单增李斯特菌P60单克隆抗体的制备及初步应用

Monoclonal antibodies against Listeria monocytogenes

目的制备并筛选单增李斯特菌P60特异性单克隆抗体,初步建立其快速检测方法。方法以体外表达并纯化的李斯特菌P60蛋白为抗原免疫Balb/c小鼠,采用杂交瘤技术制备抗李斯特菌P60的McAb,鉴定其抗体亚型,并对单抗的腹水效价、稳定性及亲和力进行分析。用纯化的单克隆抗体包被酶标板,结合生物素化处理的P60多克隆抗体,夹心ELISA筛选单增李斯特菌特异性单抗,并以重组P60为标准品,建立标准曲线。结果获得了4株可稳定分泌P60单克隆抗体的杂交瘤细胞株。ELISA检测表明,筛选的6C2单克隆抗体株可特异性与单增李斯特菌(4b)P60反应。结论建立的夹心ELISA方法可对单增李斯特菌进行特异性鉴别和定量检测。

The aim of this study was to prepare monoclonal antibodies against P60 and develop a double-antibody sandwich enzyme-linked immunosorbent assay,which would be used in the detection of Listeria monocytogenes.The purified recombinant protein of P60,produced by prokaryotic expression,was used to immunize the Balb/c mice.The hybridoma cell lines against P60 were obtained by fusing spleen cells from the immunized mice with mouse Sp2/0 myeloma cells.Specific McAb against L.monocytogenes was screened by ELISA.The isotype of the McAb was identified,and then the titers,stability and the affinity of ascites were analyzed.Sandwich ELISA was established using purified ascites and biotinylated P60 polyclonal antibody to detect the p60,which obtained from supernatant of Listeriacultures.The standard curve was established for quantitative determination using the recombinant P60 as the standard against the corresponding the value of OD450.Results showed that 6C2 McAb could be recognized specifically by L.monocytogenes,and the ELISA method based on 6C2 McAb could be used in identification and quantitative detection of samples contaminated with L.monocytogenes.