Oct 07, 2025
Synthetic peptide-based indirect ELISA using S1c (of the sequence CPVAIHADQLTPTWRVYSTC) as the antigen, performed on regular polystyrene plates
Forked from a deleted protocol
- Brian Andrich Pollo1,2,
- Ruby Anne King3,1,
- Fresthel Monica M. Climacosa4,1,
- Salvador Eugenio C. Caoili1
- 1Biomedical Innovations Research for Translational Health Science (BIRTHS) Laboratory, Department of Biochemistry and Molecular Biology, College of Medicine,University of the Philippines Manila;
- 2School of Medicine, The Manila Times College of Subic;
- 3Department of Science and Technology - Philippine Council for Health Research and Development (DOST-PCHRD);
- 4Department of Medical Microbiology, College of Public Health, University of the Philippines Manila
- Brian Andrich Pollo: corresponding author;
- Coronavirus Method Development Community
Protocol Citation: Brian Andrich Pollo, Ruby Anne King, Fresthel Monica M. Climacosa, Salvador Eugenio C. Caoili 2025. Synthetic peptide-based indirect ELISA using S1c (of the sequence CPVAIHADQLTPTWRVYSTC) as the antigen, performed on regular polystyrene plates. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrw753lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 06, 2025
Last Modified: October 07, 2025
Protocol Integer ID: 229158
Keywords: SARS-CoV-2, COVID-19, Spike protein, Synthetic peptides, Enzyme-Linked Immunosorbent Assay, ELISA, Antibody detection, Serologic tests, Protein A, Peroxidase, TMB substrate, Peptide antigens, Immunoassay methods, Polystyrene microplates, Carbonate-bicarbonate buffer, Tween 20, Skimmed milk, Blocking agents, Phosphate-Citrate Buffer, Absorbance spectrophotometry, Diagnostic assay development, Immunodiagnostics, Seroprevalence studies, based indirect elisa, indirect elisa, antibody detection in serum sample, immunosorbent assay, linked immunosorbent assay, primary antibody incubation, standard elisa plate, immobilized antigen, antibody detection, antigen, serum sample, synthetic peptide, milk in pbst, based indirect enzyme, elisa, cpvaihadqltptwrvystc, peptide s1c, enzyme, indirect enzyme, sequence cpvaihadqltptwrvystc, regular polystyrene plates this protocol, polystyrene plate, assay
Funders Acknowledgements:
Dissertation grant
IDC 211 grant
Abacov project
NIH faculty grant
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Abstract
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.
Troubleshooting
Coat
overnight incubation at 4°C
100µL/well, Carbonate-bicarbonate buffer in water, pH 9.6
Wash
3x at 5 min interval
0.05% Tween 20 in 1X PBS (PBST)
freeze
Block
*leave plates at RT for 15min before blocking*
incubation at 37°C for 1hr
200µL/well, 5% skimmed milk in PBST
Wash
3x at 5 min interval
0.05% Tween 20 in 1X PBS
Primary antibody
incubation at RT for 1hr
100µL/well, 1:100 sera in dilution buffer (0.05% skimmed milk in PBST)
Wash
3x at 5 min interval
0.05% Tween 20 in 1X PBS
Secondary antibody
incubation at RT for 1hr
50µL/well, 1:2000 Protein A-peroxidase (0.5ug/ml) in dilution buffer
Wash
3x at 5 min interval
0.05% Tween 20 in 1X PBS
Substrate
incubation at RT for 1hr
50µL/well, 0.1mg/mL TMB in Phosphate Citrate Buffer in water, pH 6.0
Stop
50µL/well, 1M H2SO4
read at 450nm