禽戊型肝炎病毒间接ELISA抗体检测方法的建立与应用

doi:10.7668/hbnxb.20195453

摘要/Abstract

摘要：

为了针对禽戊型肝炎病毒(aHEV)建立一种快速、准确的抗体检测方法,以大肠杆菌原核表达系统表达的CaHEV-GDSZ01株的ORF2蛋白作为包被抗原,对相关反应条件进行优化后确定其最佳工作条件,以建立检测aHEV鸡血清抗体的间接ELISA 方法,并对该方法进行特异性、敏感性和重复性试验,与Western Blot方法进行对比后,初步应用于检测鸡临床血清样本。结果显示,ORF2蛋白被成功表达、纯化,并经Western Blot试验证实,可与aHEV阳性鸡血清发生特异性反应,建立的ELISA的最佳反应条件为:ORF2蛋白以200 ng/孔4 ℃包被过夜(12 h),5%脱脂乳37 ℃封闭1~2 h,血清1∶1 600稀释、室温孵育1 h,兔抗鸡IgY-HRP 1∶5 000稀释、37 ℃孵育90 min,TMB底物37 ℃显色20 min后终止。该ELISA方法可检出稀释51 200倍后的aHEV阳性血清,且不与NDV、AIV-H5、AIV-H9、ALV、REV和MDV等其他鸡源性病毒的阳性血清发生交叉反应;批内、批间重复性试验的变异系数均小于10%,与Western Blot方法的总体符合率达95.12%。通过该方法检测广东不同地区养鸡场的临床血清样本,总阳性率达66.34%。成功建立了一种操作简便、有效,且特异性、灵敏性和重复性均良好的aHEV血清学检测方法,可为基层监测、防控aHEV的感染提供一定的技术支持。

关键词: 禽戊型肝炎病毒, ORF2蛋白, 间接ELISA, 血清学检测, 流行病学调查

Abstract:

The aim is to establish a rapid and accurate method for detecting Avian hepatitis E virus(aHEV)antibodies.The ORF2 protein of the CaHEV-GDSZ01 strain was expressed in an E.coli prokaryotic expression system and utilized as the coating antigen.The reaction conditions were optimized to determine the optimal working conditions,leading to the establishment of an indirect ELISA method for detecting aHEV chicken serum antibodies.The specificity,sensitivity and repeatability of the method were tested,and compared with Western Blot method,chicken clinical serum samples were preliminarily detected.The results showed that ORF2 protein was successfully expressed and purified,and Western Blot assay confirmed that ORF2 protein could specifically react with aHEV-positive chicken serum.The optimal reaction conditions for ELISA were as follows:ORF2 protein was coated with 200 ng per well at 4 ℃ overnight(12 h);blocked with 5% nonfat milk at 37 ℃ for 1-2 h;serum was diluted at 1∶1 600,incubated at room temperature for 1 h;rabbit anti-chicken IgY-HRP was diluted at 1∶5 000,incubated at 37 ℃ for 90 min;TMB was incubated at 37 ℃ for 20 min.The ELISA method could detect the positive sera of aHEV diluted 51 200 times,and did not cross-react with positive sera of other chicken-borne viruses such as NDV,AIV-H5,AIV-H9,ALV,REV,and MDV.The intra-batch and inter-batch coefficients of variation were both less than 10%,rate between the Western Blot method was 95.12%. Clinical serum samples from chicken farms in different areas of Guangdong were tested by this ELISA method, and the total positive rate reached 66.34%.In conclusion,this study successfully established a simple,effective,specific,sensitive and reproducible serological detection method for aHEV infection,which can provide technical support for the surveillance,prevention and control of aHEV infection at grassroots.

Key words: Avian hepatitis E virus, ORF2 protein, Indirect ELISA, Serologic detection, Epidemiologic survey

中图分类号:

S858.31

张玉倩, 刘春红, 吕志航, 廉春杨, 张雪莲. 禽戊型肝炎病毒间接ELISA抗体检测方法的建立与应用[J]. 华北农学报, 2025, 40(4): 225-231. doi: 10.7668/hbnxb.20195453.

ZHANG Yuqian, LIU Chunhong, LYU Zhihang, LIAN Chunyang, ZHANG Xuelian. Establishment and Application of Indirect ELISA Method for Detecting Antibodies Against Avian hepatitis E virus[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(4): 225-231. doi: 10.7668/hbnxb.20195453.

http://www.hbnxb.net/CN/Y2025/V40/I4/225

图/表 13

参考文献 24

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项目Item	抗原浓度Antigen concentration
项目Item	20 ng/μL	10 ng/μL	5 ng/μL	2 ng/μL	1 ng/μL	0.5 ng/μL	0.1 ng/μL
阳性OD₄₅₀ Positive OD₄₅₀	2.195	2.189	1.889	1.498	1.095	0.851	0.625
阴性OD₄₅₀ Negative OD₄₅₀	0.134	0.115	0.082	0.055	0.055	0.046	0.043
P/N	16.438	19.035	23.037	27.236	19.909	18.703	14.523

项目Item	抗原浓度Antigen concentration
项目Item	20 ng/μL	10 ng/μL	5 ng/μL	2 ng/μL	1 ng/μL	0.5 ng/μL	0.1 ng/μL
阳性OD₄₅₀ Positive OD₄₅₀	2.195	2.189	1.889	1.498	1.095	0.851	0.625
阴性OD₄₅₀ Negative OD₄₅₀	0.134	0.115	0.082	0.055	0.055	0.046	0.043
P/N	16.438	19.035	23.037	27.236	19.909	18.703	14.523

项目Item	血清稀释度Dilution of serum
项目Item	1∶400	1∶800	1∶1 600	1∶3 200
阳性OD₄₅₀ Positive OD₄₅₀	1.166	1.040	0.898	0.721
阴性OD₄₅₀ Negative OD₄₅₀	0.054	0.048	0.039	0.039
P/N	21.79	21.67	23.03	18.73

项目Item	血清稀释度Dilution of serum
项目Item	1∶400	1∶800	1∶1 600	1∶3 200
阳性OD₄₅₀ Positive OD₄₅₀	1.166	1.040	0.898	0.721
阴性OD₄₅₀ Negative OD₄₅₀	0.054	0.048	0.039	0.039
P/N	21.79	21.67	23.03	18.73

项目 Item	4 ℃包被过夜(12 h) Coating at 4 ℃ overnight (12 h)		37 ℃包被2 h Coated at 37 ℃ for 2 h
项目 Item	PBS	CBS	PBS	CBS
阳性OD₄₅₀ Positive OD₄₅₀	0.479	1.388	0.492	1.043
阴性OD₄₅₀ Negative OD₄₅₀	0.041	0.045	0.045	0.045
P/N	11.67	30.84	11.04	23.43

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