Establishment Of Indirect ELISA And Colloidal Gold Immunochromatography Test Strip Detection Method For Duck Blast Virus

Duck plague（DP）is a septicemic fatal infectious disease transmitted by contact transmission in ducks,geese and other wild geese,which is caused by duck plague virus（DPV）.The disease can cause tissue hemorrhage,lymphadenopathy,etc.,and has the characteristics of acute disease and high mortality.It has caused huge economic losses to the duck industry,and is one of the important diseases threatening the duck industry.Vaccination with duck plague vaccine is an important measure to prevent duck plague.However,in recent years,due to various reasons such as feeding management,biosafety measures,and improper immunization timing,duck plague immunization has often failed.It is particularly urgent to establish effective detection methods for rapid detection of duck plague antibody levels and infection in duck flocks.This study selected the g B protein of DPV for truncated expression,purification,and identification,and obtained the recombinant g B’protein;Three monoclonal antibodies were prepared by immunizing mice with recombinant g B’protein;An indirect ELISA method for detecting DPV antibodies was established using recombinant g B’protein as the coating antigen;And a double antibody sandwich colloidal gold immunochromatographic test strip was established using the prepared monoclonal antibodies.The main research content is as follows:（1）Based on the g B gene sequence of the DPV-CHv strain on Gen Bank,a pair of specific primers were designed using Primer Premier5.0 software.The C-terminus truncated fragment（g B’）of DPV-g B was amplified by PCR,connected to the p ET-32a plasmid,and transformed into BL₂₁（DE3）receptive cells.The recombinant g B’protein was induced and expressed for 8 hours under a condition of 0.7 m M IPTG and 25℃,and purified using Ni NAT agarose purification resin.Five mice were immunized with recombinant g B’protein,and the No.1 mouse with the highest titer was selected for cell fusion.Subsequently,three ascitic monoclonal antibodies were obtained through steps such as hybridoma cell screening and induction of ascitic monoclonal antibodies:1A1,2A11,and 4E4,with titers of 1:1.28×10⁵,1:1.28×10⁵,1:2.56×10⁵ respectively,the heavy chain subtypes are Ig G1,Ig G1,Ig G2b,and the light chain subtypes are allκchain.Three monoclonal antibodies were purified using octanoic acid saturated ammonium sulfate method and Protein A+G resin to obtain highly purified monoclonal antibodies.（2）An indirect ELISA method for DPV antibodies was established using recombinant g B’protein as the coating antigen and optimized.The optimized condition is that the antigen coating concentration is 2μg/m L;the optimal sealing solution is 5%skimmed milk powder;the optimal dilution of the sample to be tested is 1:100,and the optimal incubation time is 30minutes;The optimal dilution of the second antibody is 1:12000,and the optimal incubation time is 30 minutes;the optimal substrate color development time is 15 minutes.The negative and positive critical values of the established ELISA method are OD₄₅₀≤0.347 and OD₄₅₀≥0.396,respectively;the coefficient of variation of inter and intra batch repeated experiments is below 5%,indicating good reproducibility;there was no cross reaction with DHV,AIV,DTMUV,MDPV,Du CV positive serum;DPV positive serum with a dilution of 1:1600 can be detected;sixty clinical samples were selected for testing,and the coincidence rate between the test results and the neutralization test reached 93.3%.（3）Preparation of colloidal gold solution by trisodium citrate reduction method,and its macroscopic observation,determination of absorption value at 400-600 nm,and identification by transmission electron microscopy scanning.The prepared 1A1 monoclonal antibody was coupled with colloidal gold as a gold labeled antibody,and the optimal coupling conditions were p H 7.5 and antibody concentration 20μg/m L;4E4 monoclonal antibody and goat anti mouse Ig G were sprayed onto nitrocellulose membrane as detection lines（T-line）and quality control lines（C-line）,respectively.The optimal concentrations for both T-line and C-line optimization were 1mg/m L.This method has no cross reactivity with five viruses:DHV,AIV,DTMUV,MDPV,and Du CV,with a detection line of 10^4.52TCID₅₀/m L,indicating good specificity and sensitivity of the method.94 clinical samples were tested simultaneously with PCR method,and the coincidence rate of the two methods reached 98%.In conclusion,an indirect ELISA for the detection of duck blast antibodies and a colloidal gold immunochromatography test strip for the detection of duck blast virus were established,which can provide effective tools for monitoring the immune effect of duck plague vaccine and rapid diagnosis of duck blast virus.