一种新型H5N1禽流感病毒血凝素抗原快速检测试剂的建立

Establishment of a New Rapid Antigen Detection Test for HA of H5N1 Avian Influenza Virus

利用5株广谱特异性抗H5亚型血凝素单克隆抗体和酶联免疫渗滤技术成功地建立了一种适于现场检测H5亚型禽流感病毒血凝素蛋白的抗原快速检测试剂H5-HA(Ag)Dot-ELISA。该试剂对41株代表当前亚洲地区流行的各种遗传变异亚系H5N1禽流感病毒检测均为阳性,对多数毒株的分析灵敏度优于0.1个血凝滴度(HA titer),其中部分优于0.01个血凝滴度;比较该试剂与早期开发的同类ELISA试剂,发现前者对后者未能检出的H5N1新变异株检测均为阳性;利用该试剂和商品化Directigen Flu A(BD)试剂检测两株H5N1病毒株,提示前者灵敏度高于后者;该试剂对一株H5N1病毒的检测灵敏度与标准RT-PCR相当;该试剂对24株非H5亚型病毒检测均为阴性,显示出良好特异性。以上结果提示,此研究建立的H5N1病毒抗原快速检测试剂在H5禽流感现场检测上具有较好的应用前景。

We used five H5 specific broad spectrum McAbs to develop a rapid antigen detection test ＂H5-HA（Ag） Dot-ELISA＂ which combines immune-filtration with double antibody ＂sandwich＂-like principle for detection of hemagglutinin antigen （Ag） of H5 avian influenza virus. It was shown that the analytical sensitivity of HS-HA （Ag） Dot-ELISA in detecting 41 strains of human or avian HSN1 cultured viruses was at least 1 HA titer, some even up to 0.01 HA titer, and 0.1 HA titer for most strains. Comparison of this kit with our earlier developed HS-ELISA, it showed that the former had a higher detection rate than the latter in detecting these 41 H5N1 viruses. It also displayed higher sensitivity than BD commercialized Directigen Flu A kit when detecting two HSN1 viruses. It also had an equivalent sensitivity for HSN1 strain ＂Ck/HK/Yu22/02＂ when compared with standard RT-PCR. This kit also had a nice specificity and showed no cross-reactivity with 24 strains of non H5 virus. These results suggested that HS-HA（Ag） Dot- ELISA will be very useful in diagnosing H5N1 virus in the field.