Preparation Of Monoclonal Antibody Against Envelope Protein Of Duck Tembusu Virus And Development Of ELISA Kit

At present,the outbreak of duck tembusu virus(DTMUV)disease has brought serious economic losses to the duck industry in Jiangsu,Chongqing,Fujian,Zhejiang,Jiangxi,Shandong and other provinces.A number of methods for detecting DTMUV disease have been reported.However,there is no commercial detection kit to detect the disease quickly and easily.Based on the recombinant E protein of the CQW1 strain of DTMUV(Gen Bank: KM233707.1),this study prepared anti-DTMUV monoclonal antibody and developed a detection kit.The study laid the foundation for the commercial development of the kit.1.Preparation of monoclonal antibody against E protein of DTMUVThe two hybridoma cells A6E7 and D1D7 were prepared by hybridoma technology using the DTMUV-E protein as immunizing antigen.After purification by saturated ammonium sulfate purification and affinity chromatography,the concentrations of monoclonal antibodies A6E7 and D1D7 were 0.97 mg/m L and 1.09 mg/m L,respectively.Subclass analysis showed that both monoclonal antibodies had heavy chain types of Ig G1 and light chain types of κ.Indirect IFA and WB assays showed that both A6E7 and D1D7 specifically recognized DTMUV and did not cross-react with GPV,DPV and DHAV-1.By analyzing the epitopes recognized,the result indicated that A6E7 and D1D7 recognize the same epitope.Further analysis of the relative affinity of monoclonal antibodies for E protein revealed that A6E7 has a higher relative affinity than D1D7,and A6E7 can react better with E protein.2.Development of DTMUV-E protein antibody detection kitThe monoclonal antibody A6E7 was labeled with horseradish peroxidase to prepare an enzyme-labeled monoclonal antibody.Furthermore,the E protein was used as a coating antigen to establish a direct blocking ELISA method.By optimizing the experimental conditions,the results showed that the optimal concentration of E protein was 3.75μg/m L,and the optimal dilutions of serum and enzyme-labeled monoclonal antibody were 1:2 and 1:400,respectively.The time optimization results under the constant temperature of 37 °C showed that the optimal antigen coating time,blocking time,serum and enzyme-labeled monoclonal antibody incubation time and color development time were 2 h,1 h,1 h,1 h and 10 min,respectively.On this basis,the kit was prepared and assembled.Specificity assay analysis showed that the kit did not cross-react with positive serum against DHAV-1,DHAV-3,DPV,RA,Du CV,respectively.By detecting 60 serum samples of known background,the kit has an accuracy of 100% and its sensitivity is good.Moreover,the kit has good repeatability and stability and can be stably stored at 4°C for 7 months.324 clinical duck serum samples were detected by kit and indirect ELISA.The results showed that the coincidence rate of positive and negative results of the kit was 75.76% and 94.96%,respectively.The coincidence rate of the two methods was 91.05%.