Establishment Of An ELISA For Detecting Antibody Against Avian Reticuloendotheliosis Virus And Its Application

Avian reticuloendotheliosis is one of the important tumorigenic and immunosuppressive diseases in poultry.It shows a kind of pathological syndrome in chickens,ducks,geese and other birds caused by the reticuloendotheliosis virus(REV)of the retroviridae.Infection of REV can cause tumor,dwarf syndrome,glandular gastritis,acute reticulocyte tumor,lymphoid tissue and other tissues to develop chronic tumors.When the virus infects major immune organs such as thymus and bursa of Fabricius,it causes these tissues and organs atrophy,which decrease the immunity of poultry and even lead to immunosuppression,and facilitate the secondary infection of other viruses and bacteria.RE is the third avian viral tumor disease except Marek's disease virus(MDV)and Avian leukosis virus(ALV).It is one of the most serious chicken diseases in recent years.With the rapid development of the poultry industry,the immunosuppressive diseases is paid more attention.In particular,REV has brought huge economic losses to the poultry industry in China.The rapid diagnosis methods for REV infection will be very useful in the REV control.There are currently kits for detecting REV antibodies abroad,but they were too expensive to use in the farms in China.In this thesis,we will establish a more specific and effective method for detecting antibodies to REV1.Establishment of an ELISA with synthetic peptide for detecting antibody to REVIn this study,we used DNA Star software to comprehensively analyze the secondary structure,accessibility,hydrophilicity and plasticity of the amino acid sequence of the REV envelope protein gp90,and selected four peptides(REV-gp90-pl/p2/p3/p4).These peptides were coated to the ELISA plates and reacted with positive REV polyclonal antisera.The results showed that all peptides reacted with REV polyclonal antisera,but the reactivity was different.Among them,REV-gp90-peptide4 showed better reactivity with the positive REV sera.Combined with that we analyzed the epitopes of REV monoclonal antibodies prepared in our laboratory,we synthesized the peptide(REV-gp90-p5)which contained both sequences of REV-gp90-p4 and epitopes recognized by monoclonal antibody.The peptide REV-gp90-p5 was found much better to react with the REV polyclonal antisera.Subsequently,we compared the naked peptide REV-gp90-p5 and the peptide ligated with KLH to react with the positive REV sera.We found that the naked peptide as the antigen coated to the ELISA plates showed more specific and reactivity.Peptide REV-gp90-p5 could be good candidate antigen for REV antibody detection.Coated plates with the peptide REV-gp90-p5,the optimal ELISA condition was determined by different factors including the optimal coating concentration of the antigen peptide,the blocking solution and blocking time,the primary antibody concentration,the incubation time of the primary antibody and the HRP-labeled rabbit anti-chicken IgG,the color development time of the TMB substrate.The specificity and the reproducibility of the mothed were tested according to established ELISA procedures.Cross-reactivity demonstrated that the peptide ELISA method established in this study only reacted with REV polyclonal antisera,but not with the sera to other avian viruses(ALV-A,ALV-J,CAV,MDV,NDV).In addition,the variation coefficient between the batch and the batch of the method was less than 10%,indicating that the method had good repeatability.2.Preliminary application of peptide ELISA Kit for detection of REV antibody38 sera samples of chickens challenged with REV in the laboratory was evaluated for the peptide ELISA.The results were verified by indirect immunofluorescence at the same time.Among them,10 positive samples were detected by the peptide ELISA,and 15 samples showed positive in IFA,including 10 samples which were positive in peptide ELISA.The sera samples from a chicken farm in Jiangsu Province were detected by the peptide ELISA.154 positive samples were found in 160 random samples.Only 14 positive samples could be found by IDEXX REV ELISA kit.However 132 in 160 samples showed positive in IFA analysis at the same time.Take together,the peptide ELISA established in this study was higher specificity and sensitivity than the commercial kit,which lays a foundation for the diagnosis of avian reticuloendotheliosis in China.