Development Of An Indirect ELISA Kit For Detection REV Antibody Based On Antigen Epitopes

Reticuloendotheliosis(RE)is an avian tumorous disease caused by reticuloendotheliosis virus(REV),including acute reticuloma,stunting syndrome,chronic lympho-adenoma and a series of pathological syndromes.REV damages gravely organism's immune function by targeting lymphocytes or reticuloendothelial cells,which causes organism's cellular immunosuppression and humoral immunosuppression,resulting in secondary infecting other pathogens.At present,there are not effective control measures for RE,we can only eliminate RE positive chickens by antigen and antibody detection.In order to accurately know the degree of REV infection,reduce poultry farming losses.The best detection method is ELISA.The ELISA kits have advantage of simple operation high sensitivity and strong specificity.Because REV is highly variable,the existing kits not only can't detect accurately but they are also expensive.The existing kits are unfit for large-scale clinical detection.Therefore,it is great significant to research rapid effective and lower price ELISA kit for detection REV antibody.REV Gp90 protein is on the surface of REV envelope protein,containing linear epitopes and conformational epitopes.REV Gp90 protein is immunodominant protein which can inspire hosts to produce antibodies.Tandem-epitopes contain more epitope information,can provide more rapid and more accurate detection.In this subject,according to REV molecular virology and immunology,we analysis and forecast the epitopes of REV-Gp90 protein,connect conservative epitope gene.The tandem epitope gene not only is high conservative,but it has also great antigenicity.The gene fragments synthesized by biotechnology company were linked with the expression plasmids pET-32 a and then transformed into E.coli BL21 strain,conditions for optimizing induced expression,SDS-PAGE indicated that the target protein of 36 KD in size was expressed and the expressed fusion protein existed in the pattern of inclusion body.After purification and renaturation of large amount of expressed fusion protein,sensitivity and specific were analyzed by ELISA and Western Blot.The results showed that the expressed fusion protein was great sensitive and specific.The purified and renatured recombinant protein was used to coat the 96 pores ELISA board as antigen,which was incubated overnight at 4?.A series trials were performed for the groped optimal reaction conditions of the indirect ELISA and the results were obtained as follow: the best coating concentration of antigen was 20?g per Milliliter;the best dilution times of serum were 200 times and the best reaction time was 1.5h;the best coating buffer was CBS;the best blocking buffer was 5% skim milk powder and the best blocking time was 2h at 37?;the ideal dilution times of enzyme-linked secondary antibodies were 8000 times and the best reaction time was 45min;the optimal developing time was 10 min.Through the detection of 50 SPF chicken serum samples,the critical value of optical density at 450 nanometers wavelength was 0.269;Through the detection of 4 REV positive serum samples,the coefficients of variation of the reproducibility of intra-assay and intra-assay were 3.49%-6.36% and 6.10%-9.41% respectively,which were less than 10%.According to the serum test of ALV-J,MDV,GyV3,CAV,NDV,the results showed that there were no cross reactions with other serums.So,the established indirect ELISA method with the advantages of good repeatability and specificity offered a foundation to REV diagnostic kit.This kit and IDEXX kit tested a little of clinical samples which come from the chicken farms in Shandong province separately,the positive rates of this kit and IDEXX kit were 47.97% and 41.89% respectively;and compared with IDEXX kit,ours is more sensitive.A lot of chicken serums from the region of Shandong province were detected by this method.The results showed that the positive rate was 45.5%.So,the positive rate of REV is high in Shandong province,REV infections need to be solved.The subject built a foundation for REV antibody rapid diagnosis kits.The kit will be more available for molecular epidemiology investigation of REV.The technical assistance was provided to support the development of poultry enterprise.