庆大霉素单克隆抗体的制备及试剂盒的配制

Preparation of monoclonal antibody and establishment of detection method by ELISA for gentamicin

目的建立庆大霉素直接竞争酶联免疫吸附分析方法。方法应用戊二醛法制备庆大霉素完全抗原,通过杂交瘤技术筛选分泌特异性庆大霉素抗体的杂交瘤细胞株,并建立庆大霉素竞争酶联免疫吸附分析检测方法。结果获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,建立了庆大霉素竞争酶联免疫吸附分析检测方法,该方法操作简单具有良好的线性、特异性和精密度;庆大霉素质量浓度在1.5625～50.0000 ng/mL范围内,呈现良好的线性,r2=0.9913,50%抑制浓度为(IC50)为7.37 ng/mL,检测限(LOD)为1.54 ng/mL,该试剂盒与链霉素等8种药物无交叉反应。结论获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,研制的庆大霉素竞争ELISA检测试剂盒具有良好的线性、特异性和精密度。

Objective To establish an enzyme-linked immunosorbent assay （ELISA） for gentamicin. Methods Glutaraldehyde was used as cross linker in the preparation of full antigens of gentamicin. Hybridoma cell lines that produced monoclonal antibody against gentamicin were established by the hybridoma technique. An enzyme-linked immunosorbent assay （ ELISA ） for gentamicin had been established. Results Three hybridoma cell lines which could stably secrete monoclonal antibody against gentamycin were screened out through indirect ELISA. A procedure for competitive enzyme-linked immunoanalysis of gentamicin with good linearity, specificity and accuracy was established with purified monoclonal antibody against gentamicin. The linear range in gentamicin detection was between 1. 5625 - 50. 0000 ng/mL with a coefficient greater than 0.98. The concentration for half inhibition was 7.37/mL, and the limit of detection was 1.54 ng/mL. The kit showed no cross reaction with streptomycin as well as seven other drugs. Conclusions Three hybridoma cell lines which could stably secrete monoclonal antibody against gentamycin were screened out. A gentamycin detection kit was developed according to our protocol in competitive enzyme-linked immunoanalysis with good linearity, specificity and accuracy.