摘要
用表达禽流感病毒 (AIV)核蛋白基因的杆状病毒感染Sf9昆虫细胞 ,以其表达产物制备抗原 ,建立了以杆状病毒系统表达的AIV核蛋白为抗原的禽流感间接酶联免疫吸附试验诊断技术 (rNP_ELISA)。其抗原最适包被量为 0 .6 μg/孔 ,待检血清最适稀释度为 1:2 0 0 ,酶标抗体使用浓度为 1:10 0 0。根据对 140份SPF鸡血清检测结果的统计分析 ,确定其判定标准为OD值大于 0 .15的血清样品为阳性。用rNP_ELISA检测 15 0份SPF鸡血清、194份非免疫鸡血清及 30份其它 15种鸡疫病阳性血清均为阴性 ;检测A型AIV15个不同亚型 (H1～H15 )毒株的特异性血清均为阳性 ;对人工接种AIV的SPF鸡第 3天即能检出抗体 ,到第 16 2天试验结束时检测仍为阳性。其批内和批间重复试验的变异系数分别在 2 .9%～ 7.2 %和 3 .4%～ 9.8%之间。对3138份鸡血清进行监测 ,rNP_ELISA与全病毒间接ELISA(AIV_ELISA)、琼脂扩散试验 (AGP)及血凝抑制试验 (HI)的符合率分别为 99.9%、97.1%、98.8% ;并能 10 0 %检出AGP阳性及疑似、HI阳性的血清样品。试验证明 ,rNP_ELISA是检测A型禽流感病毒血清特异性抗体的一种特异、敏感、微量、快速。
An enzyme_linked immunosorbent assay(ELISA)employing recombinant nucleoprotein (rNP)expressed in baculovirus system was developed for detection of antibody against avian A influenza virus (AIV)in chicken sera.40_well polystyrene micro titration plate was coated with the rNP antigen (0.6μg/100μl/well)for 15h at 4℃. The working titer of enzyme_labeled anti_chicken Ig conjugate was 1:1000 and that of serum samples was 1:200. The samples with optical dense(OD)greater than 0.15 were judged as positive based on the data from 140 sera of specific_pathogen_free(SPF)chickens. The 344 serum samples from SPF chickens(150 serum samples),non_immunized chickens(194 serum samples)and the chickens(30 serum samples)infected by other 15 infectious pathogens than AIV were negative in the rNP_ELISA. On the other hand, the rNP_ELISA was able to reveal all serum samples against 15(H1～H15)AIV subtypes,which were positive in agar gel precipitation(AGP)and hemagglutinatin_inhibition(HI)tests as well. Furthermore,the rNP_ELISA was able to detect the serum antibody from chickens infected with AIV strain A/Turkey/England/69(H 3N 2)on as early as 3rd day post_inoculation(DPI)and the positive detection could last 162 DPI. The rNP_ELISA was found more sensitive than AGP and HI tests. The variant coefficients of the OD value within assay and between assays ranged from 2.9 to 7.2%,and from 3.4 to 9.8% respectively. The total of 3138 serum samples were evaluated by rNP_ELISA,as well as whole virus antigen ELISA(AIV_ELISA),AGP and HI tests.The correspondence between rNP_ELISA and AIV_ELISA was 99.9%,between rNP_ELISA and AGP was 97.1% and between rNP_ELISA and HI was 98.9%. The results above showed that the rNP_ELISA was rapid,sensitive,economic and specific for serologically diagnosis of AIV infection in chickens.
出处
《中国预防兽医学报》
CAS
CSCD
2000年第3期182-185,共4页
Chinese Journal of Preventive Veterinary Medicine
