本试验用H9亚型禽流感病毒FT株作为免疫原，免疫6周龄BALB/c雌性小鼠。加强免疫后取脾细胞与SP2/0细胞融合，采用ELISA方法和HI方法筛选，阳性细胞克隆经有限稀释法克隆培养，最后获得6株抗H9亚型禽流感病毒特异性单克隆抗体（McAb），分别命名为1D10、4A7、4E4、4F3、4H12、5B10。其中1D10、4E4是抗H9亚型禽流感病毒HA特异性的单克隆抗体，2株阳性杂交瘤细胞的培养上清及腹水对禽流感病毒FT株的HI效价分别为24、29 和 25、210。6株阳性杂交瘤细胞的培养上清及腹水对禽流感病毒FT株的ELISA效价分别为103、104、105、105、106、105 和106、107、108、108、109、108。特异性试验表明该6株单抗均不与其他常见的禽类病毒和H5、H7亚型禽流感病毒发生交叉反应。单抗5B10和4F3亚类皆为IgA，4H12和4A7亚类为IgG1，4E4亚类为IgG2a，1D10亚类为IgG2b。相加间接ELISA试验表明，单抗4F3和5B10对应于同一抗原表位，另4株针对不同抗原表位。6株单抗对禽流感H9亚型FT毒株均没有中和活性。 选取效价高的单克隆抗体4E4利用HiTrapTM Protein G亲和层析柱纯化后，作为建立双抗体夹心ELISA方法的捕获抗体。确定夹心ELISA试验中单抗的最适工作浓度为5μg/ml，标准阴阳性抗原稀释倍数为1:10。酶标多抗的最佳稀释度为1：300，最适浓度为9μg/ml。捕获单抗的最适包被条件为37℃包被1h后4℃过夜，选用3%BSA作为封闭液，最佳封闭时间为1.0h。抗原的反应时间为1.0h，酶标抗体最佳作用时间为1.0h，最佳显色时间为15min。 用建立的双抗体夹心ELISA方法与NDV、IBV、IBDV、ILTV和FPV以及H5、H7亚型禽流感病毒进行交叉反应性试验，结果表明：H9亚型禽流感病毒双抗体夹心ELISA方法与其他禽类病毒及H5亚型禽流感病毒均没有明显的交叉反应性，说明该方法对H9亚型流感病毒具有高度的特异性。优化好的ELISA反应体系检测近年来H9亚型禽流感病毒不同分离毒株的覆盖率很广，检测18个已经分离到H9亚型禽流感病毒的临床病料全部显强阳性反应，表明建立的双抗体夹心ELISA方法特异性良好，具有很高的精确性和敏感性。重复性实验表明，同一样品的板内和板间的变异系数均小于10%。 将包被的酶标板及酶标抗体和标准阴阳性抗原组装成简易试剂盒，同时保存于室温、4℃和－20℃，定期进行检测，结果表明：试剂盒在室温保存时极不稳定，保存期限不超过1个月，4℃可保存3个月，在－20℃稳定保存4个月以上。

In this experiment, six hybridoma cell stains were developed by confusion SP2/0 cells and spleen cells of immunized mouse with AIV FT strain (H9N2). Six positive hybridoma cell stains 1D10、4A7、4E4、4F3、4H12、5B10 were determined by ELISA and HI, which ELISA titers of cell supernatants and ascites were 103、104、105、105、106、105 and 106、107、108、108、109、108 respectively. The HI titers of cell supernatants and ascites of 1D10 and 4E4 were 24、29 and 25、210.The McAbs were specific to H9 AIV and not reacted with H5、H7 AIV. The subtypes of 1D10、4A7、4E4、4F3、4H12、5B10 were IgG2b, IgG1，IgG2a, IgA，IgG1，IgA respectively. Additivity ELISA indicated that 4F3 and 5B10 were corresponding to the same antigen epitope, but the other 4 mAbs were different epitopes. These strains had no neutralization titers against AIV FT strain. 4E4 was used as coating McAb for antigen capture ELISA after purified by HiTrapTM Protein G. The optimal working condition was listed as below. (1) capture McAb was diluted to 5μg/well, H9 AIV antigen was diluted to 1:10 and the HRP-labeled polyclonal antibody was diluted to 1:300（9μg/ml）. (2) The optimal condition of coating was 2.0h in 37℃ and then overnight in 4℃.The best blocking reagent was 3% BSA and the optimal blocking time was 1.0h.The reaction time for capturing antigen and HRP-labled polyclonal antibody were both 1.0h. The reaction time for TMB was 15min.The result proved that the H9 AIV antigen capture ELISA had excellent specificity. The sandwich ELISA based on McAbs was experimented with specificity、sensitivity and repetition.The data demonstrated that:(1)NDV、IBDV、IBV、ILTV、FPV and H5 AIV were detected by the sandwich ELISA and the result proved that there was no crossing-reaction between the antigen capture ELISA and other avian viruses and H5、H7 AIV except H9.It proved that the H9 AIV antigen capture ELISA had excellent specificity.(2) The result proved that the H9 AIV antigen capture ELISA had excellent specificity. (3)CV of intra-plate and inter-plate was smaller than 10%. The simple ELISA kits which consisted of coated microplate and reactive solutions were kept at room temperature,4℃and－20℃seperately.The detection results showed that kits were not stable at room temperature and the period of validity was only one month.At the same time,the period of validity at 4℃was 3 months and over 4 months at－20℃.