PREPARATION, CHARACTERIZATION, AND PROPERTIES OF MONOCLONAL-ANTIBODIES AGAINST THE LAC CARRIER PROTEIN FROM ESCHERICHIA-COLI
PREPARATION, CHARACTERIZATION, AND PROPERTIES OF MONOCLONAL-ANTIBODIES AGAINST THE LAC CARRIER PROTEIN FROM ESCHERICHIA-COLI
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DOI：
10.1073/pnas.79.22.6894
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发表时间：
1982-01-01
期刊：
影响因子：
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通讯作者：
KABACK, HR
中科院分区：
文献类型：
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作者：
CARRASCO, N;TAHARA, SM;KABACK, HR
采用小鼠骨髓瘤细胞与免疫小鼠脾细胞体细胞融合制备了从大肠杆菌膜中纯化的紫胶载体蛋白单克隆抗体。通过固相放射免疫测定和免疫印迹实验证明，一些克隆产生的抗体与纯化蛋白发生反应；克隆培养上清抑制乳糖在分离膜囊中的主动运输。选择5个稳定的克隆进行扩增、正式克隆和产生腹水，每个克隆在体内分泌的抗体也抑制乳糖运输。来自杂交瘤4B1的抗体是一种IgG2a免疫球蛋白，可以抑制乳糖在用纯化的乳糖载体重组的蛋白脂质体和右侧外膜囊中的主动运输。该抗体不影响膜囊泡产生质子电化学梯度，也不改变含有lac载体的囊泡结合对硝基苯基- α - d -半乳糖吡喃苷的能力。为了达到50%的运输活性抑制，无论膜中lac载体的数量如何，都需要2至3倍摩尔的lac载体抗体。因此，给定抑制程度所需的抗体浓度与膜中lac载体的数量成正比。抗体诱导的抑制在几秒钟内发生，这表明抗原表位在膜表面是可接近的。
Monoclonal antibodies directed against the lac carrier protein purified from the membrane of E. coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an immunized mouse. Several clones produce antibodies that react with the purified protein as demonstrated by solid-phase radioimmunoassay and by immunoblotting experiments; culture supernatants from the clones inhibit active transport of lactose in isolated membrane vesicles. Five stable clones were selected for expansion, formal cloning and production of ascites fluid, and the antibodies secreted in vivo by each clone also inhibited lactose transport. Antibody from hybridoma 4B1, an IgG2a immunoglobulin, inhibits active transport of lactose in proteoliposomes reconstituted with purified lac carrier and in right-side-out membrane vesicles. The antibody has no effect on the generation of the proton electrochemical gradient by membrane vesicles nor does it alter the ability of vesicles containing the lac carrier to bind p-nitrophenyl-.alpha.-D-galactopyranoside. To achieve 50% inhibition of transport activity, a 2- to 3-fold molar excess of antibody to lac carrier is required, regardless of the amount of lac carrier in the membrane. Thus, the concentration of antibody required for a given degree of inhibition is proportional to the amount of lac carrier in the membrane. Antibody-induced inhibition occurs within seconds, suggesting that the epitope is accessible on the surface of the membrane.