Development Of McAbs Against H9 AIV And Establishment Of Antigen Capture ELISA For Detecting H9 Avian Influenza Virus

In this experiment, three hybridoma cell stains were developed by confusion SP2/0 cells and spleen cells of immunized mouse with AIV A/Chicken/Shandong/6/1996 (H9N2). Three positive hybridoma cell stains 1F4C4,9F12D1,2C5H12 were determined by HI and ELISA, which HI titers of cell supernatants and ascites were 26,24,21 and 212,210,27 respectively. The McAbs were specific to H9 AIV and not reacted with H1-H8 and H10-H15 subtype avian influenza viruses and other avian disease viruses. The subtypes of the three McAbs were IgG2b, IgG2a, IgG2b respectively. These McAbs had high neutralization titers against some strains of H9 AIV.A11/C6 and BD12 were used as coating and HRP-labeling McAbs respectively for antigen capture ELISA after antibody pairing test. Based on these antibodies, the antigen capture ELISA were developed for detecting H9 AIV. The optimal working condition was listed as below. (1) capture McAb was diluted to 1:4000(0.0785μg/well), H9 AIV antigen was diluted to 1:10 and the HRP-labeled BD12 was diluted to 1:3000. (2)The reaction time for capturing antigen and HRP-labled BD12 were 1.5h and 1.0h separately in the assay. (3)0.05mol/L pH9.6 Na2CO3-NaHCO3 buffer and 3% skimmed milk-PBS solution were the best choice for coating buffer and blocking buffer.The sandwich ELISA based on McAbs was experimented with specificity,sensitivity and repetition. The data demonstrated that: (1)NDV,IBDV,IBV,ILTV,FPV and H1-H15 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 others 14 subtypes AIV except H9. It proved that the H9 AIV antigen capture ELISA had excellent specificity. (2) The sensitivity of the antigen capture ELISA was twice better than that of HA test. The minimum dectectable concerntration by this assay was 7.6ng. (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 4 months and over 5 months at -20℃.