Maxisorp polystyrene plates

96-well MaxiSorp polystyrene plates (NUNC, Thermo Fisher Scientific, Rochester, NY) were coated with 2 μg/mL recombinant Ag85B (BEI Resources) in 100 μL PBS and incubated at 4°C overnight. Plates were washed 4 times with PBS containing 0.05% Tween 20 (Sigma-Aldrich, wash buffer) and blocked with 200 μL 5% NFDM at 37°C for 2 hours, washed 4 times with wash buffer and serum samples serially-diluted in PBS/1% FCS (ELISA diluent), were added to wells and incubated at 37°C for 1 hour. Wells incubated with ELISA diluent or naïve mouse serum were used as negative controls. Plates were then washed 6 times and biotinylated goat anti-mouse IgG antibody (Southern Biotech, Birmingham, AL) diluted in ELISA diluent was added, and plates were incubated at room temperature and left for 45 minutes. Plates were washed again 6 times and 100 μL streptavidin-alkaline phosphatase (GE Healthcare UK Limited) diluted in ELISA diluent was added at room temperature for 30 minutes. Plates were subsequently washed 8 times and developed with 100 μL pNPP AP substrate (BioFx Laboratories, Owing Mills, MD) at room temperature for 15–25 minutes. Color development was stopped with 50 μL Stopping Solution (BioFx Laboratories) and plates were read at 405 nm on a Synergy HT Multi-Mode Microplate Reader (BioTek). Data are expressed as mean endpoint antibody titers ± SEM.

Reactivity of immune sera against chimeric antibodies was determined by solid-phase ELISA as previously described (27 (link)). Briefly, 96-well Maxisorp polystyrene plates (Nunc, Roskilde, Denmark) were coated with 10 μg/mL of chP3R99-LALA, ch1E10, or hR3 mAb and incubated overnight at 4°C. Diluted sera (1/1,000) were added and the plates were incubated for 1 h at 37°C. Peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used to determine the reactivity associated with IgG fraction using 0.4 mg/mL of ortho-phenylenediamine in citrate/phosphate buffer, pH 5 containing 0.2% of H₂O_2. Sera reactivity is expressed as OD values at 490 nm.