Establishment and application of immunoperoxidase monolayer assay for detection of the antibody against grass carp reovirus HZ08

In order to develop an immunological method for detection of the antibody against grass carp reovirus HZ08, using the cell culture plate which infected with the the GCRV HZ08 as the antigen, and serum collected from the vaccine immune grass carp used as the antibody, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibody against grass carp reovirus HZ08 based on the optimized reaction condition.The specificity, sensitivity, reproducibility and using for clinical application of IPMA were evaluated, and compared with that of ELISA.The results showed that in the optimized IPMA, the virus was diluted at 1×10³ copies/μL after 72 hours, HRP-IgG was diluted at 1:1000, and grass carp serum samples were diluted at 1:100, there was no cross-reaction with serum from grass carp against other pathogeny. The sensitivity test showed that the positive serum could be detected at 1:800. Reproducibility tests proved that the established IPMA had good reproducibility. The IPMA method had 95.7% positive coincident rate and 87.5% negative coincident rate with ELISA method. The results of the IPMA for detection of the immunized grass carp serum showed that the antibody titer reached peak value in the 2nd week post-inoculation.A total of 126 field serum samples were examined using this method, the average positive rate was 72.2%. The IPMA is a highly specific, sensitive, and stable method with high repeatability, and it offered a convenient tool for the epidemiological investigation, vaccine immunization effect assessment, and antigen and antibody detection.