Anti human igg fc hrp

Ni-NTA HisSorb 96-well plates (Qiagen) were coated with 50 μl of either His-tagged RBD (Acro Biosystems, SPD-S52H6, AA 306-527) or His-tagged NTD (Acro Biosystems, S1D-C52H6, AA13-303) at a concentration of 2 μg/ml overnight at 4 °C; all subsequent steps were performed at room temperature. Plates were blocked with 3% milk; plasma was diluted 1:500 in PBS for RBD ELISA and 1:1500 for NTD ELISA prior to adding 100 μl per well for 2 h. Plates were washed with PBS-T three times, followed by incubation with secondary anti-human IgG Fc HRP (Southern Biotech) at 1:4000 for 1 h. Plates were washed three additional times with PBS-T, followed by addition of TMB substrate for 10 min. The reaction was stopped with the addition of 3 M HCl; and plates were read at an OD 450. Anti-RBD antibody CR3022 (Creative Biolabs) was used to make a standard curve with 1:2 dilutions in the range of 0.5 μg/ml to 0.03 μg/ml, utilizing a 4-parameter fit to interpolate sample concentrations. Anti-NTD antibody SPD-M121 (Acro Biosystems) was used to make a standard curve with 1:2 dilutions in the range of 0.5 μg/ml to 0.03 μg/ml, utilizing a 4-parameter fit to interpolate sample concentrations.

To measure the blocking capacity of each antibody representative of each competition group, Prn2-coated plates were first incubated with isotype, 1E7, 1F2, 2B1, 2E9, or a cocktail of all four antibodies (2 μg/ml each) as mouse IgG2a for 1 h at room temperature. Then sera from six different baboons (29 ) 26 days after infection with B. pertussis D420 were serially diluted (starting 1:3 and doing additional six 1:5 dilutions) and incubated for 1 h at room temperature. Sera antibodies were detected with antihuman IgG-Fc HRP (Southern Biotech). To measure baboon sera binding to Prn1, Prn2, Prn1-Δc-term, and Prn1-ΔR1, plates were coated with each pertactin variant at 2 μg/ml overnight at 4 °C, and ELISAs were performed as aforementioned. The inverse EC₅₀ to each pertactin variant was determined, and the EC₅₀ fold change to Prn2 was reported. Experiments were repeated once.

To assess binding of the antibodies by Western blot, 100 ng of purified pertactin-1 were boiled and ran by SDS-PAGE gel. The gel was transferred to polyvinylidene fluoride membranes and blocked with PBS-T with 5% milk for 1 h at room temperature. Isolated antibodies were incubated with membrane at 0.5 mg/ml in the blocking solution for 1 h. After several washes, membranes were incubated with antihuman IgG Fc-HRP (SouthernBiotech) at 1:8000 dilution, for 1 h at room temperature. After several washes, membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and imaged. Western blot with 3G4 was performed by running 100 ng of purified Prn1 and Prn2, and B. pertussis TohamaI and D420, B. bronchiseptica and B. parapertussis cells that were grown overnight, washed, and 10 μl of cells were used after an absorbance at 600 nm was adjusted to 4.