Development And Preliminary Application Of Colloidal Gold Test Strips For Detection Of Antibodies Against H9 Subtype Avian Influenza Virus And Mycoplasma Gallisepticum

H9 subtype Avian influenza?AI?and Mycoplasma gallisepticum?MG?infection are important respiratory infections that are harmful to the poultry industry.Chickens,ducks geese,pigeons,quails and other birds can be infected.Vaccine immunization is the main means to prevent and control the occurrence and epidemic of H9 subtype avian influenza.Contrast to the prevention and control of Mycoplasma gallisepticum infection,only some breeder farms use vaccines.Serological tests are especially important for rapid diagnosis after infection and quantitative detection of antibody levels after vaccine immunization.However,traditional serological methods often require professional procedures,which are cumbersome and time-consuming.Colloidal gold detection is simple,fast and low cost.It can be widely used at the grassroots level without the need of professional technicians and expensive equipments.The laboratory screened and obtained a strain of monoclonal antibody against a variety of avian IgY Fc,which can react with chicken,duck,goose and other poultry serum,laying a foundation for the establishment of colloidal gold strip methods for the detection of various avian serum antibodies.At the same time,the quantitative detection methods can be established by combining the signal recognition of immune quantitative tachometer.In this study,a colloid gold test strip for quantitative detection of H9 subtype avian influenza virus antibodies and a colloid gold test strip for detection of Mycoplasma gallisepticum antibodies were respectively prepared,which could be used for qualitative detection and rapid diagnosis of Mycoplasma gallisepticum infection.The strips can also be quantitatively tested to monitor antibody levels after vaccination.The main research results are as follows:1.Expression and purification of AIV-HA1₉ protein and MG-vlhA1.2 proteinRecombinant prokaryotic expression plasmids of AIV-HA1₉ protein and MG-vlhA1.2 protein were constructed and expressed in E.coli BL21?DE3?.The recombinant proteins were 61KDa and 92KDa,respectively.Identified by Western blot,AIV-HA1₉ and MG-vlhA1.2 protein have good reactogenicity.2.Preparation and purification of monoclonal antibody against various avian IgY Fc fragmentsHybridoma cell line 10A1 was resuscitated,which can produce monoclonal antibody against various avian IgY Fc fragments,and injected to mice to collect ascites.Purified by caprylic acid-ammonium sulfate method,high-purity and high-activity mouse monoclonal antibody against a variety of avian IgY Fc fragments was obtained.3.Development and preliminary application of colloidal gold test strip for quantitative detection of antibodies against H9 subtype AIVIn this study,indirect method was used to prepare colloidal gold test strip for quantitative detection of antibodies against H9 subtype avian influenza virus.The colloidal gold-labeled anti-avian IgY Fc monoclonal antibody was optimally labeled with9?g McAb/mL colloidal gold,and the optimal pH was 8?L 0.1mol/L K₂CO₃ solution per mL of colloidal gold.The optimal reaction system for the test strip was:the absorbent pad was selected for H5073;the standard pad Fusion4;the sample pad Ahlstrom8964;coating solution PB buffer?pH7.2?+3%methanol;sample pad blocking solution selects sodium tetraborate buffer?pH9.0?as base fluid;C-line coated goat anti-mouse IgG?0.5mg/mL?,T-line coated AIV-HA1₉ protein?0.8mg/mL?;the spray amount of the gold-labeled antibody was 6?L/cm.The test strip has no cross-reaction with other important pathogen positive sera of poultry,and has high specificity.Combined with colloidal gold immunoassay,the HI titer was X axis,the?T/C?signal ratio was Y axis.The standard curve equation was y=0.3784x-0.7239,while the correlation coefficient R² is 0.9877,and the detection limit of HI titer is 3log₂.Both intra-assy and inter-assay coefficients of variation were less than 10%;It can be stored for at 4°C at least half a year.Taking HI test as comparison,the quantitative detection coincidence rate of clinical serum samples of immunized chickens,ducks and geese was 81.78%,indicating that this strip could be used for quantitative detection of various avian clinical samples.4.Development and preliminary application of colloidal gold test strip for detection of antibodies against Mycoplasma gallisepticumUsed for qualitative or quantitative detection of antibodies against Mycoplasma gallisepticum,the optimal reaction system of the colloidal gold test strip was established as follows:the absorbent pad was selected as H5073;the standard pad Fusion4;the sample pad Ahlstrom8964;the coating solution PBS buffer?pH7.4?+3%methanol;the sample pad blocking solution PB buffer?pH6.0?as the base solution,and the C line was coated with goat anti-Mouse IgG?0.5mg/mL?,while T-line was coated with MG-vlhA1.2protein?0.6mg/mL?.The amount of gold-labeled antibody was 7?L/cm.The test strips have no cross-reaction with other important pathogen positive sera of poultry and have high specificity.Combined with colloidal gold immunoassay,the SPA titer was X-axis,and the?T/C?signal ratio was Y-axis.The standard curve equation was y=0.3325x+1.0681,and the correlation coefficient R² was 0.9938;the detection limit was1:2^-2,in other words,that the sensitivity was 8 times higher than the SPA detection.Both intra-assy and inter-assay coefficients of variation were less than 10%;It can be stored at4°C for at least half a year.Taking SPA test as comparison,qualitative detection coincidence rates of clinical serum samples of chicken,duck and goose were 80.58%,85.48%and 83.72%,respectively;The conformance rate of the quantitative detection of clinical serum samples of immunized chickens was 85.38%,indicating that this strip could be used for rapid diagnosis of MG infection in poultry and the quantitative detection of antibody level after vaccine immunization.