Sharp A H, Campbell K P
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Circ Res. 1987 Oct;61(4 Pt 2):I37-45.
High-affinity antibodies specific for the 1,4-dihydropyridine Ca2+ channel blockers have been produced in sheep and affinity purified using a dihydropyridine-Sepharose affinity column. Dihydropyridine-Sepharose affinity matrix was synthesized by reaction of aminohexyl-Sepharose with an affinity analogue of nifedipine, dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridine-dicarbo xylate. Residual amine groups were then blocked by carbodiimide-catalyzed acetylation. [3H]Nitrendipine-binding activity in serum was specifically absorbed by the dihydropyridine-Sepharose affinity column. The bound antibody was eluted with diethylamine (pH 11.5) in 10% dioxane or with a low-affinity dihydropyridine ligand (diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate), pH 7.4. Thirty-six milligrams of highly pure IgG antibody, as demonstrated by sodium dodecyl sulfate-gel electrophoresis, was isolated from 50 ml hyperimmune sheep serum. The affinity-purified anti-dihydropyridine antibodies have been shown to have high affinity (Kd approximately 0.1 nM) and specificity for the 1,4-dihydropyridine Ca2+ channel blockers and, therefore, exhibit dihydropyridine-binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+ channel blockers. Immunoblot staining of an azidopine-bovine serum albumin conjugate with affinity-purified antidihydropyridine antibodies demonstrated that the anti-dihydropyridine antibodies recognize the 1,4-dihydropyridine Ca2+ channel blockers when covalently coupled to protein and, therefore, should be useful in the identification and purification of receptors covalently labelled with 1,4-dihydropyridine Ca2+ channel blockers.
已在绵羊体内产生了对1,4 - 二氢吡啶钙离子通道阻滞剂具有特异性的高亲和力抗体，并使用二氢吡啶 - 琼脂糖亲和柱进行亲和纯化。二氢吡啶 - 琼脂糖亲和基质是通过氨基己基 - 琼脂糖与硝苯地平的亲和类似物1,4 - 二氢 - 2,6 - 二甲基 - 4 - (2 - 异硫氰酸苯酯) - 3,5 - 吡啶二羧酸二甲酯反应合成的。然后通过碳二亚胺催化的乙酰化反应封闭残留的胺基。血清中的[3H]尼群地平结合活性被二氢吡啶 - 琼脂糖亲和柱特异性吸附。结合的抗体用10%二氧六环中的二乙胺（pH 11.5）或低亲和力二氢吡啶配体（1,4 - 二氢 - 2,4,6 - 三甲基 - 3,5 - 吡啶二羧酸二乙酯，pH 7.4）洗脱。通过十二烷基硫酸钠 - 凝胶电泳证明，从50毫升超免疫绵羊血清中分离出了36毫克高纯度的IgG抗体。已证明亲和纯化的抗二氢吡啶抗体对1,4 - 二氢吡啶钙离子通道阻滞剂具有高亲和力（Kd约为0.1 nM）和特异性，因此表现出与1,4 - 二氢吡啶钙离子通道阻滞剂的膜受体相似的二氢吡啶结合特性。用亲和纯化的抗二氢吡啶抗体对叠氮平 - 牛血清白蛋白偶联物进行免疫印迹染色表明，抗二氢吡啶抗体在与蛋白质共价偶联时能识别1,4 - 二氢吡啶钙离子通道阻滞剂，因此可用于鉴定和纯化用1,4 - 二氢吡啶钙离子通道阻滞剂共价标记的受体。
