Preliminary establishment and application of a blocking ELISA based on CD2v protein of African swine fever virus
In order to establish a blocking enzyme-linked immunosorbent assay(ELISA)method for specific detection of CD2v antibodies against African swine fever virus(ASFV),CD2v eukaryotic protein was used as coating antigen,and a monoclonal antibody against the CD2v protein of ASFV was used as blocking antibody.After optimization of the reaction conditions,sensitivity and specificity test,a specifically blocking ELISA method was developed.The results showed that the optimal antigen coating concentration was 20 ng/well,the optimal serum dilution was 1∶1,and the optimal dilution of the enzyme-labelled antibody was 1∶10 000.The prelimary judge criteria of blocking ELISA method was as follows:the cut off values of average percentage inhibition(PI)≥41.23％was taken as CD2v antibody positive result,the PI value＜41.23％was CD2v antibody negative result.The specificity test showed that this method had no cross-reactions with positive sera with pseudorabies virus,porcine circovirus 2,porcine reproductive and respiratory syndrome virus,porcine parvovirus and classical swine fever virus,respectively.The sensitivity test showed that a 128-fold dilution of positive serum can be detected by this method.The intra-assay and inter-assay variation coefficients were both less than 10％.The coincidence rate of this method to 64 pig sera was 96.88％.In conclusion,the blocking ELISA based on ASFV CD2v protein established in this study can be applied to the identification of low-virulent ASFV strain with CD2v deletion combining with the serological diagnosis method of p30 protein and other antigens of ASFV.This method provides an effective detection technology for the epidemiological investigation and epidemic monitoring of low-virulent ASFV strain with CD2v deletion in China.
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