Horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) diluted 1:7500 in 2.5% BLOTTO was then added to all wells and incubated for 1 h at room temperature. All reactions were detected using TMB Microwell ELISA substrate (Kirkegaard and Perry Laboratories, Gaithersburg, Md.). The substrate was allowed to react for 10 min at room temperature, and then the reaction was stopped by adding an equal buy Target Selective Inhibitor Library volume of 1 M H3PO4. Optical densities (OD) at 450 nm were determined with a Spectra Max 190 Plate Reader (Molecular Devices, Inc., Palatine, IL). End point titer values were determined as the reciprocal

of the highest dilution that had an absorbance value greater than or equal to 0.1 above the background value. End point titers

of antigen-specific antibody responses were determined for each individual animal. The geometric mean titers (GMTs) were determined for each group of mice. Standard errors were calculated for log-transformed titers. Statistical analyses were performed with SPSS version 10.0 for Windows (SPSS, Inc., Chicago, IL). Antibody titers or levels of antibodies between groups were compared by using the Kruskal–Wallis test followed by the Mann–Whitney U rank sum test. Animals immunized with 100 μg of KLH and either a 6 or 20 μg dose of full-length NSP4 as an adjuvant. Both doses of NSP4 exhibited a statistically significant (p = 0.04 Mann–Whitney U Test) 6-fold increase in KLH-specific serum IgG titers (GMT = 72,839) compared to the Compound Library purchase group of mice receiving KLH alone (GMT = 11,494) ( Fig. 1A) and so the lower dose was chosen for future experiments. In addition, those animals also showed significantly higher (p = 0.05, Mann–Whitney U Test) (>30-fold increase) KLH-specific fecal IgA antibody responses (GMT = 2302 ng/ml) compared to the antigen alone group (GMT = 71 ng/ml) ( Fig. 1B). Serum IgG and fecal IgA specific antibody levels decreased approximately 20-fold and 30-fold, respectively, when mice were inoculated with KLH co-administered with NSP4 compared to mLT (GMT; IgG = 1,447,738;

IgA = 74,083 ng/ml). those When full-length NSP4 was given with TT (10 μg), it enhanced serum TT-specific total immunoglobulin (GMT = 11,143) responses (17-fold increase) to a greater extent than to those seen with KLH, when compared to the antigen alone group (Fig. 2A). However, in contrast to the enhanced fecal antibody responses observed when KLH was given as the antigen, there was no significant increase (p > 0.05, Mann–Whitney U Test) of TT-specific fecal antibody response in the group of animals that received NSP4 and TT as compared to TT alone ( Fig. 2B). In contrast to the observations with KLH and TT, NSP4 did not enhance serum antibody responses to OVA (GMT = 28,963) compared to the antigen alone (GMT = 15,521) group (Fig. 2C). However, a significantly higher level (11-fold increase; (p = 0.