Glenney J R, Zokas L, Kamps M P
Molecular Biology and Virology Laboratory, Salk Institute for Biological Studies, San Diego, CA 92138.
J Immunol Methods. 1988 May 9;109(2):277-85. doi: 10.1016/0022-1759(88)90253-0.
Phosphotyrosine coupled to KLH, BSA, and OVA was used for the production and screening of antibodies to phosphotyrosine. 800 hybridomas secreting antibodies that bound to phosphotyrosine were detected by ELISA. The most reactive 100 of these 800 were tested subsequently for their ability to bind phosphotyrosine-containing proteins on Western blots. Eight stable hybridoma cell lines were selected for further study, cloned by limiting dilution, and grown as ascites. These antibodies were purified by three different methods, and it was found that affinity chromatography on phosphotyrosine-affigel provided the most rapid and effective method. Many phosphotyrosine-containing proteins were detected by using these antibodies in Western blotting and immunoaffinity purification procedures. Binding of anti-phosphotyrosine antibody could be competed by phosphotyrosine or phenylphosphate but not by phosphoserine, phosphothreonine, or free phosphate. These antibodies should be useful for the identification and purification of proteins phosphorylated on tyrosine residues in transformed and growth factor-treated cells.
与钥孔血蓝蛋白（KLH）、牛血清白蛋白（BSA）和卵清蛋白（OVA）偶联的磷酸酪氨酸用于磷酸酪氨酸抗体的制备和筛选。通过酶联免疫吸附测定（ELISA）检测到800个分泌与磷酸酪氨酸结合抗体的杂交瘤。随后对这800个中反应性最强的100个进行检测，以确定它们在蛋白质免疫印迹法中结合含磷酸酪氨酸蛋白的能力。选择了8个稳定的杂交瘤细胞系进行进一步研究，通过有限稀释法克隆，并作为腹水培养。这些抗体通过三种不同方法进行纯化，发现磷酸酪氨酸琼脂糖亲和层析提供了最快速有效的方法。在蛋白质免疫印迹法和免疫亲和纯化程序中使用这些抗体检测到许多含磷酸酪氨酸的蛋白质。抗磷酸酪氨酸抗体的结合可被磷酸酪氨酸或苯磷酸盐竞争，但不能被磷酸丝氨酸、磷酸苏氨酸或游离磷酸盐竞争。这些抗体对于鉴定和纯化在转化细胞和生长因子处理细胞中酪氨酸残基磷酸化的蛋白质应是有用的。
