FIG. 3.

Effects of duration of agitation on IgG binding to antigens of M. avium subsp. paratuberculosis and M. avium subsp. avium by EVELISA. Eighteen milligrams of M. avium subsp. paratuberculosis (solid circles) or M. avium subsp. avium (open circles) bacilli were suspended in 600 μl of 80% ethanol, agitated by vortex at room temperature for various intervals, and centrifuged, and 50 μl of the supernatant was inoculated into each well of a 96-well microtiter plate. After evaporation at room temperature overnight, the plate was used in an ELISA. After incubation of the wells with NC (dotted line) or PC (solid line) serum, IgG binding was detected by using biotin-labeled anti-bovine IgG antibody and HRP-labeled streptavidin. Each point represents the mean ± standard deviation (n = 3) of absorbance at 415 nm. This experiment was repeated twice with similar results.