Detection Of H5 Subtype Avian Influenza Virus By Gold Immunochromatographic Assay And Dot-immuogold Filtration Assay

H5 subtype avian influenza viruses(AIVs),which are mostly the highly pathogenic pathogens,can cause a large-scale death of poultry and bring huge losses for the poultry industry.Meanwhile,the H5AIVs can also cause human infection and death.The H5 AIVs circulating in China were mainly clustered into Clade 2.3.2,Clade7.2 and Clade2.3.4.4 respectively,according to the phylogenetic analysis of the HA genes.A new subclade of viruses within the Clade 2.3.2,which was designated as Clade 2.3.2.1e,were continuously detected since 2015 in several provinces of China.The big differences of antigenic characteristics existed in the different branches of H5 AIVs,which bring great challenges for diagnosis of disease based on the monoclonal antibodies(MAbs).In this study,we aimed to prepare the MAbs using the representive virus of Clade7.2,Clade2.3.4.4 and Clade 2.3.2.1e as immunogens,and then establish the detection method of Colloidal gold strip and Dot-immunogold filtration assay(DIGFA)based on the broad reactivities MAbs.In this study,the immunogenic mixture prepared with the vaccine seed virus of the Clade7.2CK/LN/S4092/2011(H5N1)(Re-7),Clade2.3.4.4 DK/GZ/4/2013(H5N1)(Re-8)and Clade2.3.2.1e DK/AH/S1246/14(H5N1)(Re-10)were immunized into 4-week-old female BALB/c mice.After 4times of immunization,the aseptic spleen cells and myeloma cells(SP2/0)were fused under the action of polyethylene glycol(PEG).Screened by indirect ELISA method and HI method,6 strains of hybridoma cells,which could stabilily secrete MAbs against hemagglutinin(HA)protein,were obtained and designated as 1B10,2A10,2C8,3E6,4G9 and H2B1 respectively.The MAbs 1B10,2A10,2C8 and H2B1 could widely cross-react with the current circulating H5N1 AIVs.Two good reactivity monoclonal antibodies 2A10 and 2C8 were selected.To prepare the the gold labeled antibody,the MAb 2A10 was labeled with colloidal gold particles of 20nm size and purified by centrifugation of 10000 rpm.The colloidal gold labeled MAb 2A10 diluted by 8 times was dispensed onto the conjugate pad,MAb 2C8 was dispensed on the bottom of the NC membrane as the test line with a concentration of 1.5mg/mL,and Goat anti-mouse IgG was dispensed at the upper position as the control line with a concentration of 1 mg/mL.The colloidal gold strip and assembled and used to detect the H5 subtype AIVs circulating in China.The results showed that the colloidal gold strip has good broad-spectrum reaction with H5 AIVs,and good specificity without cross-reaction with other subtypes AIVs.The dectection sensitivity was viral samples with the HA titer of 2~4.In order to further improve its sensitivity,we prepared the gold standard silver staining immune osmotic card by pointing the Mab2C8 and Goat anti-mouse IgG to the osmosis card.The sensitivity was increased to detect the viral samples with the HA titer of 2~2.In conclusion,6 anti-HA MAbs 1B10,2A10,2C8,H2B1,3E6 and 4G9 were prepared by hybridoma technology in this study.The colloidal gold strip and DIGFA based on the MAbs for detection of H5 subtype AIVs were initially established.Compared to colloidal gold strip,the DIGFA was more sensitive.