Pnpp substrate

HCV antigen-specific cross-reactive and transgene-specific IgG antibodies were measured in sera collected from immunized mice using 96-well plates coated overnight (at 4 °C) with antigens from HCV (core, NS3 or NS5), HIV (nef), EBOV (gp) at 1 μg/mL or sonicated Mtb H37 Ra (1 × 10⁶ cfu/well) in 1× PBS. The next day, after blocking with 1% BSA at room temperature for 1 h, serial dilutions of serum samples (starting from 1:100) were added to the 96-well plate in 3 replicates and incubated at room temperature for 2 h. After application of serum, anti-mouse IgG labeled with alkaline phosphatase (AP) (Southern Biotech, Birmingham, AL, USA) was added and plates were incubated for 1 h. Color was developed by adding PNPP substrate (Southern Biotech). Plates were washed with 1× PBST (1× PBS with 0.1% Tween-20) after each incubation step. Absorbance was read using a FluoStar Optima ELISA Reader (BMG Labtech GmbH, Ortenberg, Germany), and OD values from HCV antigen coated plates, corrected for background from OD values in SOD coated plates, were plotted in the graphs shown here.

Detection of IgE and IgG1 levels in the serum of naïve and infected mice was determined as follows: Purified mouse anti-IgE (clone R35–72, BD Biosciences, Franklin Lakes, NJ) or a commercial IgG1 ELISA kit (SouthernBiotech) was used for coating. As secondary reagents anti-mouse IgE-AP or IgG1-AP (SouthernBiotech), followed by development with pNPP substrate (SouthernBiotech) was applied. For detection of parasite-specific IgE or IgG1, a 10-20 μg/mL NES protein suspension (Supplementary Figure S2) was coated on 96-well polystyrene plates overnight (4°C), blocked with 3% BSA/PBS for 2 h and then incubated for 2 h with serum dilutions. Parasite-specific antibodies were determined using the secondary reagents described above. For Nb-LSA1a-specific ELISA, 96-well polystyrene plates were coated with a 10-20 µg/mL Nb-LSA1a suspension. Absorption was measured at 405 nm on a Multiskan FC photometer (Thermo Fisher) and blank wells were used for background subtraction.