Crawford G, Slemmon J R, Salvaterra P M
J Biol Chem. 1982 Apr 10;257(7):3853-6.
The newly developed monoclonal antibody technology was applied to the production of antibodies selective for Drosophila melanogaster choline acetyltransferase (EC 2.3.1.6). Two stable cell lines, 1C8 and 1G4, were isolated from NS-1/spleen cell hybrids by employing a choline acetyltransferase enzyme activity-screening method. Both cell lines were cloned twice and were maintained in continuous culture and as ascites tumors. Purified antibody was isolated from ascites fluids by pH elution after adsorption to Protein A-Sepharose. Both antibodies eluted from the Protein A-Sepharose as a single subclass, IgG1, and directly inhibited choline acetyltransferase activity. Scatchard analysis of titration data for choline acetyltransferase antibody-enzyme interaction generated linear curves for both antibodies: KA for 1C8 was 2.77 X 10(7) M-1 and KA for 1G4 was 0.78 X 10(7) M-1. Inclusion of the choline acetyltransferase substrate acetyl-CoA at 10 times the KM in the antibody-enzyme reaction mixture substantially reduced the level of inhibition observed with both antibodies; choline, however, exhibited no protective effect. Neither antibody reacted with choline acetyltransferase-containing extracts of vertebrates or other insect neural tissues. We conclude that the two antibodies are nonidentical, monoclonal, and highly selective for D. melanogaster choline acetyltransferase, both reacting at or near the acetyl-CoA binding region of the enzyme-active site.
新开发的单克隆抗体技术被应用于生产对黑腹果蝇胆碱乙酰转移酶（EC 2.3.1.6）具有选择性的抗体。通过采用胆碱乙酰转移酶活性筛选方法，从NS-1/脾细胞杂交体中分离出两个稳定的细胞系，1C8和1G4。这两个细胞系都进行了两次克隆，并以连续培养和腹水瘤的形式保存。在吸附到蛋白A-琼脂糖后，通过pH洗脱从腹水液中分离出纯化的抗体。从蛋白A-琼脂糖上洗脱下来的两种抗体均为单一亚类IgG1，并直接抑制胆碱乙酰转移酶的活性。对胆碱乙酰转移酶抗体-酶相互作用的滴定数据进行Scatchard分析，两种抗体均产生线性曲线：1C8的KA为2.77×10⁷ M⁻¹，1G4的KA为0.78×10⁷ M⁻¹。在抗体-酶反应混合物中加入10倍于KM的胆碱乙酰转移酶底物乙酰辅酶A，可显著降低两种抗体所观察到的抑制水平；然而，胆碱没有保护作用。两种抗体均不与脊椎动物或其他昆虫神经组织中含胆碱乙酰转移酶的提取物发生反应。我们得出结论，这两种抗体是不同的单克隆抗体，对黑腹果蝇胆碱乙酰转移酶具有高度选择性，均在酶活性位点的乙酰辅酶A结合区域或其附近发生反应。
