3 3 5 5 tetramethylbenzidine tmb reagent

Antisera collected from blood samples harvested at Day 7 (D7), Day 21 (D21), and Day 35 (D35), were used to quantify elicited IgG antibody titres against each immunizing antigen by enzyme-linked immunosorbent assay (ELISA). In brief, 96 well flat-bottom plates were coated with 5 µg/well of the immunizing antigen in carbonate-bicarbonate buffer (pH 9.6) at 4°C overnight for protein capture and immobilization. The following day, the plates were washed three times with 250 µL of PBS containing 0.05% Tween 20 (PBST). All wash steps mentioned henceforth were performed 3 times with 250 µL of PBST. Wells were blocked with 250 µl of 5% skim milk in PBST (w/v) for 45 minutes at 37°C and washed. 100 µL of antisera diluted in 2.5% skim milk in PBST was added in duplicate in 2-fold dilutions going from 1 in 250 to 1 in 512,000 and wells were incubated for 2 hours at 37°C and washed. 100 µL of goat anti-mouse IgG secondary antibody [conjugated to horseradish peroxidase (HRP)] (Sigma Aldrich, USA) diluted 1 in 15,000 in 2.5% skim milk in PBST was added to each well and incubated for 1 hour at 37°C and washed. Plates were developed using 50 μL of 3,3,5,5′-tetramethylbenzidine (TMB) reagent (Sigma Aldrich, USA) for 20 minutes in the dark. Development was quenched with 25 μL of 3 M H₂SO₄ and OD₄₅₀ readings taken using a 96 well plate reader and plotted.

The FcγRs RI, RIIA, RIIB, RIIIA (158V), and RIIIA (158F) were produced by MedImmune and coated on high binding Costar^TM 96-well plates (Fisher Scientific) at a concentration of 2 μg/ml in PBS (pH 7.4) at 4 °C overnight. Immune complexes were prepared at a final antibody concentration of 15 μg/ml. 3-fold dilutions were made in PBS (pH 7.4), added to wells, and incubated for 2 h at 25 °C. Bound mAb or BiSAb was detected with goat anti-human Fc-HRP (Abcam, San Francisco, CA) using 3,3′,5,5′-tetramethylbenzidine (TMB) reagent (Sigma). Data were analyzed using GraphPad Prism.