Peptide-based ELISA for Antibody Detection

Streptavidin-coated polystyrene 96-well microtiter plates (Pierce, Thermo Scientific) were blocked with 0.1% PBST supplemented with 1% w/v sodium caseinate (Sigma-Aldrich) for 1 h at RT before being coated with biotinylated 18-mer overlapping peptides synthesized based on consensus E2 glycoprotein sequence (Kam et al, 2012b (link)) for 1 h at RT. Mouse serum diluted 1:500 (mouse) or human plasma diluted 1:2,000 in 0.1% PBST and 0.1% w/v sodium caseinate were added and incubated for 1 h at RT followed by the addition of relevant HRP-conjugated goat secondary IgG (Santa Cruz, cat# sc-2005) to detect bound antibodies. Reactions were developed using 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich) and terminated by Stop reagent (Sigma-Aldrich). Absorbance at 450 nm was measured using TECAN Infinite® M200 microplate reader and analyzed using Magellan™ software.