荧光免疫吸附法定量检测单核细胞增生李斯特菌

Fluorescence immunosorbent assay for quantitative detection of Listeria monocytogenes

目的研制荧光免疫吸附法定量检测单核细胞增生李斯特菌。方法采用双抗夹心免疫吸附法筛选7株抗单核细胞增生李斯特菌的单克隆抗体,获得最佳配对抗体10E7H6和10A11。以生物素化的10A11为检测抗体,10E7H6为捕获抗体,建立了基于链霉亲和素标记荧光微球为检测探针的荧光免疫吸附法检测单核细胞增生李斯特菌。结果该方法中最佳捕获抗体浓度为10μg/mL,最佳检测抗体浓度为5μg/mL,反应最佳pH为7.4;在该条件下,单核细胞增生李斯特菌最低检测限为10~5 CFU/mL,比传统酶联免疫吸附法提高了两个数量级;与李斯特菌属内其他几种主要李斯特菌有一定的交叉反应,与其他非李斯特菌属的主要致病菌无显著交叉反应。结论该方法可用于纯培养液中单核细胞增生李斯特菌的检测,也可用于李斯特菌属内其他几种主要李斯特菌的免疫学快速筛查检测。

OBJECTIVE Establishment of fuorescence immunosorbent assay for quantitative detection of Listeria monocytogenes.METHODS The coupled mAbs named 10E7H6 and 10A11 were screened from seven strains of anti-L.monocytogenes mAbs using a sandwich enzyme-linked immunosorbent assay(ELISA).The fluorescent immunoassay was established for L.monocytogenes detection by using biotinylated 10A11 mAbs as detection antibody,10E7H6 mAbs as capture antibody,and streptavidin-labeled fluorescent microspheres as detection probes,respectively.RESULTS The optimum concentrations of capture and detection antibodies were 10μg/mL and 5μg/mL,respectively,and the optimum reaction pH was 7.4.Under these conditions,the limit of detection of the proposed method for L.monocytogenes detection was 10^5 CFU/mL,which improved by two orders of magnitude compared to conventional ELISA;and it has a certain cross-reaction with several other Listeria but no significant cross-reactivity with other pathogenic bacteria.CONCLUSION The method can be used for the detection of L.monocytogenes in pure culture solution,and can also be used for rapid immunological screening test of several other Listeria species in the genus Listeria.