辣根过氧化物酶标记JWA抗体及其在免疫检测反应中的应用

Preparation and Immuno Application of the Monoclonal Antibody of JWA Labeled with Horseradish Peroxidase

目的:运用辣根过氧化物酶标记JWA单克隆抗体,并且将标记的抗体应用于ELISA法和western blot等免疫检测反应中。方法:运用高碘酸盐-四氢硼化钠氧化还原体系活化辣根过氧化物酶,活化的辣根过氧化物酶按1∶1标记JWA单克隆抗体;将HRP标记的抗体分别运用直接和双夹心ELISA测试;同时将标记单克隆抗体运用于western blot检测。结果:辣根过氧化物酶成功标记JWA单克隆抗体。其中HRP-JWA(4C9)抗体在直接法ELISA试验中和多肽的结合能力高于HRP-JWA(7C3);其在450 nm处的最大吸光度值为0.96。双夹心ELISA实验中,预先包被JWA(4C9)单抗,检测抗体使用HRP-JWA(7C3)可以得到较好的实验结果,在450nm处的最大吸光度值为1.30。在western blot实验HRP-JWA抗体浓度1.0μg·m L-1可以检测出SGC7901细胞中目的蛋白。结论:运用氧化还原方法成功标记JWA单克隆抗体,并可以初步应用于免疫反应的检测。

Objective: To label JWA monoclonal antibody with horseradish peroxidase(HRP) and apply it in ELISA and western blot immune detection. Methods: Using sodium periodate and sodium borohydride redox reaction, HRP was activated; The activated HRP was used to mark JWA monoclonal antibody in tubes; The HRP labeled antibody was respectively applied in direct, sandwich ELISA and western blot detection. Results: HRP was successfully labeled on JWA monoclonal antibodies. In the direct method of ELISA test, HRP-JWA(4c9) antibodies combined polypeptide better than the combination of HRP-JWA(7c3); Its max OD450 was 0.96. While in double sandwich ELISA experiment, pre-coated by JWA(4c9) antibody, using the HRP-JWA(7c3) antibody as detected antibody could get good results, the max OD450was1.30. HRP-JWA could also identify the target protein in SGC7901 cells in western blot experiments.Conclusion: JWA antibody has been successfully marked with HRP, which could be used preliminarily in ELISA and western blot.