基于大片吸虫分泌排泄产物层析组分的牛片形吸虫病间接ELISA诊断方法的建立

doi:10.16431/j.cnki.1671-7236.2021.08.034

摘要/Abstract

摘要： 为建立更敏感、特异的牛片形吸虫病早期诊断方法，本试验将收集的大片吸虫分泌排泄产物（Fasciola gigantica excretory-secretory products，FgESP）进行凝胶过滤层析并从中筛选出检测效果最优的UV280吸收峰，从此峰对应的组分中筛选出诊断效果最优的层析组分后将其作为抗原，通过棋盘滴定试验优化抗原包被浓度、血清稀释度、酶标二抗稀释度、显色时间等，确定临界值，建立牛片形吸虫病间接ELISA诊断方法。对建立的方法进行敏感性和特异性试验，检测试验感染0~14周的水牛血清和临床160份水牛血清样本，并与已有的诊断抗原FgESP（阴阳临界值为0.320）进行诊断效果比较。结果显示，层析组分F22具有较优的检测效果。间接ELISA的最佳反应条件为：F22抗原包被浓度0.157 μg/mL，待测血清与酶标二抗的稀释度分别为1：400和1：40 000，显色时间为25 min。应用建立的方法检测15份水牛阴性血清，确定D_{450 nm}阴阳性临界值为0.408。比较F22与FgESP发现，二者特异性相似，但F22作为抗原的敏感性高于FgESP。检测试验感染大片吸虫的水牛0~14周血清，结果表明，从感染第2周开始F22-IgG水平极显著高于FgESP-IgG水平（P<0.01），因此，F22比FgESP诊断效果更优。对160份水牛血清检测发现，F22阳性检出率为71.25%，FgESP阳性检出率为63.75%。上述结果表明，相比于FgESP，以F22作为诊断抗原建立的大片吸虫病间接ELISA诊断方法敏感性更高，可更有效地进行牛大片吸虫病的早期诊断。

关键词: 大片吸虫; 诊断抗原; ELISA

Abstract: To establish a more sensitivity, specificity diagnostic method that could be used for the early diagnosis of bovine fascioliasis, Fasciola gigantica excretory-secretory products (FgESP) was chromatographed to screen UV280 chromatographic peak.While UV280 chromatographic peak showing the best detection effect was screened out, the best chromatographic component under corresponding chromatographic peak was further screened as diagnostic antigen.The conditions of coating antigen, antibody dilution, dilution of enzyme-labeled secondary antibody and reaction time were optimized by checkerboard titration test, and the indirect ELISA diagnostic method for detection of bovine fascioliasis was established.Sensitive, specific, clinical tests and 0-14 weeks serum IgG antibody of buffalos experimentally infected Fasciola gigantica were performed on the established indirect ELISA assay.Meanwhile, serum IgG antibody of 160 buffalos were also detected, then result was compared with that of the existing diagnostic antigen (FgESP, the cut-off value was 0.320).The results showed that chromatographic component F22 worked well.The optimal reaction conditions of the indirect ELISA were as follows:The coating antigen of F22 was 0.157 μg/mL, the testing serum dilution and the HRP conjugated anti-bovine IgG dilution were 1:400 and 1:40 000 respectively, the incubation time was 25 min.The established method was used to detect 15 buffalo negative serums, while the cut-off value of D_{450 nm} was 0.408.Comparision of F22 and FgESP indicated that they were comparable in specificity, while F22 showed higher sensitivity.Upon established indirect ELISA, it was found that from the second week anti-F22-IgG was significantly higher than that of anti-FgESP-IgG in the period of 0-14 weeks infected buffalo serum (P<0.01).Therefore, F22 showed better diagnostic effect than FgESP.The positive rate of F22 was 71.25% and FgESP was 63.75% in 160 serum samples detection of buffalos.All above suggested that indirect ELISA based on F22 showed better diagnostic effect than FgESP, which could apply to early diagnosis of fascioliasis in the future.

Key words: Fasciola gigantica; diagnostic antigen; ELISA

中图分类号:

S855.9

靳纬坤, 袁湘祥, 王锦辉, 侯林静, 郑梦伟, 吴正姣, 张为宇, 邸文达. 基于大片吸虫分泌排泄产物层析组分的牛片形吸虫病间接ELISA诊断方法的建立[J]. 中国畜牧兽医, 2021, 48(8): 3010-3018.

JIN Weikun, YUAN Xiangxiang, WANG Jinhui, HOU Linjing, ZHENG Mengwei, WU Zhengjiao, ZHANG Weiyu, DI Wenda. Establishment of Indirect ELISA for Bovine Fasciolasis Diagnosis Based on Chromatographic Component of Fasciola gigantica Excretory-secretory Products[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 3010-3018.

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