| Since 2014,duck farms in many southern provinces such as Jiangsu and Zhejiang have experienced outbreaks of diseases mainly in young day-old meat ducks with symptoms such as short beak,large tongue,and stunted growth,which is called"duck short-beaked dwarf syndrome"Compared to other parvoviruses in waterfowl,the disease is infected for a longer period of time and infects the host more extensively.According to survey statistics,the mortality rate of meat ducks infected with this disease is as high as 6%,and because of the shrinkage of the upper and lower beaks and the larger tongue,the diet of sick ducks is difficult,compared with healthy ducks,the weight loss rate of sick ducks is up to 40%,causing serious economic losses to duck farms.The researchers detected the organs of the diseased duck and isolated a plant with a homologous similarity rate of 92.7%-99.7%with the new goose parvovirus,It can be determined that duck-derived goose parvovirus is the main pathogen causing duck short-beaked dwarf syndrome.In this experiment,purified and concentrated duck-origin goose parvovirus(D-GPV)was used to immunize BALB/C mice,and hybridoma cell technology was used to fuse with myeloma cells with mouse splenocytes with a serum titer of more than1:100000,using blocking ELISA and indirect immunofluorescence technology screening for fused clones,hybridoma cells that secrete D-GPV monoclonal antibodies and generate high blocking rates were selected.The experimental results showed that the cell supernatant of a total of 7 hybridoma cells could specifically recognize D-GPV-infected DEF cells and obtain a blocking rate of more than 80%,which were named respectively 2F4、11F11、16D9、24G4、35G5、37D8、R9D11.The monoclonal antibody of the above hybridoma cell lines was prepared by the method of induced ascites in sensitized mice,and then the ascites prepared by the above seven hybridoma cells was comprehensively analyzed by blocking ELISA,IFA,Western Blot and virus neutralization experiments.The results of ELISA and IFA blocking of all ascites were consistent with the results of the corresponding cell culture supernatant,and both could specifically identify D-GPV-infected GEF cells,and all of them could obtain a blocking rate of more than 80%.The results of Western blot showed that the above seven monoclonal antibodies could recognize the structural proteins VP1,VP2 and VP3 of D-GPV.All but 37D8 have the ability to neutralize D-GPV,of which 2F4 has the strongest neutralization ability,and was selected as the target monoclonal antibody for the subsequent establishment of a blocking ELISA detection method.A D-GPV blocking ELISA antibody detection method was established using D-GPV as coated antigen,D-GPV positive serum as blocking antibody,and D-GPV monoclonal antibody as primary antibody.Through multiple detection and optimization of reaction conditions,the final conditions were as follows:the antigen coating was diluted at 1:3000;Block with 5%skim milk,37°C for 1 h;D-GPV-negative and positive serum was diluted 1:10,monoclonal antibody was diluted1:10~6,and HRPase secondary antibody was diluted at 1:4000;All serum and antibody incubation conditions were 37°C;TMB color rendering time is 10 min.Using duck D-GPV-negative serum as the serum to be tested,and laboratory-collected D-GPV-negative serum as negative control serum,The criteria for determining the method of blocking ELISA after calculation are:When PI≥21.13%,the serum samples were judged to be positive for duck-derived goose parvovirus antibody,negative when PI≤13.32%,and suspicious when PI ranged from 13.32%to 21.13%.The established blocking ELISA method was used to detect a variety of avian disease-positive serum to verify whether the method was specific,and the results showed that the method had a blocking effect on duck-derived goose parvovirus-positive serum.and did not block the positive serum of duck plague virus,duck tambusu virus,duck hepatitis 1 and 3 duck virus,H5 subtype avian influenza virus,H9 subtype avian influenza virus,duck reovirus,adenovirus type 2and duck egg reduction syndrome,indicating that the detection method has strong specificity.The sensitivity test results showed that when the positive serum of duck-derived goose parvovirus was diluted to 4056 times,the blocking rate was still greater than 21.13%,and compared with the IFA result,it was at least 32 times more sensitive,indicating that the detection method had better sensitivity.Through the repeatability test of the same batch and different batches coated with enzyme plates,the results showed that the coefficient of variation of the same batch of enzyme plates was between 0.85%-8.88%,less than 10%,and the coefficient of variation of different batches of enzyme plates was between 0.91%-8.42%,less than10%,indicating that the detection method had good repeatability.In order to verify the accuracy of the method in one step,24 clinical serum samples were tested by IFA and the established blocking ELISA,and the results showed that the results of IFA and blocking ELISA were consistent with the negative and positive controls,indicating that the established blocking ELISA method was suitable for clinical sample detection.In summary,a monoclonal antibody against duck-derived goose parvovirus was successfully prepared in this experiment,and a blocking ELISA method with strong specificity,sensitivity and reproducibility was successfully established under these conditions,which provided an effective detection method for the detection of duck-derived goose parvovirus antibody... |
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