抗李斯特氏菌溶血素单克隆抗体制备及检测LMO的初步应用

目的制备抗李氏溶血素单克隆(LLO)抗体,初步应用单克隆抗体检测LMO。方法以重组LLO为免疫原,以天然LLO为筛选蛋白,通过淋巴细胞杂交瘤技术,制备抗LLO单克隆抗体;以ELISA、Westernblot等方法对单克隆抗体进行鉴定;以阻断ELISA法进行检测LMO的探索。结果获得了1株稳定分泌抗LLO单克隆抗体的杂交瘤细胞株2C4,经鉴定此单抗为IgG1型,属κ链抗体,杂交瘤细胞染色体数目在85～103。ELISA和Westernblot实验表明此单抗对LLO有较好的结合能力,对其他菌分泌的溶血素则无特异结合能力,用阻断ELISA检测LMO的检测时间和最低检测菌数分别为增菌后5h和9.5×105个/mL。结论研制了一株稳定分泌抗LLO单克隆抗体的杂交瘤细胞系,初步建立了阻断ELISA检测LMO的方法。

Preparation of monoclonal antibodies against listeriolysin O and its preliminary use in the detection of Listeria monocytogenes

In the preparation of these monoclonal antibodies(McAbs),the purified recombinant listeriolysin(LLO)was used as antigen to immunize BALB/c mice,and the anti-LLO McAbs were detected with natural LLO by indirect ELISA.In these ways of preparation of McAbs,one strain of hybridoma cell line steadily secreting anti-LLO antibodies was obtained and named as 2C4.This McAb belonged to IgG1 subtype and the κ chain antibody,and the chromosomal numbers of the hybridoma cells were 85-103.As demonstrated by ELISA and Western blot assay,this antibody showed stronger binding ability to natural LLO,but no any binding capacity with hemolysins secreted by other bacteria.The detection time used to block the detection of Listeria monocytogenes(LMO)with ELISA and the lowest number of LMO to be detected were 5 hrs and 9.5 × 105/ml,respectively.