FIG. 4.

Effects of quantity of bacteria on IgG binding to antigens of M. avium subsp. paratuberculosis analyzed by EVELISA. After M. avium subsp. paratuberculosis was suspended in 80% ethanol to adjust the cell density to 4,000 μg of M. avium subsp. paratuberculosis/50 μl, agitated by vortex at room temperature for 30 seconds, and centrifuged, the supernatant was collected. After the supernatant was serially diluted in 80% ethanol, 50 μl of the diluted supernatant was inoculated into each well of a 96-well microtiter plate and allowed to evaporate at room temperature overnight before being used in an ELISA. After incubation of the wells with NC (hatched bars) or PC (solid bars) serum samples, IgG binding was detected by using biotin-labeled anti-bovine IgG antibody and HRP-labeled streptavidin. Each bar represents the mean ± standard deviation (n = 3) of absorbance at 415 nm. This experiment was repeated twice with similar results.