应用尿素溶性抗原制备抗猪囊虫单抗的研究

Study on Prepration of Monoclonal Antibody Against Cysticercus Cellusae with Urea-soluble Antigen

目的 为提高抗猪囊虫单抗的特异性。方法 使用猪囊虫尿素溶性抗原 ( Ag- u)并用水溶性抗原 ( Ag- w)作对照分别免疫 BABL / c小鼠 ,取其脾细胞分别与 SP2 / 0骨髓瘤细胞融合 ,EL ISA法进行筛选。同时筛选出的阳性克隆扩大培养 ,取上清单抗分别与包虫、血吸虫、肝吸虫、肺吸虫、姜片虫和丝虫抗原进行交叉反应性测定。结果 Ag- u和 Ag- w的阳性克隆率分别为 13.5 % ( 13/ 96 )和 12 .5 % ( 12 / 96 ) ,特异性阳性克隆率分别为 30 .8% ( 4/ 13)和 8.3% ( 1/ 12 )。 SDS- PAGE分析 Ag- u和Ag- w的条带数分别为 9和 17,分子质量 >97.4ku的条带比率分别为 6 6 .7% ( 6 / 9)和 2 9.4% ( 5 / 17)。免疫组化显示来自 Ag-u的 2株单抗定位于囊虫的头节 ,而来自 Ag- w的 1株单抗定位于囊虫的囊壁。结论 Ag- u用于制备单抗的阳性克隆得率与Ag- w没有明显的差异 ,而特异性阳性克隆的得率是 Ag- w的 4倍 ,亦即 Ag- u不仅有良好的免疫原性 。

Objective To promote the specificity of monoclonal antibody (McAb) against cysticercus cellulosea. Methods We first applied the Agu to immunize BABL/c mouse, and fused the spleen cells of the immunized mouse with SP2/0 myeloma cells. Enzymelinked immunosorbent assay (ELISA) was used to screen the McAb which the hybridoma cells produced. The McAb crossreactions were tested with antigens from hydatid, schistosoma, clonochis sinensis, paragonimus, fasciolopsis and filaria. The McAbs made by Agw were served as control. Results The results showed that the positiveclone rate of hybridoma cells made by Agu and Agw were 13.5%(13/96) and 12.5%(12/96) respectively. Two specific positiveclone rates were 30.8% (4/13) and 8.3%(1/12) respectively. SDSPAGE showed there were 9 and 17 bands in the gel of the Agu and Agw, and the band ratios of the bands, whose molecular weight were more than 97.4ku, were 66.7%(6/9) and 29.4%(5/17). Immunohistochemical analysis indicated that two McAbs from Agu located on the scolex, and one McAb from Agw located on the ground membrane of cyst wall. Conclusion The results suggest that Agu is the same as Agw in immunogenicity, however, is superior to Agw in specificity. Agu might have better specificity than Agw dose in preparation of McAb against cysticercus cellulosae and could be expected to enhance the probability to get McAbs.