Hohmann A, Hodgson A J, Di W, Skinner J M, Bradley J, Zola H
Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, South Australia.
J Histochem Cytochem. 1988 Feb;36(2):137-43. doi: 10.1177/36.2.3335772.
A mouse monoclonal antibody, FMC55 (an IgG1), to alkaline phosphatase was prepared and evaluated in immunostaining. Clones producing antibody to alkaline phosphatase were selected using a micro-ELISA which identified antibodies forming active soluble complexes (APAAP) with the enzyme. Conditions that influenced the formation of the complex were investigated by using a quantitative assay in which the complex was captured by a bridging anti-mouse antibody. The ratio of FMC55 to enzyme had a major influence on the activity of the complex. Although all complexes had some activity, those that contained excess antibody had reduced ability to bind to anti-mouse antibody because of competition with excess unlabeled antibody. The optimal complex was formed with 3 micrograms of FMC55 per unit of enzyme. This complex contained neither free enzyme nor free antibody. The molecular weight by gel permeation chromatography was 600,000, giving a composition of two enzyme and two antibody molecules or one enzyme and three antibody molecules. The size of the complex was not altered by adding excess antibody or excess enzyme. Immunoblotting showed that FMC55 bound only to the Mr 140,000 homodimeric form of alkaline phosphatase. The APAAP complex was used in combination with biotin-streptavidin-peroxidase reagent to detect two antigens labeled with two different mouse monoclonal antibodies in the same tissue preparation.
制备了一种针对碱性磷酸酶的小鼠单克隆抗体FMC55（一种IgG1），并在免疫染色中进行了评估。使用微量酶联免疫吸附测定法（micro-ELISA）筛选出产生针对碱性磷酸酶抗体的克隆，该方法可鉴定与该酶形成活性可溶性复合物（碱性磷酸酶抗碱性磷酸酶复合物，APAAP）的抗体。通过一种定量测定法研究影响复合物形成的条件，在该测定法中，复合物由桥接抗小鼠抗体捕获。FMC55与酶的比例对复合物的活性有主要影响。尽管所有复合物都有一定活性，但那些含有过量抗体的复合物由于与过量未标记抗体的竞争，与抗小鼠抗体结合的能力降低。每单位酶与3微克FMC55形成最佳复合物。该复合物既不含有游离酶也不含有游离抗体。凝胶渗透色谱法测得的分子量为600,000，表明其组成为两个酶分子和两个抗体分子或一个酶分子和三个抗体分子。添加过量抗体或过量酶不会改变复合物的大小。免疫印迹显示FMC55仅与分子量为140,000的碱性磷酸酶同型二聚体形式结合。碱性磷酸酶抗碱性磷酸酶复合物与生物素 - 链霉亲和素 - 过氧化物酶试剂联合使用，以在同一组织标本中检测用两种不同小鼠单克隆抗体标记的两种抗原。
