A RAPID, SENSITIVE METHOD FOR DETECTION OF ALKALINE-PHOSPHATASE CONJUGATED ANTI-ANTIBODY ON WESTERN BLOTS
A RAPID, SENSITIVE METHOD FOR DETECTION OF ALKALINE-PHOSPHATASE CONJUGATED ANTI-ANTIBODY ON WESTERN BLOTS
复制标题
DOI：
10.1016/0003-2697(84)90320-8
复制
发表时间：
1984-01-01
影响因子：
2.9
通讯作者：
GOTSCHLICH, EC
中科院分区：
文献类型：
--
作者：
BLAKE, MS;JOHNSTON, KH;GOTSCHLICH, EC
建立了一种快速、灵敏的Western blots检测抗体-抗原复合物的方法。采用Towbin等人的方法将抗原分离并印迹到硝化纤维素上。剩余的附着位点被阻断，用单月桂酸聚氧乙烯轨道烷（Tween 20）洗涤硝化纤维素。然后将印迹与抗血清或杂交瘤上清反应进行检测。抗原-抗体反应完成后，清洗印迹，用偶联到碱性磷酸酶上的抗抗体处理。磷酸酶与磷酸吲哚酚反应生成靛蓝时释放的H+离子将四氮唑盐还原成双双胍，以此检测碱性磷酸酶的存在。与先前描述的技术相比，该方法的优点是：使用Tween 20可以用考马斯蓝染色，碱性磷酸酶反应的底物长时间稳定，反应产物形成强烈的蓝色，不褪色，分辨率非常好，几乎没有增宽，反应对picogram抗原量敏感，反应是定量的。
A rapid, sensitive method was developed to detect antibody-antigen complexes on Western blots. The methods of Towbin et al. 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 H+ 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: use of Tween 20 allows the blot to be stained with Coomassie blue, the substrates of the alkaline phosphatase reaction are stable for long periods of time, the reaction products form an intense blue color which does not fade, the resolution is extremely good with little to no band broadening, the reaction is sensitive to picogram quantities of antigen and the reaction is quantitative.