抗恶性疟原虫重组HRP-Ⅱ单克隆抗体的制备与鉴定

PRODUCTION AND IDENTIFICATION OF MONOCLONAL ANTIBODIES AGAINST RECOMBINANT HRP-Ⅱ OF PLASMODIUM FALCIPARUM

采用恶性疟原虫重组富组氨酸蛋白 (HRP- )融合表达蛋白免疫 BAL B/ c小鼠 ,取脾细胞与 SP2 / 0细胞融合 ,经 3次 EL ISA筛选 ,共获得 6株分泌抗恶性疟原虫重组 HRP- 单克隆抗体的杂交瘤细胞株 (1B11、1D10、2 E7、3A3、3F9、4G5 ) ,用有限稀释法进行克隆、亚克隆及扩大培养 ,并用杂交瘤细胞经腹腔接种 BAL B/ c小鼠制备腹水。 6株杂交瘤细胞分泌的单克隆抗体 (Mc Ab)经琼脂双扩散鉴定均为 Ig G1亚类 ,Dot- EL ISA及 Western blot分析显示 6株 Mc Ab都能与重组 HRP- 抗原发生特异性反应 ,但其中只有 2 E7和 3A3在 Dipstick免疫胶体金反应中能与恶性疟原虫培养上清中的天然 HRP-

BALB/c mice were immunized by recombinant HRP Ⅱ antigen of Plasmodium falciparum expressed as a fusion protein with β galactosidase. Spleen cells from the immunized BALB/c mice were fused with mouse myeloma cell SP2/0. Six hybridoma cell strains (1B11, 1D10, 2E7, 3A3, 3F9 and 4G5) secreting McAbs against HRP Ⅱ of P. falciparum were screened by ELISA. The hybridoma cells were cloned, subcloned and cultured in a large scale. BALB/c mice were injected intraperitoneally with 10 6 hybridoma cells. The subtype of the monoclonal antibodies secreted by the hybridoma cells was analyzed by double immuno diffusion. All of the six strains of McAb were IgG1. Dot ELISA and Western blot analysis demonstrated that all of the six McAbs strongly and specifically reacted with the recombinant HRP Ⅱ. However, only two of them (2E7 and 3A3) reacted with the native HRP Ⅱ from supernatant of cultured P. falciparum in the test of dipstick colloidal gold immunoassay.