Immunochemical Studies of Bovine and Human Choline‐O‐Acetyltransferase Using Monoclonal Antibodie
Immunochemical Studies of Bovine and Human Choline‐O‐Acetyltransferase Using Monoclonal Antibodie
复制标题
使用单克隆抗体对牛和人胆碱-O-乙酰转移酶进行免疫化学研究
作者：
A. Levey;D. Rye;B. Wainer
翻译后摘要：牛和人的胆碱乙酰转移酶（ChAT，EC 2.3.1.6，乙酰辅酶A：胆碱-O-乙酰转移酶）的免疫化学性质进行了研究，使用六种单克隆抗体（AB 1，AB 5，AB 6，AB 7，AB 8和AB 9）与酶反应。在含有人或牛ChAT的十二烷基硫酸钠-聚丙烯酰胺凝胶的“Western”印迹上，除AB 1外的所有抗体均特异性结合68，000和70，000 MW的两种蛋白质。酶被特异性地吸附到固定化抗体上，并且不能通过低pH和/或高盐浓度洗脱，尽管酶在免疫吸附剂上保留活性。在十二烷基硫酸钠的存在下，从固定化的AB 1中洗脱由在Western印迹研究中所见的相同的两种蛋白质组成的纯牛酶。虽然活性酶不能通过标准条件从固定化抗体洗脱，但游离抗体和固定化抗体的各种组合在竞争脱离结合的酶中是有效的。游离抗体AB 1从固定化AB 1定量洗脱活性酶。抗体从各种免疫吸收剂中洗脱酶的不同能力反映了酶和抗体的有趣特性。
Abstract: Immunochemical properties of bovine and human choline acetyltransferase (ChAT, EC 2.3.1.6, acetyl‐CoA:choline‐O‐acetyltransferase) were studied using six monoclonal antibodies (AB1, AB5, AB6, AB7, AB8, and AB9) reactive with the enzyme. All antibodies except AB1 bound specifically to two proteins of 68,000 and 70,000 MW on “Western” blots of sodium dodecyl sulfate‐polyacrylamide gels containing human or bovine ChAT. The enzyme was specifically absorbed to immobilized antibody and could not be eluted by low pH and/or high salt concentrations, although the enzyme retained activity on the immunoabsorbent. Pure bovine enzyme consisting of the same two proteins as seen in the Western blotting studies was eluted from immobilized AB1 in the presence of sodium dodecyl sulfate. Although active enzyme could not be eluted from immobilized antibodies by standard conditions, various combinations of free and immobilized antibodies were effective in competing off bound enzyme. Free antibody AB1 quantitatively eluted the active enzyme from immobilized AB1. The different capacities of the antibodies to elute enzyme from various immunoabsorbents reflect interesting properties of both the enzyme and the antibodies.