白念珠菌果糖二磷酸醛缩酶单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against fructose-bisphosphate aldolase of Candida albicans

目的制备抗白念珠菌果糖二磷酸醛缩酶(Fba1)单克隆抗体,用以研究胞壁蛋白Fba1在侵袭性念珠菌感染(IC)中的致病作用。方法取重组白念珠菌Fba1蛋白免疫BALB/c小鼠的脾细胞与骨髓瘤细胞Sp2/0融合,筛选分泌抗体的杂交瘤细胞,有限稀释法克隆化。用ELISA测定单克隆抗体的效价,western blot和免疫荧光技术鉴定其特异性,用IsoStrip鉴定其抗体类型。结果筛选到1株稳定分泌抗Fba1单克隆抗体的杂交瘤细胞,抗体类型为IgG2a,轻链为κ型。鉴定结果表明,ELISA效价为1×106,能与念珠菌裂解物中天然Fba1和基因重组Fba1特异性结合。免疫荧光检测表明,该株单克隆抗体与白念珠菌、光滑念珠菌、近平滑念珠菌和热带念珠菌均发生反应。结论建立了1株持续分泌高效价抗念珠菌属特异性Fba1单克隆抗体的杂交瘤细胞株,为研究Fba1在IC中的致病作用提供了实验材料。

Objective To prepare the monoclonal antibody against Candida albicans fructose-bisphosphate aldolase（Fba1）,and investigate the pathgenic role of Fbal in the infection of invasive candida.Methods After BALB/c mice were immunized with recombinant Fba1,the spleen cells were hybridized with myeloma cell Sp2/0.The hybridized cells secreting antibodies against Fbal were selected for cloning by limiting dilution assay.The titer and the specificity of monoclonal antibodies were determined by ELISA,western blot and immunofluorescence assay respectively.The isotype of antibodies was identified by IsoStrip reagent.Results A hybridoma cell clone secreting anti-Fba1 monoclonal antibody steadily was selected.The isotype of the monoclonal antibody was IgG2a and its light chain belonged to κ-type.The titer of the monoclonal antibody was 1∶106 determined by ELISA.western blot showed that the monoclonal antibody could specifically bind with natural and recombinant Fbal.Immunofluorescence assay indicated that it could react with the spore and hypha of Candida Spp.Conclusion The hybridoma cell line which continuously secret monoclonal antibody against Candida Spp Fba1 with high titer and specificity was successfully prepared.It will provide some experimental basis for studying the disease mechanism of IC to Fba1.