抗-p30单克隆抗体杂交瘤细胞株的建立

Establishment of Hybridoma Cell Lines Producing Monoclonal Antibodies against-p30 Antigen

作者用纯化的p30抗原免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞融合。融合率为93.95％,抗体阳性率为21.01％。经克隆化,获得两株分泌抗-p30单克隆抗体的杂交瘤细胞株E_8与G_1。染色体计数:E_8为98.7±6.54;G_1为97.7±7.77。PAGE于γ区出现浓染的蛋白带。免疫组织化学染色和ELISA抑制试验证实该两株细胞分泌的抗体具有较好的种属特异性和器官特异性。均属IgG_2。都不能使人精子凝集和制动。

BALB/c mice were immuniaed with thepurified p30 antigen. SP2/0 myeloma cellsand immune spleen cells were fused with50% PEG(Sigma, MW 3350-4000) .ffe cellfusion rate was 93.95%, and the antibodyproducing rate 21.01%. The technique of limiting dilution wasused for cloning of the hybridoma cells. Twohybridoma cell lines E_8 and G_1 secretingMcAb against p30 antigen were obtained. Thenumber of chromosome of both E_8 and G_1cell lines was 98.7 ±6.54 and 97.7 ±7.77,re-spectively. Results of the PAGE of the asci-tes generated by the hybridoma cell lines E_8and G_1 showed a thick protein band at theγ-region which was absent from the ascitesgenerated by the SP2/0 myeloma cells.Theimmunoglobulin of the McAb E_8 and G_1belonged to IgG_2 subclass.The results of theimmunohistochemical method using thehorseradish peroxidase conjugated antibodyshowed that both E_8 and G_1 McAb onlyreacted with epithelia cells of the normalhuman prostatic glands and tbeir ducts,butdid not cross-react with other thirty-fourdifferent kinds of normal human tissues.The results of inhibiting ELLSA test showedthat both E_8 and G_1 McAb were only inhibi-ted by the human seminal plasma and p30,weakly inhibited by the adult male's urine.but were not inhibited by other eightdifferent kinds of human body fluids andsecretions as well as semens from eightdifferent species of animals. It was concludedthat McAb of E_8 and G_1 were organ andspecies specific. Human sperms cannot beagglutinated and immobilized by anti-p30McAb of E_8 or G_1 as demonstrated bythe TAT and the SIT test.