Development And Preliminary Application Of Colloidal Gold Test Strips For Detection Of Serum Antibodies In Chicken And Duck Against H5 And H7 Subtypes Avian Influenza Virus

The H5 and H7 subtypes of avian influenza virus（Avian influenza virus,AIV）infection with poultry results in a high mortality rate,and endangers the development of the breeding industry.Many poultry and wild poultry are susceptible to infection in the natural environment,and some strains can infect humans.Vaccination of vulnerable poultry could effectively prevent and control the spread of AIV.Therefore,the monitoring of antibody levels is important for the assessment of immune status.Colloidal gold test strips are convenient and specific,so they are widely used to detect antibody levels in many chicken farms.In view of the lack of universal colloidal gold products for the detection of AIV antibody insusceptible poultry such as duck and goose,this research used a strain of monoclonal antibody against a variety of avian IgYFc as a colloidal gold marker,which can react with chicken,duck,goose and other poultry serum.Two types of colloidal gold test strips were studied using this monoclonal antibody,aiming to detect serum antibodies of H5 and H7 subtypes AIV of chicken and duck,which could be used not only for rapid diagnosis of H5 and H7 subtypes AIV infection,but also applicab1e to monitor antibody levels after vaccination.The main research results are as follows:1.Expression and identification of two fusion proteinsThe recombinant plasmids p GEX-KG-HA1₅ and p GEX-KG-HA1₇were respectively expressed in E.coli BL21（DE3）and the two expression products were purified to obtain the recombinant protein HA1₅ and HA1₇,with the size of 63KDa and 61KDa,respectively.AIV-HA1₅and AIV-HA1₇ protein were identified by Western blot,which showed that they have good reactogenicity and specificity.2.Purification of monoclonal antibody against various avian IgYFc fragmentsThe monoclonal antibody was purified mice ascites by caprylic acid-ammonium sulfate method.The ascites was produced by injecting mice with hybridoma cell line 10A1,which can produce monoclonal antibody against various avian IgYFc fragments.After purification high-purity and high-activity mouse monoclonal antibody was obtained.3.Establishment of colloidal gold detection method for H5 subtype AIV antibodyMouse anti-avian IgYFc fragment gold-labeled monoclonal antibody is used as the indicator,goat anti-mouse Ig G（0.1mg/m L）is marked as line C,AIV-HA1₅ protein（0.5mg/m L）is marked as line T,and the labeled amount of monoclonal antibody is 8μg Mc Ab/m L colloidal gold,the standard gold p H is 9μL 0.01mol/L K₂CO₃ solution per milliliter of colloidal gold,water-absorbing pad CH37K,gold standard pad Fusion4,serum sample pad XQ-Y7,the diluent of the protein on the C line and T line is the coating solution3（based on PB with p H 7.2 as the basic buffer）,the gold standard spray volume is 5μL/cm.The test strip has good specificity and reproducibility,and can be stored for 6 months at4°C.A total of 152 samples of clinically immunized chicken and duck serum were tested,and 140 samples were consistent with the HI method.The total coincidence rate is 92.11%（140/152）.A total of 14 samples of unimmunized chicken and duck serum were tested,the test results of strips were all negative,indicating that this test strip can be used to determine the antibody titer of serum samples of chicken and duck.4.Establishment of colloidal gold detection method for H7 subtype AIV antibodyMouse anti-avian IgYFc fragment gold-labeled monoclonal antibody is used as indicator,goat anti-mouse Ig G（0.1mg/m L）is marked as line C,AIV-HA1₇ protein（0.3mg/m L）is marked as line T,and the labeled amount of monoclonal antibody is 8μg Mc Ab/m L colloidal gold,the standard gold p H is 9μL 0.01mol/L K₂CO₃ solution per milliliter of colloidal gold,water-absorbing pad CH37K,gold standard pad Fusion4,sample pad NJ-Y8,the diluent of the protein on the C line and T line is the coating solution 2（TBS with p H 8.2 as the basic buffer）,the gold standard spray volume is 6μL/cm.The test strip has good specificity and reproducibility,and can be stored for 3 months at 4°C.A total of167 samples of clinically immunized chicken and duck serum were tested,and 158 samples were consistent with the HI method.The total coincidence rate is 94.61%（158/167）.A total of 14 samples of unimmunized chicken and duck serum were tested,and the test results of strips were all negative,indicating that this test strip can be used to determine the antibody titer of chicken and duck serum samples.