Establishment And Primary Application Of Doubleantibody Sandwich ELISA Diagnostic Kit For Detecting Hemagglutinating Encephalomyelitis Virus

Porcine hemagglutinating encephalomyelitis(PHE)is a highly contagiousdisease in piglets caused by porcine hemagglutinating encephalomyelitis virus(PHEV).The main clinical features are vomiting,exhaustion and obvious neurological symptoms.Piglets,specially 1 to 3 weeks after birth,are the most susceptible group,but the adult pigs could rarely be infected.Piglets all died in the end if infected.There is still no specific drugs and vaccine against the virus,as a result,the faster we diagnose the disease,the smaller risks bring to farmers.At present,there are a variety of diagnostic methods for PHE,but most of them more or less have some drawbacks,which may impede the diagnosis of disease.In the study,based on the preparation of the monoclonal antibody against PHEV N protein and its polyclonal antibody,a double-antibody sandwich ELISA diagnostic kit was successfully assembled,providing accurate and timely diagnosis in the outbreak of PHE.The content mainly divides into following parts:1.Construction of expression vector of N gene and purification of N protein.According to the sequence of N protein,the primer of N protein can be designed via Primer 5.After cloned into a prokaryotic expression vector p ET-28 a,we can acquire a recombinant plasmid p ET-28a-N.With the inducing of IPTG,the fusion protein N(about 55KDa)can be highly expressed in Escherichia coli BL21.Fusion protein could interact with the pig serm conteining antibody against PHEV,according to the identification of SDS-PAGE and Western-blot.Through the purification via Ni-NTA affinity chromatography and the determination of the concentration of recombinant N proteins,the average concentration of fusion proteins were 1659.3ug/m L?1139.1ug/m L and 726.8ug/m L respectively.2.Preparation of monoclonal antibodies against PHEVBefore the fusion between spleen mononuclear cells and murine myeloma cells(SP2/0),Balb/c mice should be immunized subcutaneously with purified recombinant N proteins.In the following time,HAT medium killed all kinds of cells but hybridomas,which provided four single-secreting anti-PHEV Mc Ab after screening by ELISA in the end.The four single-secreting anti-PHEV Mc Ab were named:2H2(Ig G1)?2A1(Ig G1)?1E2(Ig G2a)and 4D4(Ig G1)respectively.According to the ELISA,these 4 Mc Ab titers ranged from 1:12800 to 1:51200.And the result of Western-Blot showed that except the 2A1 identified non-structural protein N,others all just only could recognize the protein HE.By indirect ELISA and IFA,4 Mc Abs were able to react with PHEV specifically.3.Preparation of PHEV negative and positive control sera and polyclonal antibodyThe positive control sera were collected from healthy rabbits which were injected with the PHEV-67 N subcutaneously at first.After purification and detection of positive serum,polyclonal antibody of PHEV can be acquired.The negative control sera came from healthy rabbits which were free of PHEV.Also,ELISA results revealed that the negative sera were free of PEDV?PRV?TGEV and PCV2.4.Establishment of the method of the PHEV antigen capture ELISA,and the assembly and validation of the ELISA kitThe double-antibody sandwich ELISA detection method was established with the PHEV polyclonal antibody,coated with the enzyme labeled plate and the PHEV Mc Ab,as the detection antibody through a series of Optimization programs:the optimal concentration of Mc Ab were 0.5?g/m L(37?,1h);The dilution of polyclonal antibody was defined as 1:8000(4?,12h);The best sealing buffer was 5%BSA(37?,2h);The TMB substrate should be added and incubated at room temperature for about 15 min.The specificity test showed that the ELISA detection had no cross-reactivity with other related pathogens of swine diseases.The lowest concentration of PHEV could detect of the ELISA detection was 63.12 ng/m L.Based on the established ELISA method,the assembly and quality evaluation of kit was completed.It suggested that The variation coefficient intra-assay and inter-assay were 4.8% to 9.6% and 3.2% to 8.9%,respectively,proving the repeatability was qualified.There was no obvious variation of detecting results after an assay which put the ELISA plate coated polyclonal antibody in 37? for 6 days.The result revealed that the ELISA kit stored at 4 ? could be maintained for about 6 months.The diagnostic sensitivity,specificity and accuracy of the PHEV-N ELISA kit all were 90% above,compared with the RT-PCR on serum samples.This study successfully established a double-antibody sandwich ELISA detection method and completed the assembly of ELISA kit and quality evaluation,which is of great significance for the nationwide PHE diagnosis and epidemic surveillance.Results showed that the sensitivity,repeatability and stability of the ELISA kit were all qualified.