抗大肠杆菌_(506)株36kD蛋白单抗的制备及其在人精子上的免疫定位

用纯化的36kD蛋白免疫Balb/c小鼠。取免疫脾细胞与小鼠骨髓瘤细胞SP2/0以PEG进行细胞融合,融合率为86％。采用ELISA筛选出杂交瘤细胞阳性孔。经过4～7次有限稀释克隆化,共获得7株能稳定分泌36kD相应MCAb的杂交瘤细胞。将杂交瘤细胞分别注射小鼠腹腔,测定腹水McAb的效价为1:5×10~3～1:1×10~5,抗体亚型均为IgG1。用荧光显微镜观察,结果发现EC2作用的人精子头颈部出现明显荧光反应,提示共同抗原存在于人精子头颈部。这些结果有助于进一步探讨细菌感染引起男性不育的机制以及研制细菌避孕疫苗等工作。

Production of Murine Monoclonal Antibodies (McAbs) Reactive to a 36kD Protein of Escherichia coli506 Strain and Immunological Localization of Cross-antigens in Human Sperm

Balb/c mice were immunized by intraperitoneal injection of purified 36kD protein. Spleen cells from one immunized mice were harvested and fused with myeloma line SP2/0 in the presence of polyethylene glycol. The resulting hybrid cells were screened according to their ability to bind to 36kD protein in plate ELISA. Hybrids secreting reactive antibodies were subcloned 4 to 7 times by limiting-dilution cloning and 7 hybrids were selected. Hybrid cells were grown as ascites in the peritoneal cavity of silica H primed Balb/c mice. McAbs were purified from ascitic fluid. It was showed that all the 7 clones were of the IgGl isotype and the liters were between 1 : 5×103- 1 : 1 ×105. Immunological localization of the cross-reactive antigens on human sperm with the 36kD protein was tested by means of indirect immunofluorescence. It was found that EC2 could react with the head and neck parts of human sperm. These results expand our understanding on the machanisms of male infertility induced by bacterial infection and have a great significance in preparing a potential effective immuno-contraceptive vaccine from bacteria in the near future.