Establishment and application of indirect ELISA method for the detection of pigeon circovirus antibodies
In order to establish an indirect ELISA method for detection of pigeon circovirus(PiCV)antibodies,PiCV Cap gene fragment with a size of 828 bp was cloned into pGEX-4T1 vector,and the solu-ble Cap recombinant protein was obtained by low temperature induction,which was verified by Western-blot and found to react specifically with both anti-GST tag antibodies and anti-Cap protein antibodies.The purified Cap recombinant protein was used as coating antigen,an indirect ELISA method for detec-tion of PiCV antibody was established by optimizing each reaction condition through checkerboard titration method.The results showed that the optimal antigen coating concentration was 0.25 mg/L,5％BSA as a blocking solution,incubation at 37 ℃ for 2 h,the optimal dilution of the serum to be tested was 1∶800,the incubation time was 45 min,the optimal dilution of rabbit anti-pigeon IgG-HRP secondary an-tibody was 1∶8 000,the incubation time was 30 min,and the optimal color development time was 15 min.When the serum sample D450≥0.341,it was judged as positive.The positive serological results of pigeon adenovirus,pigeon herpesvirus,pigeon paramyxovirus type Ⅰ and rotavirus were negative,and the posi-tive serum diluted to 1∶12 800 could still be detected,with intra-assay and inter-assay coefficients of variation of less than 10％.The coincidence rate of indirect ELISA method with conventional PCR method was 88.2％.The indirect ELISA method was used to detect 86 clinical serum samples,and the posi-tive rate was 17.4％.The indirect ELISA method is highly specific,sensitive and repeatable,which pro-vides technical support for the detection of antibody levels after PiCV vaccine candidate immunization andPiCV epidemiological investigation.
国家科技期刊平台
NSTL
万方数据
