The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the ELISA. Diagnostic tests with high sensitivity is necessary both to distinguish infected vaccinated animals and disease control programs for identifying the carrier animals. To detection of FMD virus, the current strategies are mainly based on capture antibody (sandwich) ELISA test. Applying laying pullets as animal bioreactor for production specific egg yolk antibody (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. In the present study, we aimed to produce a concentrated and purifies
IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized by inactivated FMDV serotype virus and then, on days ۱۴, ۲۱, and ۲۸ following vaccination, the eggs and sera were collected and then, the
IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using polyethylene glycol ۶۰۰۰–ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography and dialyzed. The purified
IgY concentration estimated by Bradford assay, confirmed it presents by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving optimum concentration of antigen/antibody (sera) in sandwich ELISA, checkerboard titration test was setup base on indirect ELISA results. Eventually, ۱۱۹ previously confirmed samples (including ۸۰ positive and ۳۹ negative) by both Real time PCR (quantitative PCR, qPCR) and with a commercial ELISA kit, used for evaluation of sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit was ۱۰۰% and ۹۸%, respectively. According to this, the present developed capture ELISA kit based on
IgY has high sensitivity and specificity for FMD virus detection and it could be used in future as both commercial detecting and serotyping applications.
