Development Of Colloidal Gold Test Strip For H7 Subtype Avian Influenza Virus

Avian influenza virus(AIV)belongs to the genus of influenza A virus in the Orthomyxoviridae family.According to the difference of surface hemagglutinin and neuraminidase,it can be divided into 16 different HA subtypes and 9 NA subtypes.Since 2013,H7N9 avian influenza viruses have been found in China,including highly pathogenic and low pathogenic viruses.The H7N9 avian influenza virus has a high mortality rate after infecting humans.Most of the human infection cases have close contact with poultry on the live poultry market.Therefore,it is very necessary to develop a rapid detective technology for H7N9 avian influenza virus.In this study,the H7N9 subtype avian influenza virus A/Chicken/Huizhou/HZ-3/2017(HP)purified by differential centrifugation was used to immunize BALB/c mice.After screened by immunoperoxidase monolayer assay(IPMA)and indirect ELISA,25 hybridoma cells that stably secreted monoclonal antibodies(McAbs)specific for H7N9 subtype AIV were established,18 of which(2A2,2H6,3A8,3E5,3H3,7A2,7H7,9B6,10A8,11B8,12F11,13C10,14C2,15C5,16D2,16F2,17A2,and 21E2)recongnized HA protein,while McAb 12F12 and the other 6 McAbs(7D11,8A6,11A8,13A6,15E11 and 18A2)reacted to NA protein and NP protein,respectively.The IPMA titer of the McAb ascites were determined between 1×10-4-1×10-6.The subtype identification results showed that heavy chain subtypes of the most McAbs were IgG1(except for 10A8 was IgG2a,and 2A2 was IgG2b),and all the light chain were ? chain.The results of the virus response spectrum test showed that 18 H7-HA McAbs and 1 H7-NA McAb recongnized the H7N9 subtype AIV isolated from 2013 to 2017,but did not react with other subtypes of influenza viruses.6 H7-NP McAbs reacted with some other subtypes of influenza viruses.Hemagglutination inhibition(HI)and virus micro-neutralization test results showed that the HI titer of 16 H7-HA McAbs were between 4 log2-13 log2,and the neutralization titer were between 1:500-1:16 000,the other two H7-HA McAbs did not show any HI or neutralizing activity.Western-blot analysis results showed that 15 of the 18 hybridoma cell culture supernatants showed a specific protein band near 63 KDa,while the other 3 did not have a band,indicating that the some of the 18 H7-HA McAbs recongnized linear epitopes while the other did conformational epitopes.Preliminary peptide scan results showed that the antigenic sites recognized by McAbs 2A2,2H6,3A8,3H3,7H7,9B6,11B8,12F11,13C10,14C2,]5C5,16D2,17A2 and 21E2 were GVTSACRRSGSSFYAEMK(142-159 aa).The antigenic site recognized by McAb 10A8 was YKSTQSAIDQITGKLNRL(381-398 aa),while the other three McAbs 3E5,7A2,and 17A2 do not react with the polypeptide and recognize conformational epitopes.Sixteen strains of HA McAbs with HI and neutralizing activity were labeled with colloidal gold,and paired McAbs 9B6 and 10A8 were selected by Dot-blot.The gold-labeled antibody was prepared by labeling the McAb 10A8 with 30 nm colloidal gold particles.The detection line and the control line were sprayed on the nitrocellulose membrane at 0.8 mg/mL McAb 9B6 and 1.3 mg/mL rabbit anti-mouse IgG,respectively.By systematically optimizing the test paper parameters,a rapid detective strip for H7 subtype avian influenza virus was established.The test results of the characteristics of the colloidal gold immunochromatographic strip showed that the detection limit of the test strip for H7N9 subtype AIV allantoic fluid was 2.4 log10EID50/0.1 mL,and it had no cross-reactivity with other subtypes of AIV and common poultry viruses,indicating that the strip had good specificity.The strip could detect the H7N9 subtype AIV isolated from different years of 2013-2017,and could also detect the H7-Re2 antigen strain used for the trivalent inactivated AIV vaccine,indicating that the strip had broad spectrum.The results of detecting H7N9 subtype AIV allantoic fluid with the strip were 2-8 hemagglutination units higher than the hemagglutination test,indicating that the strip had better sensitivity.The shelf life test results showed that the detection limit of the test strips for the H7N9 subtype AIV allntoic fluid did not change after stored for 6 months,indicating that the strip have good stability.The H7 subtype avian influenza virus colloidal gold immunochromatographic strip developed in this study is convenient,fast and reliable,and provides a rapid and simple method for H7 subtype avian influenza virus monitoring.