犬腺病毒2型抗原的制备及间接ELISA的建立  

Purification of Canine Adenovirus Type-2 and Development of an Indirect ELISA Detection Method for Anti-canine Adenovirus Antibodies

作　　者：葛艳华[1,2] 姜骞[2] 刘家森[2] 司昌德[2] 郭东春[2] 林欢[2] 曲连东[2] 

机构地区：[1]东北农业大学动物医学院,哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150001

出　　处：《实验动物科学》2010年第5期40-44,共5页Laboratory Animal Science

摘　　要：用MDCK细胞增殖犬腺病毒2型弱毒疫苗株,病毒培养上清液经差速离心和酒石酸钾密度梯度离心浓缩、纯化后作为诊断抗原,建立了犬腺病毒2型抗体检测的间接ELISA。确定最佳抗原包被量为0.168μg/孔,待检血清最佳稀释倍数为1:100,作用时间为60 min,羊抗犬酶标抗体稀释倍数为1:2000,作用时间为60 min,底物作用时间为37℃,10 min。判定标准为S/P值≥0.351者判为阳性,S/P值≤0.318者判为阴性。该抗原不与犬瘟热病毒、犬细小病毒、犬波特氏杆菌等3种犬常见传染病的阳性血清反应,具有良好的特异性;批内、批间重复试验,变异系数小于15%,显示具有较好的重复性;检测血清样品56份,与病毒中和试验结果比对的符合率为93.75%。本研究结果为实现犬腺病毒感染监测,进行犬腺病毒流行病学调查提供了一种简便的血清学诊断方法。Objective An indirect-ELISA detection method was developed for anti-canine adenovirus type-2（CAV-2） antibodies.CAV-2 vaccine strain was inoculated in MDCK cells;the supernatant was purified and concentrated by density gradient centrifugation and potassium tartrate density gradient centrifugation,and then used as coating antigen.In the indirect-ELISA procedure,the CAV-2 coating antigens at the optional amount of 0.168 μg per well were incubated for 60 min with the sera diluted at 1∶100.Then goat anti-canine IgG conjugated HRP at 1∶ 2000 was added and incubated for 60 min.The samples that have S/P ratios of ≥0.351 were positive and ≤ 0.318 were negative.The antigen showed no cross reaction with the canine bordetella bronchiseptica、canine distemper virus and canine parvovirus positive sera.Both in the intra-and inter-batch test,the variation coefficients were less than 15%,which indicated good inter-plate repeatability.The intra-plate reproducibility of the indirect-ELISA method had 93.75% agreement with that of the virus neutralization test.In summary,the indirect ELISA detection method is simple and rapid for monitoring CAV-2 infection and assessing vaccination in the field.

关 键 词：犬2型腺病毒 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]