Preparation Of Monoclonal Antibody Specific To Hemagglutinin Of Subtype H5 Avian Influenza Virus And Development Of A Double-Antibody Sandwich ELISA

The BALB/C mice were immmutied with the H5N1 AIV virus which seed on MDCK . We use the virus to drop the BALB/C mice nose . Anther we use the embryo allantoic cavity to seed and propagtion the H5N1AIV, then use differential centrifugation to prifive virus, we use it to emelope antigen of indirect ELISA. Through the cellulav fusion and clone, using the Hiindirect ELISA rempircal method .We secret anti-AIV H5 subtype antibody, named 2D5, 3B2 and 3E4, were obtained after fusion followed by three or four screening. The ascites of mAbs were prepared by injecting the hybridoma cells into mice abdomen. The indirect ELISA results showed that the mAbs revealed high affinity with the AIV H5 subtype at the titer of 1:51200—102400 and the HI titer of 1:160— 1;640. In addition, the affinity with the standard antigen of these mAbs was demonstrated by western blotting. Of all the mAbs, 2D5 and3E4 belong to IgG2a subset, 3B2 belong to IgG1 subset. At the same time, these mAbs have been proved to have good specificity by essay of the cross-reaction.An antigen-captured ELISA (AC-ELISA) was developed to detect AIV H5 subtype antigen directly from the mAbs ascites with purification. And the poly antibody, standard serum, which was used as the second antibody, subsequently. In the same time, we were grading-up all parameters of the experiment and screening the best reaction system. Pass to detecte AIVH5 antigen and other subtypes of AIV, which indicated that the method has good reproducibility. On the dfbasis of the test, we concerned the AC-ELISA as a sensitive, specific and stable method to AIV H5 subtype antigen detection. This method is sensitive , save time, suitable and can direct a lot of sample product, direct clinical product manifest, that thismethod monitors to the epidemic situation of AIV and vaccine usage etc. are all the leading meaning having been certainly.