Establishment And Application Of An Antigen-capture Enzyme-linked Immunosorbent Assay For Detecting Avian Reticuloendosiliosis Virus

Avian reticuloendotheliosis(RE)is a pathologic syndromes caused by reticuloendotheliosis virus(REV),which is characterized by lymphoid tissue and other tissues of chronic tumor formation.Day old chicks infected with REV often causes severe immunosuppression.If REV-infected chickens were immunized with avian vaccines,the protective effect of the vaccine would seriously reduce,and may cause failure of the vaccination.REV can be transmitted vertically through eggs and it is one of the viruses that need to be eradicated in breeders.Use of SPF chicken embryos infected with REV would gave rise to attenuated vaccines contamination.In recent years,epidemiological investigations shown that REV infection has been quite common in China,and REV contamination in vaccines has also been reported continuously.Therefore,REV monitoring is very important for the prevention and control of the disease.At present,the methods used for REV detection includes virus isolation combined with indirect immunofluorescence identification assay(IFA),reverse transcription polymerase chain reaction(RT-PCR)and real-time fluorescence quantitative(RT-q PCR).To date,virus isolation combined with IFA is the standard method for the detection of REV contamination in attenuated vaccines by Chinese Veterinary Pharmacopoeia.However,these assays have high requirements for the operation of testing equipment and personnel,and there are limitations in promoting these assays in enterprises.Therefore,a simple and rapid antigen capture enzyme-linked immunosorbent assay,AC-ELISA was established for detection of REV in this study.Firstly,according to the published REV IBD-C1605 genome sequence on Genbank,the primers to amplify the gp90 gene of REV were designed and synthesized using Primer Premier 5.0 software.The gp90 gene was amplified from the genome of REV IBD-C1605strain as the template.After cloning and sequencing,the purified product of gp90 gene was recovered and ligated to p ET-32a(+)to construct the prokaryotic expression vector,the induced protein was identified by SDS-PAGE and Western blot,and the rabbit polyclonal antibody against REV-gp90 protein was obtained by immunizing New Zealand female white rabbits with an antibody potency of 1:10~7.Then,rabbit anti REV-gp90 protein polyclonal antibody was used as the capture antibody,and the previously prepared mouse anti REV-gp90protein monoclonal antibody 1A12D was used as the detection antibody to establish the AC-ELISA assay.The optimal coating concentration of the multiple antibody was 1?g/m L,the optimal dilution ratio of the monoclonal antibody was 1:4000,p H 7.4 PBST as the coating solution overnight at 4°C,1%BSA as the blocking solution,and the optimal dilution ratio of1:10000 for the enzyme-labeled antibody.The specificity test showed that the established AC-ELISA in this study had specific binding reaction with REV,and had no cross-reaction with subgroup J avian leukemia virus(ALV-J),Marek's disease virus(MDV),avian hepatitis E virus(a HEV)and C4 Fowl adenovirus(FAd V-4).The limitation of this detection method was about 195 TCID₅₀units of IBD-C1605(REV).In order to explored the feasibility of the establishion of the AC-ELISA for vaccine contamination and other scenarios,different doses of REV were mixed into avian attenuated vaccine to artificially simulate the situation of REV contaminated avian attenuated vaccine in production.According to the requirements of the appendix of the Chinese veterinary Pharmacopoeia,the samples for passage of second generation after the contaminated vaccine were inoculated into cells,then the cell culture supernatant was detected by AC-ELISA and real-time fluorescence quantitative(RT-q PCR).The cells were fixed and detected by indirect immunofluorescence(IFA).The results showed that when the artificial simulated REV contamination doses were 1000 TCID₅₀,100 TCID₅₀and 10 TCID₅₀,the detection results of RT-q PCR,IFA and AC-ELISA were completely consistent.RT-q PCR had the highest sensitivity with 100%positive detection when the contamination dose was 1 TCID₅₀,while IFA and AC-ELISA detected 7/8 and 5/8 positives respectively,giving consistent results for the determination of vaccine contamination.In conclusion,rabbit polyclonal antibody against REV-gp90 protein was prepared,and an AC-ELISA for detecting REV was established,so it had good sensitivity and specificity.It shows a good application prospect in artificial vaccine REV contamination experiment,and provides more detection technology reserves for REV eradication and monitoring.