Establishment and Application of Indirect ELISA Method for Detecting Antibodies Against Avian hepatitis E virus

doi:10.7668/hbnxb.20195453

Abstract

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

CLC Number:

S858.31

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.

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References 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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