Blake M S, Johnston K H, Russell-Jones G J, Gotschlich E C
Anal Biochem. 1984 Jan;136(1):175-9. doi: 10.1016/0003-2697(84)90320-8.
A rapid, sensitive method has been developed to detect antibody-antigen complexes on "Western blots." The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which had been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.
已开发出一种快速、灵敏的方法来检测“蛋白质免疫印迹”上的抗体 - 抗原复合物。采用H. 托宾、T. 施泰林和J. 戈登的方法将抗原分离并印迹到硝酸纤维素膜上。封闭剩余的结合位点，并用聚氧乙烯山梨醇单月桂酸酯（吐温20）洗涤硝酸纤维素膜。然后将印迹膜与待测抗血清或杂交瘤培养上清反应。抗原 - 抗体反应完成后，洗涤印迹膜并用与碱性磷酸酶偶联的抗抗体处理。通过磷酸酶对磷酸吲哚酚的反应形成靛蓝时释放的氢离子将四唑盐还原为二亚甲臜来检测碱性磷酸酶。该方法相对于先前描述的技术的优点是：（1）使用吐温20可使印迹膜用考马斯亮蓝染色；（2）碱性磷酸酶反应的底物长时间稳定；（3）反应产物形成强烈的蓝色且不褪色；（4）分辨率极高，几乎没有条带展宽；（5）该反应对皮克量的抗原敏感；（6）该反应是定量的。
