抗牛安氏隐孢子虫单克隆抗体的研制与初步鉴定

PREPARATION AND CHARCTERIZATION OF MONOCLONAL ANTIBODIES AGAINST CRYPTOSPORIDIUM ANDERSONI

通过流行病学调查分离到奶牛隐孢子虫虫株,经实验室鉴定为牛安氏隐孢子虫(Cryptosporidium andersoni),经蔗糖漂浮法收集和蔗糖密度梯度离心、滤器滤过、多次低速离心提纯后,通过冻融和超声波处理的全卵囊抗原免疫Balb/c小鼠4次,取阳性小鼠脾细胞与SP2/0骨髓瘤细胞融合.经间接ELISA方法筛选,有限稀释法3～4次克隆,获得5株分泌抗牛安氏隐孢子虫(C.andersoni)单克隆抗体的杂交瘤细胞株(1A3,3B10,3F4,4B3,4F11).5株单抗的染色体数目平均在96～98条之间,上清效价分别在1:100和1:12 800之间,腹水效价分别在1:12 800和1:204 800之间.采用免疫荧光法对各株的单抗腹水进行初步鉴定,发现其中4株为抗卵囊壁单抗,1株为抗子孢子单抗.对其中3株单抗的腹水提纯后进行SDS-PAGE电泳分析,其重链分子量约为52.7kDa,轻链分子量约为24kDa.

A cryptosporidial strain was indentified as Cryptosporidium andersoni which isolated from dairy farm in Zhengzhou. The oocyts of C. andersoni were purified by discontinuous sucrose gradients, then treated by filtration and centrifugation, freezethawed and sonicated. BALB/c mice were immunized for four times with purified oocyst antigens of C. andersoni. Spleen cells of the mice were selected to fuse with SP2/0 cells. The positive wells were selected by indirect ELISA, and after 3 to 4 generations of clane by limited dilution, 5 hybridized cell clones （1A3, 3B10, 3F4, 4B3, 4Fll） against C. andersoni were obtained whose chromosome numbers ranged between 96 and 98. The supernant titers ranged from 1 ： 100 to 1 ： 12 800, and ascites titers between 1：12 800 and 1：204 800. The monoclonal ascites were examined with indirect assay, and 4 of 5 were against oocyst wall, the other one was against sporozoite. Three ascites clones were purified and passed through SDS-PAGE electrophoresis, the results shown that the heavy chains were approximately 52.7 kDa and light chains was about 23, kDa.