Cleaveland J S, Kiener P A, Hammond D J, Schacter B Z
Bristol-Myers Squibb, Wallingford, Connecticut 06492.
Anal Biochem. 1990 Nov 1;190(2):249-53. doi: 10.1016/0003-2697(90)90188-f.
A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.
已开发出一种用于蛋白质酪氨酸激酶的96孔微量滴定酶联免疫吸附测定（ELISA）。该测定使用几种被酪氨酸激酶磷酸化的底物之一、抗磷酸酪氨酸抗体和过氧化物酶偶联的二抗。通过监测450nm处吸光度的变化来监测显色情况，显色取决于时间、酶、ATP和底物浓度。用磷酸苯酯抑制抗磷酸酪氨酸抗体的结合，证明了ELISA对磷酸酪氨酸的特异性。ELISA获得的结果与用[32P]ATP直接磷酸化底物获得的结果相比具有优势。蛋白激酶抑制剂星形孢菌素和K252a显示出可滴定的酪氨酸激酶活性抑制作用。该测定是传统方法的一种快速、非放射性替代方法，也适合自动化。
