ELISA-based Comparison of mAb Binding

An ELISA method was used to compare binding efficiency of mAbs according to a published method [47] (link). LPS from E. coli 055:B5 (Sigma) and Leptospira licerasiae[48] (link) were used as specificity controls. B. melitensis LPS was dissolved (2 µg/ml) in 0.06 M sodium carbonate buffer (pH 9.6); 100 µl was used to coat wells by incubation overnight at 4°C. For the assay, Superblock blocking buffer (Thermo Fisher Scientific, IL, and USA) was used for blocking, TBST was used for washing the plate and 1% Superblock blocking buffer in TBST was used to dilute antibodies and conjugates. LPS-coated plates were blocked (for 1 hr at 22°C followed by three washes. mAbs were diluted (2 µg–0.02/ml) and added 200 µl per well in duplicate and incubated overnight at 4°C. The wells were washed three times with TBST and 200 µl of diluted (1∶5,000) goat anti-mouse immunoglobulin G (IgG) antibody conjugated to horseradish peroxidase was added to each well and incubated for 1 hr at room temperature. The plate was washed four times and 100 µl of chromogenic substrate (TMB Microwell Peroxidase Substrate system, KPL, Gaithersburg, MD) was added to each well and the reaction was stopped by the addition of 100 µl of 2N H₂SO₄. The plate was read using a microplate reader (Spectramax Plus, Molecular Devices, Sunnyvale, CA) at wavelength 450 nm.