Development And Nreliminary Annlication Of Double Antibody Sandwich ELISA Kit For Detection Of Porcine Delitacoronavirus

Porcine deltacoronavirus(PDCoV),a novel swine enteric coronavirus,can cause severe vomiting,diarrhea and dehydration in piglets.In 2012,it was first detected in fecal samples of pigs in Hong Kong,and outbreaks of PDCoV were reported in pig farms in several states in the United States in early 2014.Subsequently,PDCoV was detected in pigs from many countries.PDCoV outbreaks were also noticed in swine herds from Henan,Hebei,Anhui,Jiangsu,Jiangxi and some other provinces of China,and the detection rate of PDCoV is as high as 20%in China.Especially in diarrhea piglets,the detection rate of PDCoV is high up to 60%,causing huge economic losses to pig industry.Currently,there is no effective vaccine or commercial detecting kits to prevent and detect PDCoV.Thus,it is urgent to develop a rapid,accurate and efficient diagnostic kit.In this study,monoclonal antibody and polyclonal antibody of PDCoV N protein prepared in our laboratory were used to establish a double-antibody sandwich ELISA antigen detection kit,and its clinical application was evaluated.Experiments are as follows:1.Preparation of monoclonal antibody against PDCoV N proteinIn order to obtain high-purity PDCoV N protein monoclonal and polyclonal antibody,ammonium sulfate precipitation was used to purify PDCoV N polyclonal and monoclonal antibody,respectively.Indirect ELISA and direct ELISA,SDS-PAGE and Western blot were used to identified the purity of antibodies.The results showed that the ELISA antibody titer of high-purity PDCoV N protein monoclonal and polyclonal antibody are 4×105 and 6.4×104,respectively.The purified polyclonal antibodies were labeled with horseradish peroxide(HRP)by sodium periodate method.These antibodies provided the material basis for the development of double-antibody sandwich ELISA kit.To establish detection PDCoV antigen double antibody sandwich ELISA,the purified monoclonal antibody was used as coating antibody,and HRP labeled polyclonal antibody was used as detection antibody.Reaction conditions,such as concentration of coating antibody,the type of blocking solution,concentration of blocking solution,incubation time,concentration of detection antibody,incubation time of detection antibody and incubation time of chromogenic solution time were optimized,and PDCoV double antibody sandwich ELISA was established.The optimal reaction conditions are as follows:the coating concentration of N monoclonal antibody was 100 ng/well,the optimal coating condition was 4℃ overnight,the blocking solution concentration was 1%BSA 37℃for 2 h,the antigen incubation time was 1 h,the optimal working concentration of HRP-labeled polyclonal antibody was 1:8000,the incubation time of HRP-labeled antibody was 50 min,and the color development time of TMB solution was 10 min.The double antibody sandwich ELISA assay was used to detect 50 PDCoV negative clinical samples(feces,intestinal tissue and small intestine contents)of pigs.The critical value of the assay was determined as OD450=0.196 for negative and positive PDCoV samples,taking the mean value of the sample plus 3 times of the standard deviation(X+3SD)as the critical value.The method was used to detect some swine common virus pig,like epidemic diarrhea virus(PEDV),pig infectious gastroenteritis virus(TGEV),pig sappi virus(PSV),pig pseudo rabies virus(PRV),pigs,parvovirus(PPV)and pig reovirus(MLRV),and all of them were negative,which demonstrated that the method had good specificity.The coefficients of variation of both the in-batch and inter-batch repeated tests were less than 7%,indicating that the method had good repeatability.Based on the above experiment,a double antibody sandwich ELISA antigen detection method was established,providing a foundation for the later preparation of double antibody sandwich ELISA kit.2.Assembly and evaluation of PDCoV antigen detection kitIn order to assemble the established PDCoV double-antibody sandwich ELISA antigen detection method into the detection kit,the required reagents and materials were prepared,optimized,and the finished kit was assembled,and the kit was evaluated.Different antigens were detected by the assembled kit,and the results showed that the kit only responded to PDCoV,but did not respond to PEDV,TGEV,PSV,PRV,PPV and MLRV,indicating that the specificity of the kit was good.PDCoV(TCID50 106/0.1 mL)was diluted 10 times and tested with the assembled kit.The results showed that the test result was still positive when diluted to 10-4,indicating that the detection line of the kit for the virus was 100 TCID50.Then different batches and kits assembled in the same batch were used for detection,and the coefficient of variation was small,indicating that the kit had good stability and good reproducibility.Further,the kit was used for parallel detection of 20 disease materials(10 negative and 10 positive samples of PDCoV)with the established common PCR method by the laboratory.The results detected by the kit were consistent with the results of common PCR detection,and 10 positive samples were detected in both cases.In this section,the established double-antibody ELISA antigen detection method was successfully assembled into a detection kit,which provided a strong technical support for the epidemiological investigation of PDCoV infection in clinical pigs.3.Preliminary application of PDCoV antigen detection kitTo further understand the applicability of the kit for clinical disease detection,virus of different titers were mixed with PDCoV negative pig samples,and PDCoV double antibody sandwich ELISA antigen detection method was used to detect.The content of PDCoV virus was made as X axis,while OD450 value was made as Y axis.Standard curve was drawn,and the regression equation was Y=19.653+44.38*X.The standard curve can be used for the quantitative determination of PDCoV in clinical porcine diarrhea samples.At the same time,PDCoV was used to infect ST cells,and culture supernatant was collected at different infection time.The kit was used for detection,and PDCoV infection time was made as X axis,OD450 value was made as Y axis.Growth curve was drawn.The results showed that when the cells were infected for 36 h,OD450 value reached to the peak.This growth curve was consistent with the growth curve based on TCID50,showing that the kit can be used in the laboratory PDCoV on cell proliferation.Because PDCoV has extensive tissue addiction,we further detected pig tissues infected with PDCoV by this kit.The results showed that in all of the tissue samples infected PDCoV,the spleen,kidney,ileum and rectum had a high level of virus.This it is consistent with the results of RT-PCR.Finally,the kit was used to detect the antigen of 186 samples of swine diarrhea materials collected clinically.Among them,31 samples were PDCoV positive,with a positive detection rate of 17%.This provided important information for understanding the prevalence of PDCoV in pigs in Henan.In conclusion,a double-antibody sandwich ELISA kit for detecting PDCoV antigen was successfully developed in this study.This kit has high specificity and good reproducibility,which can not only be used for clinical sample detection,but also for laboratory research,providing technical support for clinical diagnosis of PDCoV and epidemiological investigation of PDCoV.