Development Of ELISA Methods For The Detection Of Antibodies Against Avian Influenza Viruses Of H5 And H7 Subtypes

Highly pathogenic avian influenza(HPAI)is a devastating disease in poultry and caused by some H5 and H7 subtypes influenza viruses.Vaccination is one of the most effective strategy for the prevention and control of HPAI.A DNA vaccine for against H5N1 avian influenza has been licensed for commercial use in China,providing a new choice for the vaccination strategy with a great potential for being used broadly in fields.However,the traditional antibody detection methods,such as hemagglutination-inhibition(HI),have been proven not effective in evaluating the immune effects of the DNA vaccine.In this study,therefore,we were to develop new ELISA methods to providing necessary tools for the clinical application of this vaccine.To obtain the antigens for the ELISA methods,the HA genes of avian influenza virus strains A/Chicken/Guizhou/4/2013(CK/GZ/4/13(H5N1)),A/Duck/Guizhou/S4184/2017(H5N6)(DK/GZ/S4184/2017(H5N6)),and A/Chicken/Guangxi/SD098/2017(H7N9)(CK/GX/SD098/17(H7N9))were cloned into the insect expression vector pfastbac1 by PCR amplification to construct recombinant plasmids pfastbac1-HAs.Then the plasmids were transposed into DH10bac competent cells to obtain plasmids rBacmid-HAs.Recombinant baculovirus was gained by lipofectamine-mediated transfection of rBacmid-HAs into SF9 cells.The antigenicity of the expressed proteins was analyzed by immunofluorescence assay(IFA)and western blot.The proteins were then expressed in High Five cells and purified visa applying affinity chromatography.The results showed that the H5HA and H7HA proteins both had good immunogenicity and could be used as antigens for developing the ELISA methods.Finally,we have developed indirect-ELISA assays for detecting antibodies against the influenza viruses of H5 and H7 subtypes.Firstly,we determined the best binding concentrations of the antigens and sera by using chessboard titration method.We then optimized all the reaction conditions of the assays,such as the selection of blocking solution,blocking time periods,and the dilutions of enzyme-labeled antibodies,using the method of controlling variables.Finally,the repeatability,specificity and sensitivity of the assays were determined through the comparisons with the performance of HI assays.The optimum working conditions of H5 subtypes were as follows:coating ELISA plate with protein of 2 ng/μL,sealing with 1%BSA for 2 h,incubating primary antibody diluting 50 times with commercial diluent for 1.5 h and incubating enzyme-labled secondary antibody diluting 2000 times for 1 h.The results showed that the indirect ELISA method of H5 subtype had good repeatability,sensitivity and specificity.The correlation equation between H5 subtype indirect ELISA method and HI was as follows:y=9.616*x-1.888,R~2=0.8246.As for The optimum working conditions of H7 subtypes,besides the concentration of coating protein was 5 ng/μL,the others were same as the former.It also had good repeatability and sensitivity,yet it had cross-reaction with other subtypes.The correlation equation with HI was y=9.139*x+0.3650,R~2=0.7873.The preliminary applications of these two assays indicate that they have the potential to be used in field practice.