Establishment And Preliminary Application Of C-ELISA Method For Detection Of Antibody Against H7 Subtype Avian Influenza Virus

Avian influenza virus(AIV)can cause avian influenza(AI)that mainly affects poultry flocks.Most AIVs cause only a mild to asymptomatic infection,but highly pathogenic avian influenza virus(HPAIV),restricted to the H5 and H7 subtypes,causes extremely high mortality rate of up to 100% in domestic bird populations.Besides,some of these viruses may occasionally infect humans.According to the WHO reports,H7N9 virus has caused a total of 1,568 human infections,resulting in 615 deaths between February 2013 and March 2019.The H7N9 virus first emerged in China in 2013 was nonpathogenic to poultry,and then mutated to HPAIV in 2017,which caused huge economic losses in the poultry industry.Since they commonly infect birds and poultry,a H5/H7 bivalent inactivated vaccine was initiated in China in September 2017 to control the threat of H7N9 influenza virus to the poultry industry and to reduce the circulating viral load in chickens.Therefore,a reliable and convenient detection method of H7-AI antibody to evaluate the antibody level is needed after vaccine immunization.Hemagglutination inhibition(HI)is the official subtype-specific test for influenza serological differential diagnosis,which is stable and easy to operate.However,this method has some problems in use,for example,red blood cell of SPF chickens is difficult to obtain in the field,and disposable 4 HAU of antigen should be accurately determined,etc.In order to avoid the defects of HI method and further increase the diversification of market products,a competitive enzyme-linked immunosorbent assay(C-ELISA)method for detecting H7-AI antibodies in poultry serum was established.Based on the previous work in our laboratory,a strain of H7-AI monoclonal antibody(m Ab),named 1H9,was selected,resuscitated and cultured to prepare mouse ascite.Then,the HI titer of it was determined,followed by purification.The m Ab was labeled with horseradish peroxidase(HRP)by sodium periodate to obtain HRP-labeled m Ab(HRP-1H9).And the H7-AIV(A/chicken/Guangdong/SD008/2017)antigen was purified by sucrose density gradient centrifugation.The optimal concentration of antigen coating and serum dilution were determined by checkerboard titration.To determine the cut-off value of C-ELISA,the ROC curves and interactive dot diagram of546 sera with clear background were plotted by using Med Calc software.In addition,chicken serums of H1--H6,H8--H15 and H7Nx(N1--N4,N7--N9)subtypes of AIV,and other serums of common diseases in poultry,including Newcastle disease virus(NDV),Infectious bronchitis virus(IBV),and Infectious bursal disease virus(IBDV),were prepared.They were used as standard serums for specificity,sensitivity and repeatability tests,respectively.Combined with the results of 1,392 field samples,the diagnostic performance of the established C-ELISA method for the detection of antibodies against H7-AI was evaluated.The results showed that the optimal concentration of antigen coating was 5 ?g/m L,serum dilution was 1/10,and HRP-1H9 dilution was 1/3,000.When 546 standard samples(178 AIVs negative samples and 368 AIVs positive serum samples)were tested,the cut-off values were determined to be 40%,with a sensitivity of 98.91% and specificity of 99.44%.Therefore,when PI is greater than 40%,the antibody against H7-AI is positive,otherwise,it's a negative serum of H7.By detecting the serums of AIVs(H1--H15),NDV,IBV and IBDV,the results showed that the PI of AIV-H7Nx(N1--N4,N7--N9)positive serums were all greater than 40%,while the other AIVs serums(H1--H6 and H8--H15),NDV,IBV and IBDV were all lower than 40%.When a positive serum was serial two-fold diluted,the PI value of serum was still greater than 40% at 320 times dilution,indicating good sensitivity.The coefficients of variation(CV)of both intra-and inter-batch repetitions were all less than 12% in repeatability experiment.According to statistical analysis,the overall coincidence rate between the C-ELISA method and the HI assay was 98.56%,indicating that the C-ELISA method had a high coincidence rate with HI test and could be used to detect field serum samples.In summary,a C-ELISA antibody detection method using a specific m Ab of H7-AI as competitive antibody was established.Compared with the traditional method,it was reacted by simultaneously added serum antibodies and competitive antibodies,which simplified the procedures,shortened the reaction time,and provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AI.