Biotome’s Epitope Mapping Pipeline Discovers Unique Diagnostic Targets for SARS-CoV-2
- lukemarshall93
- Oct 17
- 4 min read
Updated: Oct 20
Research Summary
Conventional serology assays for SARS-CoV-2 based on whole protein detection often suffer from suboptimal specificity due to cross-reactivity with seasonal coronaviruses (Fox 2022, West 2021). This study employed peptide microarray technology (Carmona 2015) to identify highly specific linear B-cell epitopes of the SARS-CoV-2 Spike protein that can distinguish between SARS-CoV-2 infection and pre-existing immunity to seasonal coronaviruses.
Key Findings
A comprehensive analysis of SARS-CoV-2 Spike protein identified 21 distinct linear B-cell epitopes.
Only 4 epitopes were highly specific for SARS-CoV-2 infection, with minimal cross-reactivity to pre-pandemic serum samples:
S005 (aa 278-298, subdomain S1A)
S010 (aa 550-586, subdomain S1C)
S019 (aa 1134-1156, subdomain S2-HR2)
S021 (aa 1248-1271, subdomain S2-CT)
Three of these epitopes are on the protein surface, while one (S021) is in the endo-domain.
The diagnostic performance of these epitopes was validated using the Luminex platform, demonstrating excellent discrimination between infected and non-infected individuals (AUC 1.00, 0.99, and 0.84 for epitopes S010, S019, and S021, respectively).
Methodology
Serum samples were taken from PCR-verified SARS-CoV-2 infected patients (n=40) and pre-pandemic controls (n=12).
Linear B-cell epitopes were mapped using pooled serum samples on medium-density peptide arrays. Each array contained the entire SARS-CoV-2 spike protein, divided amongst 1,262 12-amino acid peptides. Each array was analyzed for IgG and IgA antibody binding.
Epitopes were identified by selecting regions with ≥3 consecutive peptides above background in at least two separate pools.
Epitopes were confirmed in a secondary microarray investigation. This protocol used focused, 109 peptide arrays to test individual serum samples against the epitopes identified in phase 1.
The three peptides with the highest diagnostic potential were synthesized and tested in a bead-based immunoassay using cohort samples and a set of certified SARS-CoV-2 serology verification samples from NIBSC.
Results
The study demonstrated that pre-pandemic serum samples contain IgG antibodies reacting to the majority of Spike protein epitopes (71% of identified epitopes), indicating substantial cross-reactivity with seasonal coronaviruses. Only 4 out of 21 epitopes showed high specificity for SARS-CoV-2 infection.
The discriminatory capacity of these four epitopes was excellent, with AUC values of 0.81-0.94 for IgG detection. When implemented in a Luminex-based assay, these epitopes showed strong correlation with commercial anti-SARS-CoV-2 IgG ELISA (Spearman correlation coefficient 0.84-0.91 for S010, 0.82-0.87 for S019, and 0.65-0.71 for S021).
3D structural modeling revealed that epitopes S005, S010, and S019 are surface-exposed on the Spike protein, while S021 is in the endo-domain (Fig. 1). Epitopes S010 and S015 have been previously reported as virus-neutralizing epitopes (Poh 2020), suggesting potential applications beyond diagnostics.
Implications
This research has significant implications for the development of highly specific serological diagnostics for SARS-CoV-2 infection with minimal cross-reactivity to seasonal coronaviruses.
Peptide-based, precision diagnostics have the potential for differential diagnosis of variants of concern (VOCs), as 11 of the 21 identified epitopes contained mutations associated with VOCs. In addition, they open the potential for diagnostics that detect neutralizing antibodies.
Epitope mapping through phased peptide-microarray exploration establishes an approach for rapid development of high-specificity serology tests for future pandemic threats.
Diagnostic peptides identified through this approach have a direct, low-cost path to research and clinical use through standard immunoassay platforms (bead-based assay and ELISA).
Biotome Epitope Mapping Service
This investigation utilized Biotome’s microarray-based epitope mapping approach.
Technology: Custom peptide microarray
Screening Library: 1,262 12-amino acid peptides
Validation Library: 109 12-amino acid peptides
Samples: Pooled serum, 20µL
Detection System: High-resolution near-infrared laser fluorescence, 700 nm and 800 nm
Validation: Luminex multiplexed assay
Beyond Traditional Serology
This study identified peptide epitopes that differentiate SARS-CoV-2 infection from pre-existing immunity to seasonal coronaviruses, highlighting one of the significant advantages peptides have over protein-based serology.
Peptides are consistent antibody targets, proven effective on various diagnostic platforms (ELISA, lateral flow, chemiluminescence). They are produced via chemical synthesis rather than recombinant expression, ensuring consistency, reducing production complexity, and eliminating biological contaminants. Finally, novel peptide sequences are patentable, unlike naturally occurring proteins, providing opportunities for intellectual property protection.
References
Carmona, S. J., Nielsen, M., Schafer-Nielsen, C., Mucci, J., Altcheh, J., Balouz, V., et al. (2015). Towards high-throughput immunomics for infectious diseases: Use of next-generation peptide microarrays for rapid discovery and mapping of antigenic determinants. Molecular & Cellular Proteomics, 14(7):1871–1884.
Fox, T., Geppert, J., Dinnes, J., Scandrett, K., Bigio, J., Sulis, G., et al. (2022). Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane Database of Systematic Reviews, 11(11):CD013652.
Poh CM, Carissimo G, Wang B, Amrun SN, Lee CYP, Chee RSL, et al. (2020). Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients. Nat Commun, 11(1):2806.
West, R. M., Kobokovich, A., Connell, N., & Gronvall, G. K. (2021). Antibody (serology) tests for COVID-19: A case study. mSphere, 6(3):e00201-21.