摘要
为建立一种快速检测鸭甲肝病毒(DHAV)1型和3型(DHAV-1、DHAV-3)血清抗体的方法,本研究诱导表达了DHAV-1、DHAV-3重组蛋白VP0、VP1、VP3,并采用ELISA方法比较了3者的反应原性,结果显示DHAV-1 VP0更适合作为检测DHAV-1、DHAV-3血清抗体的通用型诊断抗原。经各反应条件优化,建立了一种基于DHAV-1 VP0重组蛋白的可同时检测DHAV-1、DHAV-3血清抗体的间接ELISA方法。优化的主要反应条件为:抗原包被浓度为0.25μg/m L,待检血清稀释度为1100,山羊抗鸭HRP-IgG 1400稀释。该方法特异性较强,只与DHAV-1、DHAV-3阳性血清反应,与鸭源小鹅瘟病毒(GPV)、鸭病毒性肠炎病毒(DEV)、H9N2亚型禽流感病毒(H9N2 AIV)、鸭坦布苏病毒(DTMUV)等阳性血清均无交叉反应;敏感性为1320;批内变异系数小于4%,批间变异系数小于6%;与商品化的ELISA抗体检测试剂盒比较,二者阳性符合率为93.3%,阴性符合率为88.9%,总符合率为95.7%,且该方法可用于临床血清样本的检测。本研究为检/监测DHAV血清抗体水平提供了技术手段。
In order to establish a rapid serological method for detecting the antibodies against Duck hepatitis A virus(DHAV)type 1 and type 3(DHAV-1,DHAV-3),the reactionogenicities of recombinant proteins VP0,VP1 and VP3 with DHAV-1 and DHAV-3 were compared by using ELISA assays.The result showed that the DHAV-1 VP0 could react with serum antibodies against both DHAV-1 and DHAV-3 serving as a universal antigen.After the optimization of reaction conditions,an indirect ELISA was established based on DHAV-1 VP0 recombinant protein for the antibodies detection of DHAV-1,DHAV-3.The mainly optimized reaction conditions were as following:the concentration of coated antigen was 0.25μg/m L,and the dilutions of serum and goat against duck HRP-Ig G were 1.100 and 1.400 respectively.The specificity assay showed that the ELISA only detected antibodies against DHAV-1 and DHAV-3 but had no cross-reaction with other serum antibodies against goose parvovirus(GPV),duck enteritis virus(DEV),H9 N2 subtype avian influenza virus(H9 N2 AIV)and duck tambusu virus(DTMUV).The sensitivity was 1.320 of DHAV-3 positive serum,and the coefficient variability of intra-batch was less than 4%and inter-batch was less than 6%.Compared with commercial DHAV ELISA antibody detect kit,the coincidences of positive and negative rate were 93.3%and 88.9%,respectively,and the total coincidence rate was 95.7%.The results suggested that the indirect ELISA could detect the clinical DHAV serum samples.This study provid a technological method to detect and/or monitor DHAV serum antibody level for drafting effective preventative measure.
作者
马波
戚海惠
张文晶
陈浩田
张琪
常蕊
刘悦
张雪莲
张桂红
王君伟
MABo;QI Hai-hui;ZHANGWen-jing;CHEN Hao-tian;ZHANG Qi;CHANG Rui;LIU Yue;ZHANG Xue-lian;ZHANG Gui-hong;WANG Jun-wei(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;College of Veterinary,South China Agricultural University,Guangzhou 510642,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第3期245-251,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金青年基金(31502049)
