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目的建立以多抗为捕获抗体和单抗为检测抗体的夹心免疫-PCR检测旋毛虫循环抗原。方法利用杂交瘤技术制备抗旋毛虫单抗;旋毛虫抗原免疫家兔,分离纯化兔血清,制备兔抗旋毛虫多抗。多抗作为捕获抗体包被酶标板,单抗为检测抗体,选择适宜多抗和单抗浓度,建立夹心ELISA方法,再利用亲和素和生物素的高结合力连接起标记了生物素的抗体和DNA片段,将抗原抗体特异反应的免疫技术同无限扩增DNA片段的基因检测技术结合,利用PCR反应对DNA片段的扩增能力,提高检测灵敏度。结果制备了兔抗旋毛虫多抗,间接ELISA法筛选出与多种寄生虫抗原无交叉反应的单抗F4C6,夹心ELISA检测旋毛虫抗原最低浓度为0.05μg/ml,免疫PCR方法检测最低浓度为0.1ng/ml。结论首次建立夹心免疫-PCR检测旋毛虫抗原的方法,检测旋毛虫抗原的灵敏度高于传统ELISA方法104倍。
OBJECTIVE: To establish a sandwich immunoassay for detection of Trichinella spiralis antigens using polyclonal antibody as capture antibody and monoclonal antibody as detection antibody. Methods Anti-Trichinella monoclonal antibody was prepared by hybridoma technique. Trichinella antigens were used to immunize rabbits and the rabbit serum was isolated and purified to prepare anti-Trichinella polymorpha. The polyclonal antibody was used as the capture antibody coated ELISA plate, the monoclonal antibody as the detection antibody, the suitable multi-antibody and the monoclonal antibody concentration were selected to establish a sandwich ELISA method, and then the high binding affinity of avidin and biotin was used to link the labeled biotin Antibodies and DNA fragments, the antigen-antibody-specific immunological technology with unlimited amplification of DNA fragments of gene detection technology, the use of PCR amplification of DNA fragments to improve the detection sensitivity. Results The anti-Trichinella multilocularis was prepared by indirect ELISA. F4C6 was screened by indirect ELISA for the detection of anti-Trichinella antigens with a minimum of 0.05μg / ml. The lowest concentration of Trichinella antigens detected by sandwich ELISA was 0.1 ng / ml. Conclusion The sandwich immuno-PCR method was established for the first time to detect Trichinella antigens, and the sensitivity of detecting Trichinella antigens was 104 times higher than that of the traditional ELISA method.