Research On Competitive Elisa Kit For Fmdv Nsps Antibody Test

Object In order to enhance the specificity and sensitivity of diagnostic reagents for FMD,a recombinant antigen and hyperimmune serum were developed with NSPs(3ABC) for the competitive ELISA kit to distinguish the infected animals from vaccinated and FMDV free animals . Materials and Method 1.NSPs-3ABC Clonnig and Expression: The 3ABC fragment was amplified with the RNA template of FMDV.After been purified, the target fragment was cloned into the BamHI and HindIII restriction sites of the plasmid pET30c(+) as pET-A-3ABC. At the same time. the purified fragment was gene modified and cloned into the same restriction sites of the plasmid PinPiontXa-1 as pPiont-B-3ABC.The expression production was then assessed by SDS-PAGE, ELISA. 2. Competitive ELISA Development:The titres of antigen, negative and positive serum, hyperimmune serum and HRP-denkey anti pig guinea was test.and assay condition was optimized. A formaula was developed to calculate the PI of samples and standards was confirmed.3.The specificity, sensitivity and reproductivity of the reagents producing protocols were studied with background known sera from reference laboratory and clinic samples in random. Positive detection rate, negative detection rate and C.V% in a batch or batches were calculated. 4.Reagents store condition and shelf life was tested following a serials tests with each component and a kit stored in different temperature. Result The expressed production was used as the antigen in ELISA with high specificity and allowed the differentiation of infected animals from vaccinated and free ones.It was shown that the optimal concentration of 3ABC protein for coating plate is 1:320, the dilution of negative and positive serum, hyperimmune serum sample and HRP-denkey anti pig guinea was 1:40,1:5000 and 1:4000 respectively.and the PI of samples below 40%were considered as negative,PI between 40% and 50% as ambiguous and PI above 50% as positive. It was also shown that the assay can detect the antibodies induced by virus after infected in 7 days,the specificity and sensitivity is high as 98.15% and 90.67%.and the test consistency of this assay is 91.2% with UBI ELISA kits for FMDV. By the way, the kit can be stored in 2-8℃for a year. Conclusion The results revealed that the competitive ELISA can differentiate the antibodies induced by virus infection from those induced by vaccinated, with good specificity and high sensitivity for the detection of FMDV 3ABC antibody in serum, and the potential use of reagents produced in this study is obvious.