Fig. 1

Schematic illustration of the cELISA immunoassay for the detection of H7 antibody. A 96-well microtiter plate coated with purified inactivated H7 whole-virus antigen is prepared. The mixture of primary competitive mAb and unknown serum sample is diluted to optimal concentration and added to the antigen coated plate. After an incubation period of 1 h, the plate is washed to remove unbound antibody and a secondary antibody labeled with horseradish peroxidase specific for targeting the competitive mAb is added. After another incubation of 30 min, the plate is washed again to remove unbound secondary antibody and then the substrate is added. If the serum sample is negative, the enzyme on secondary antibody which binds specifically the murine competitive mAb linked on the antigen-coated plate will catalyze the substrate and result in a color change. In contrast, the antibody in positive serum sample will compete with competitive mAb for capturing the same epitope of coated antigen. Subdued color change will be observed due to the species-specific property of the secondary antibody. The color intensity resulting from antigen bound mAb is inversely proportional to the amount of epitope-specific antibody present in test serum