基于基因C型鸭甲肝病毒VP1重组蛋白的鸭甲肝病毒抗体ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2014.07.017

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (7): 1148-1153.doi: 10.11843/j.issn.0366-6964.2014.07.017

基于基因C型鸭甲肝病毒VP1重组蛋白的鸭甲肝病毒抗体ELISA检测方法的建立

杨发龙^1,2，张焕容^1,2*，程方明¹，岳华^1,2，汤承^1,2

（1.西南民族大学生命科学与技术学院，成都 610041；2.动物医学四川省高等学校重点实验室，成都 610041）

收稿日期:2013-10-18 出版日期:2014-07-23 发布日期:2014-07-23
通讯作者: 张焕容，E-mail：zhrong05@163.com
作者简介:杨发龙（1976-），男，青海大通人，博士，副教授，主要从事兽医微生物与免疫学研究
基金资助:
国家高技术研究发展计划（2012AA101304）；西南民族大学研究生学位点建设项目（2014XWD-S0906）；四川省教育厅创新团队项目（13TD0057）

Development of an Indirect ELISA for Detecting Antibodies against Genotype A and C Duck Hepatitis A Virus Based on Recombinant VP1 Protein

YANG Fa-long^1,2，ZHANG Huan-rong^1,2*，CHENG Fang-ming¹,YUE Hua^1,2，TANG Cheng^1,2

(1.College of Life Science and Technology， Southwest University for Nationalities， Chengdu 610041， China;2.Key Laboratory of Sichuan Institutes of Higher Learning， Chengdu 610041， China)

Received:2013-10-18 Online:2014-07-23 Published:2014-07-23

摘要/Abstract

摘要：

以纯化的基因C型鸭甲肝病毒(DHAV-C) VP1重组蛋白作为包被抗原，建立检测DHAV-C抗体的间接ELISA方法。结果表明，试验的最佳条件：用pH9.6、0.05 mol•L^－1的碳酸盐缓冲液将包被抗原稀释成5 μg•mL^－1，4 ℃包被过夜，抗体稀释度为1∶100，HRP酶标羊抗鸭IgG稀释度为1∶5 000，封闭液为含10%脱脂乳的0.01 mol•L^－1 PBS，37 ℃封闭2 h，血清稀释液为含1% BSA的PBS，抗体反应时间为60 min，酶标羊抗鸭IgG反应时间为30 min，底物作用时间为15 min，临界值为OD_{450 nm}≥0.474。该方法重复性好，批内变异系数为2.03%~2.95%，批间变异系数为3.28%~7.38%，与鸭圆环病毒(DUCV)、鸭乙型肝炎病毒(DHBV)、鸭瘟病毒（DPV）、新城疫病毒（NDV）、禽流感病毒（AIV）、鸭抗大肠杆菌、鸭抗金黄色葡萄球菌、鸭抗沙门菌、鸭抗禽多杀性巴氏杆菌以及鸭疫里默杆菌阳性血清无交叉反应，而与基因A型鸭甲肝病毒（DHAV-A）阳性血清有交叉反应，表明该方法可以作为检测DHAV-C和DHAV-A抗体的通用ELISA方法。与中和试验相比，阳性血清检测符合率为80%，阴性血清检测符合率为100%。初步临床应用表明：该方法可用于雏鸭母源抗体和免疫后抗体消长的检测。本研究基于DHAV-C VP1重组蛋白建立的间接ELISA方法可用于基因A型和C型鸭甲型肝炎的血清流行病学调查和抗体检测。

Abstract:

The aim of this study was to establish an indirect ELISA to rapidly detect antibody of genotype C duck hepatitis A virus (DHAV-C).An indirect ELISA based on DHAV-C VP1 recombinant protein as coating antigen was established for rapid detection of DHAV antibody.The reaction conditions were optimized，including 5 μg•mL^－1 VP1 recombinant protein as coating antigen，1:100 dilution of testing serum and 1:5000 dilution of HRP conjugated goat anti-duck IgG with cut-off value 0.474 (OD_{450 nm}).The pH 9.6，0.05 mol•L^－1 bicarbonate buffer was used as coating antigen dilution buffer，the sealing buffer was 0.01 mol•L^－1 PBS buffer containing 10% skimmed milk，and the dilution buffer was PBS buffer containing 1% BSA.The coating condition was at 4 ℃ for a whole night，antibody reacted with coating antigen for 60 min，HRP conjugated goat anti-duck IgG incubated for 30 min and substrate reaction time was 15 min.The specific test showed that there were no cross reactions with positive sera against duck circovirus，duck plaque virus，duck hepatitis B virus，Newcastle disease virus，avian influenza virus，duck E.coli，duck Staphylococcus aureus，duck Salmonella anatis，duck Pasteurella multocida and Riemerella anatipestifer.However，DHAV-A antibody showed cross reaction with DHAV-C VP1 recombinant protein.The inter- and intra -assay demonstrated that the co-efficient variations were 2.03%-2.95% and 3.28%-7.38%，respectively.Compared with neutralization test，the detection coincidences of positive and negative sera were 80% and 100%，respectively.This method had been successfully used to detect maternal antibody and the growth and decline of immune antibody.The results revealed that the indirect ELISA could be used for sero-epidemiological survey and antibody detection of duck hepatitis A disease caused by DHAV-A or DHAV-C.

中图分类号:

杨发龙，张焕容，程方明，岳华，汤承. 基于基因C型鸭甲肝病毒VP1重组蛋白的鸭甲肝病毒抗体ELISA检测方法的建立[J]. 畜牧兽医学报, 2014, 45(7): 1148-1153.

YANG Fa-long，ZHANG Huan-rong，CHENG Fang-ming,YUE Hua，TANG Cheng. Development of an Indirect ELISA for Detecting Antibodies against Genotype A and C Duck Hepatitis A Virus Based on Recombinant VP1 Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(7): 1148-1153.

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基于基因C型鸭甲肝病毒VP1重组蛋白的鸭甲肝病毒抗体ELISA检测方法的建立

Development of an Indirect ELISA for Detecting Antibodies against Genotype A and C Duck Hepatitis A Virus Based on Recombinant VP1 Protein

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