Serological analysis of B. pertussis-specific antibodies.

Vaccinated and challenged mouse serological responses to RTX (9) and PT (LIST Biologicals) were determined by enzyme-linked immunosorbent assays (ELISA). High-binding 96-well ELISA plates were coated overnight at 4°C with 50 μl of purified RTX or PT in PBS at a concentration of 1 μg/ml. IgG2a monoclonal m1B7 (63) (anti-PT) and IgG2a monoclonal M1H5 (9) (anti-RTX) were obtained from Jennifer Maynard. Plates were washed with PBS plus 1% Tween 20 (PBS-T) and then blocked with 5% milk in PBS-T for 2 h at 37°C. Sera were diluted to a concentration in the linear detection range for each antigen. Plates were incubated for 2 h at 37°C with serially diluted serum samples from vaccinated groups. Following 3 PBS-T washes, goat anti-mouse IgG secondary antibody conjugated to alkaline phosphatase (AP) (Southern Biotech) was diluted at 1:2,000 in blocking buffer, added to each well, and incubated for 1 h at 37°C. Antibody standards were prepared from anti-PT or anti-RTX by 2-fold dilutions from 100 μg/ml. Plates were washed and then developed for 30 min with 100 μl p-nitrophenyl phosphate. Colorimetric signal was determined by measuring sample absorbance at 450 nm using a SpectraMax i3 (Molecular Devices). Antibody titers were determined by establishing a minimum detection limit greater than 2-fold over the standard deviation of the average of negative controls containing no antigen. Serum antibody concentrations were calculated by plotting absorbance values of samples to absorbance of known concentrations of control antibodies. Values were plotted using a logarithmic scale and fit to a four-parameter equation. Antibody concentrations were determined by plotting the calculated antibody sample concentrations to sample dilution factors and fit to a four-parameter logistic curve (64). Dilutions were accounted for by multiplying antibody concentration by dilution factor.