Evaluation of specific chicken IgY antibody value developing diagnostic capture antibody ELISA kit against Foot and Mouth disease

Abstract

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 14, 21, and 28 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 6000–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, 119 previously confirmed samples (including 80 positive and 39 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 100% and 98%, 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.