白念珠菌感染特异性Csa2蛋白双抗体夹心ELISA体系的建立与评价

Establishment and evaluation of a double antibody sandwich ELISA to detect Csa2 protein of Candida albicans

目的以白念珠菌Csa2蛋白为靶标,建立双抗体夹心ELISA检测体系,评价该方法的检测灵敏度和特异性。方法构建pPIC9K-Csa2重组表达载体,电转化毕赤酵母GS115,甲醇诱导表达、纯化重组蛋白rCsa2。rCsa2蛋白分别免疫新西兰大白兔和豚鼠制备免疫血清。以纯化兔多抗和豚鼠抗血清配对,利用棋盘滴定法,确定最佳实验条件,建立双抗体夹心ELISA检测系统。检测rCsa2和白念珠菌不同培养时间的上清,确定检测方法的灵敏度;检测其他3种念珠菌、5种曲霉、新型隐球菌和马尔尼菲青霉的培养上清,评价检测方法的特异性。结果成功构建表达载体并获得真核表达重组蛋白rCsa2,经SDS-PAGE鉴定相对分子质量(Mr)为13 300,符合预期值;Western blot法证实该蛋白可与特异性抗体结合。免疫动物获得高效价免疫血清,并以此成功建立双抗体夹心ELISA,可检测rCsa2蛋白的灵敏度约为240 pg/mL,并最早可检测到培养18 h的白念珠菌培养上清,与其他10种临床常见真菌的培养上清无交叉反应。结论建立了有较高的检测灵敏度和特异性的Csa2的双抗体夹心ELISA,为区别诊断白念珠菌感染提供新的方法。

Objective To establish a double antibody sandwich ELISA for detecting Csa2 protein in Candida albicans infection and evaluate its specificity and sensitivity. Methods A recombinant expression vector pPICgK-Csa2 was constructed and transformed into Pichia pastoris GS115. A large-scale expression of recombinant Csa2 protein （rCsa2） was optimized using methanol, and the protein was purified in P. pastoris expression system. New Zealand Rabbits and guinea pigs were respectively immunized with the purified rCsa2 to prepare polyclonal antisera. The double antibody sandwich ELISA was established by choosing the optimal dilution of coating antisera and detecting antisera. Different concentrations of rCsa2 and culture supernatants of C. albicans collected at different time points were used to evaluate the sensitivity of detection. The specificity of the sandwich ELISA was evaluated by detecting culture supernatants of other three Candida spp, five Aspergillus spp, Cryptococcus neoformans and Penicillium mameffei. Results The rCsa2 protein was successfully expressed and purified. SDS-PAGE showed that its Mr was 13 300. Western blotting demonstrated that the protein bound to specific antibody. The sensitivity of the sandwich ELISA we established using the high-titer antisera was about 2.40 pg/mL of rCsa2, and could detect Csa2 protein in the culture supernatant of C. albicans when cultured for as early as 18 hours. There was no cross-reactivity between the culture supernatants of other 10 clinically important fungi and C. albicans. Conclusion The double antibody sandwich ELISA for detecting Csa2 protein has been established with good sensitivity and specificity. Csa2 protein could be used as a new diagnostic marker of C. albicans infection.