Sigmafast p nitrophenyl phosphate substrate

A standard indirect enzyme-linked immunosorbent assay (ELISA) protocol was used for detection of the antigen-specific IgG in the immunized mouse sera. Briefly, Nunc MaxiSorp 96-well plates were coated with 5 μg/ml of purified His₆-ESAT-6-Pet-BP protein or S. Typhimurium LPS (Sigma) in 0.1 M carbonate buffer, pH 9.6 (overnight, 4°C). The plates were washed three times in wash buffer (PBS, 0.05% Tween 20), incubated with PBS-1% bovine serum albumin (BSA) for 1 h at 37°C, and washed as described above. The plates were incubated for 1 h at 37°C with 3-fold serial dilutions of mouse sera in dilution buffer (PBS, 0.05% Tween 20, 1% BSA), followed by three washes with wash buffer. The plates were incubated with a relevant AP-conjugated secondary antibody in dilution buffer for 1 h at 37°C (goat anti-mouse IgG–AP [1/10,000 dilution; Sigma] for protein- and LPS-specific total IgG or goat anti-mouse IgG1–AP, goat anti-mouse IgG2a–AP, or goat anti-mouse IgG2b–AP [1/2,000; Southern Biotech] for protein-specific IgG isotypes). The plates were washed as described above and developed using SigmaFAST p-nitrophenyl phosphate substrate (Sigma); 405-nm readings were taken at regular intervals by use of a Labsystems Multiscan MS plate reader. The IgG titers were estimated using the specified threshold OD method.

Sera were obtained by cardiac puncture post-euthanasia one day after the final (7^th) allergen challenge. Serum was separated from whole blood by centrifugation at 1,500×g for 5 min after allowing coagulation for 30–60 min at room temperature. Serum samples were stored at −20°C until analyzed. OVA-specific IgE antibody levels were determined by ELISA. Serially diluted serum samples were incubated on microtiter plates coated with 20 µg/ml OVA. IgE antibodies were detected with alkaline phosphatase conjugated anti‐mouse IgE (Rockland) and Sigma Fast™ p‐nitrophenyl phosphate substrate and quantified by measuring the optical density (OD) at 405 nm. The antibody concentrations are presented as endpoint titers defined as the reciprocal of the highest serum dilution producing an OD above background of naïve sera. The cutoff value is determined as the OD (mean+2 standard deviations) of the corresponding dilution of naive sera (34 (link), 35 (link)).

ELISA plates (96 well, Dynex, Chantilly, USA) were coated with recombinant CbpF (rCbpF; 3 pmol) in carbonate buffer pH 9.5 by overnight incubation at 4°C. Wells were blocked with 3% w/v BSA (diluted in PBS-T; PBS with 0.05% v/v TWEEN® 20, Sigma, Gillingham, UK) for 1 h. Triplicate wells were incubated with the relevant CEACAM-Fc construct diluted in 1% BSA (w/v in PBS-T) for 1 h at RT, where the final concentrations are detailed where required. Following a minimum of three washes with PBS-T, the AP-conjugated anti-IgG Fc secondary antibody was then incubated for a further hour before three more wash steps. Plates were developed using SigmaFast™ p-Nitrophenyl phosphate substrate (Sigma) and absorbance was measured at 405 nm (A405). Data underwent background subtraction and standardisation for graphical presentation.