Peptide-binding ELISA for Antibody Characterization

Immulon 4HBx 96-well plates (Thermo Fisher, Waltham, MA) were coated overnight with 100 ng/well of the following peptides at 4 °C: full-length FL-CSP, NANPx6, peptide 21 (NPDPNANPNVDPNAN)^{12 (link)}, or NPDP₁₉ peptide (KQPADGNPDPNANPNVDPN)^{21 (link)}. Plates were washed thrice with PBS-Tween (1X PBS + 0.05% tween) solution and blocked for 1.5 h at room temperature with 0.5% casein + 1% Tween solution. MAbs were plated starting between 1000 and 200 ng/ml and serially diluted threefold down the plate. After 2 h incubation at room temperature, unbound antibodies were washed away thrice with PBS-Tween solution and remaining mAbs were detected using Anti-human IgG-horseradish peroxidase (HRP) conjugate (1:5000; Southern Biotech, Birmingham, AL) as secondary antibody along with 100 µl per well of ABTS 2-component substrate (KPL, Gaithersburg, MD) for color development for 1 h. After stopping the reaction with 20% SDS, optical density at 415 nm (OD₄₁₅) was measured on a Synergy 4 microplate reader (BioTek, Winooski, VT). Concentration of mAb needed to achieve an OD of 1 using Gen5 software (BioTek, Winooski, VT).