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目的分析异尖线虫Ⅲ期幼虫抗原,寻找用于免疫诊断的有效抗原组分。方法用新鲜活异尖线虫Ⅲ期幼虫口饲感染SD大鼠,获得感染鼠血清。取异尖线虫Ⅲ期幼虫,以匀浆冻融及超声法制备Ⅲ期幼虫可溶性抗原(solubleantigens of third-stage larvae of anisakis,SAA);用SAA包板,采用ELISA检测感染鼠及其他寄生虫病患者血清特异TgG抗体,观察SAA的反应原性和特异性;以SDS-PAGE电泳分析SAA组分,并转移至NC膜,进行Western blot,分析其与感染鼠血清的反应性。结果 SAA-ELISA检测感染鼠血清的敏感度为100%,特异度为100%,与华支睾吸虫病和囊虫病患者血清的交叉反应率均为10%。SDS-PAGE分析SAA,获得2条分子质量单位为38、55ku的主蛋白带和介于10～170ku之间的10条次带。经Western blot分析,SAA有14个抗原组分能被感染大鼠血清识别,以170、130、73、38、22、15ku蛋白反应较强。SAA与血吸虫病、华支睾吸虫病、囊虫病、旋毛虫病、蛔虫病、钩虫病、弓形虫病、舌形虫病患者及健康人血清无交叉反应,70ku和93ku组分与并殖吸虫病、鞭虫病患者血清起交叉反应。结论异尖线虫Ⅲ期幼虫可溶性抗原组分复杂,其中的38.55ku蛋白可能是较理想的诊断抗原组分,其性质及诊断效果有待进一步探讨。
Objective To analyze the stage Ⅲ larvae antigen of Anisakis nematode and to search for an effective antigenic component for immunodiagnosis. Methods Infected SD rats were infected with freshly harvested Isopharyngeal Phase Ⅲ larvae to obtain the infected rat serum. The third stage larvae of anisakis (SAA) was prepared by homogenate freeze-thaw and sonication. The SAA-coated plate was used to detect the expression of soluble antigens Serum-specific TgG antibodies of patients were used to observe the reactivity and specificity of SAA. The SAA components were analyzed by SDS-PAGE electrophoresis and transferred to NC membrane for Western blot analysis. Results The sensitivity and specificity of SAA-ELISA for the detection of sera of infected mice were 100% and 100%, respectively. The cross-reactivity with SAA-ELISA was 10% in serum of patients with clonorchiasis and cysticercosis. SDS-PAGE analysis of SAA obtained two molecular mass units 38,55ku of the main protein band and 10 to 170ku between 10 sub-bands. Western blot analysis, SAA 14 antigen components can be infected rat serum to identify 170,130,73,38,22,15ku protein reaction stronger. There were no cross reactions between SAA and schistosomiasis, Clonorchiasis, Cysticercosis, Trichinellosis, Ascariasis, Hookworm, Toxoplasmosis, Paragonimiasis, whipworm disease patients serum cross-reaction. Conclusions The soluble antigen component of stage III larvae of Anisakis is complicated. The 38.55 ku protein may be an ideal antigen component for diagnosis. The nature and diagnostic value of the 38.55 ku protein remains to be further explored.