Synthetic peptide-based indirect ELISA using S1c (of the sequence CPVAIHADQLTPTWRVYSTC) as the antigen, performed on regular polystyrene plates v1

This protocol describes a synthetic peptide-based indirect enzyme-linked immunosorbent assay (ELISA) using the peptide S1c (sequence: CPVAIHADQLTPTWRVYSTC) as the immobilized antigen. The assay is designed for antibody detection in serum samples and uses regular polystyrene microplates. Plates are coated overnight at 4 °C with 100 µL/well of S1c in carbonate–bicarbonate buffer (pH 9.6), then washed thrice with 0.05% Tween 20 in PBS (PBST). After a 15 min equilibration to room temperature, wells are blocked with 200 µL/well of 5% skimmed milk in PBST for 1 h at 37 °C and washed again three times. Primary antibody incubation is performed for 1 h at room temperature using 100 µL/well of sera diluted 1:100 in 0.05% skimmed milk–PBST, followed by washing. Detection is achieved with Protein A–peroxidase (0.5 µg/mL) diluted 1:2000 in PBST, incubated for 1 h at room temperature. After a final wash, the reaction is developed with 0.1 mg/mL TMB in phosphate–citrate buffer (pH 6.0) for 1 h at room temperature, stopped with 50 µL of 1 M H₂SO₄, and absorbance is measured at 450 nm. This optimized workflow enables reliable detection of anti-S1c antibodies using accessible reagents and standard ELISA plates.