Fig. 4
a Antibody titer of the anti-rLTB.Etx40–62 antisera against LTB and Etx. ELISA was carried out to determine the LTB-specific and Etx-specific antibody titers in the anti-fusion protein antisera by coating the respective proteins (500 ng/well) as target. Anti-fusion protein antisera (1:2000) was then added followed by the addition of secondary anti-mouse HRP-conjugated antibody. The color was developed by the addition of the substrate OPD and the absorbance was measured at 490 nm. Statistical difference (p value) was calculated using ordinary two-way ANOVA as compared to preimmune serum (PI). b Immunoblot analysis of the rEtx with the anti-rLTB.Etx40–62 antisera. Purified rEtx (lane 1) was immunoblotted with anti-rLTB.Etx40–62 (1:5000 in 1× PBS) antisera. Distinct immunoreactive band indicates that the antibodies present in the anti rLTB.Etx40–62 antisera are able to efficiently crossreact with rEtx. Lane M indicates migration of protein molecular weight (kDa). c Antigen-specific antibody isotyping of the anti-rLTB.Etx40–62 antisera. Different antibody isotypes in anti-rLTB.Etx40–62 antisera (1:10,000 in 1× PBS) collected on 28 days post immunization were analyzed by ELISA using biotinylated isotype specific anti-mouse IgG1, IgG2a and IgG2b (1:5000) secondary antibodies. Pre-immune (PI) sera collected prior to immunization was included as control. Statistical difference (p value) for each isotype in the anti-fusion protein antisera was calculated using Student’s two-tailed t-test as compared to that measured in preimmune serum (PI). **p ≤ 0.005; ***p ≤ 0.001