column with 0.5 M NaCl were free of unconjugated Fib75 but were enriched in multiply substituted conjugate molecules. A high performance liquid immunoaffinity chromatography procedure based on the selective binding of conjugated RIP to immobilised affinity-purified anti-RIP antibody permitted the complete separation of the gelonin and momordin immunotoxins from unconjugated Fib75 without altering the composition, molecular integrity or cytotoxic activity of the immunotoxins.
在pH 7.5的磷酸盐缓冲液中，使等量的核糖体失活蛋白（RIPs）蓖麻毒素A链、相思豆毒蛋白和苦瓜素与蓝色琼脂糖凝胶CL-6B（固定化的汽巴克隆蓝F3GA）结合，然后用含0.5 M氯化钠的缓冲液进行定量洗脱。洗脱曲线的差异表明这些RIPs对汽巴克隆蓝F3GA染料具有不同的亲和力。将RIPs与单克隆抗体Fib75偶联导致对蓝色琼脂糖凝胶的亲和力降低。在能使Fib75-蓖麻毒素A链免疫毒素与未偶联的Fib75完全分离的条件下，用相思豆毒蛋白和苦瓜素制备的Fib75免疫毒素未能完全结合到蓝色琼脂糖凝胶柱上。用0.5 M氯化钠从柱上洗脱下来的Fib75-相思豆毒蛋白和Fib75-苦瓜素组分不含未偶联的Fib75，但富含多取代的偶联分子。基于偶联的RIP与固定化的亲和纯化抗RIP抗体的选择性结合的高效液相免疫亲和色谱法，能够将相思豆毒蛋白和苦瓜素免疫毒素与未偶联的Fib75完全分离，同时不改变免疫毒素的组成、分子完整性或细胞毒性活性。
