How Glycopeptides Improve Diagnostic Reagent Sensitivity and Specificity?

The Role of Glycopeptides in Modern Diagnostic Reagents

(1) Glycopeptides as Synthetic Antigens or Epitope Mimics

Glycopeptides (GPs) can be synthesized to mimic natural antigens or specific epitopes recognized by antibodies. This can be used to generate highly specific antigens for diagnostic assays. Synthetic GPs that are structurally similar to the natural GP antigens in a sample can be synthesized to create highly specific antigens for use in diagnostic assays to specifically detect the target biomarkers. Laboratory-made GPs can be engineered to match tumor-associated antigens (TAAs) such as Tn and STn which appear in excess on cancer cell surfaces. Diagnostic assays gain improved sensitivity and specificity through the use of synthetic GPs as antigens for detecting cancer biomarkers. Engineered synthetic GPs incorporate precise glycan structures which bind uniquely to designated receptors or glycan-binding proteins (GBPs). Special glycan structures on GPs function as antigens which allows them to selectively bind to lectins or antibodies that target these specific glycans. This can improve the specificity of diagnostic assays and reduce cross-reactivity with non-specific proteins.

Fig. 1 IgG glycans are important biomarkers and functional effectors in health and disease.^1,2

(2) Why Glycosylation Improves Antibody Specificity in ELISA, CLIA, etc?

The IgG Fc fragment contains two domains of the constant heavy chain. IgG has a conserved N-linked glycosylation site which is made of asparagine-297 (N297) and is located in the proximity of the hinge region in the Cγ2 domain. IgG Fc N297 glycans occupy a gap that develops between the two Cγ2 domains according to X-ray crystallographic studies. The N-linked glycans on IgG Fc generate the necessary steric hindrance between the two heavy chains to maintain its opened conformation. In the absence of glycans, the two heavy chains collapse and result in abolishment of binding to FcγRs and C1q. Scientists use glycosylation as a method to enhance antibody specificity in various immunoassays including enzyme-linked immunosorbent assays (ELISA) and chemiluminescent immunoassays (CLIA). Antigen glycosylation leads to structural complexity that creates more precise antibody binding sites. Specific glycans that exist on the Fc region of antibodies can determine their interaction with Fc receptors thereby enhancing the precision of antibody-target antigen binding. GPs can be used as capture or detection antigens in ELISA, which results in increased sensitivity and specificity of the assay. Engineered glycan structures on peptides can enhance antibody binding affinity which results in more precise detection of analytes. GPs enhance chemiluminescent signals specificity in CLIA because higher specificity results in diminished background noise and improved detection of low-abundance analytes. Glycosylation can also improve the stability of GPs in diagnostic assays. Peptide protection from proteolytic degradation by carbohydrate moieties maintains antigen integrity and functionality during the assay process. Assays which have prolonged incubation time or have several washing steps can benefit greatly from this process.

Fig. 2 A schematic illustration of immunoglobulin G (IgG) and the Fc glycan modification.^3,4

Use Cases of Glycopeptides in Diagnostic Applications

(1) Tumor Marker Detection (e.g., Tn, STn, sLeX antigens)

The power of GPs as tumor markers has been illustrated in a series of recent studies. A significant part of TAAs, such as Tn, STn, and sLeX (sialyl-Lewis X), are GPs and can be detected with GPs that are antigen mimics. Immunoassays based on GPs carrying Tn and STn antigens could be used to detect these antigens as cancer biomarkers with high sensitivity and specificity. The glycosylation change of α2-macroglobulin and α1-antitrypsin at the time of cancer onset has been reported in a number of papers. One report showed that α2-macroglobulin N-glycans that contain α2,6 sialylation, N-acetylglucosamine residues and tri-/tetraantennary high-mannose-type complexes were significantly elevated in colorectal cancer patients. Hepatocellular carcinoma patients experienced a substantial increase in both sialylation and fucosylation alongside glycan branching of α1-antitrypsin glycans. Researchers demonstrated that fully sialylated biantennary glycan profiles of α1-antitrypsin and α2-macroglobulin enable significant differentiation between lung cancer patients and healthy individuals. The development of a tool that combines nine tumor markers and 1688 enriched GPss (EGPs) and identifies patterns in these data with a machine learning model was introduced as Comprehensive serum GP spectra analysis. The analysis improved diagnostic accuracy and achieved an ROC-AUC of 0.935.

(2) Viral Glycoprotein Mimicry for Infectious Disease Tests

Diagnostic assays for infectious diseases utilize GPs which mimic viral GPs. Viral GPs represent surface proteins of viruses which enable viral entry into host cells and become targets for the immune system. Scientists create GPs that resemble viral GPs which can then be applied in diagnostic tests to identify viral infections. For example, GPs mimicking glycosylation sites on the SARS-CoV-2 spike protein have been used to develop immunoassays for the detection of antibodies to SARS-CoV-2. The GPs can be used in ELISA or CLIA to detect antibodies in patient samples, leading to accurate and reliable detection of past infection. Glycosylation on these peptides can also be designed to permit specific binding of antibodies to the GP, minimizing the chance of false positives or negatives. Diagnostic assays have utilized GPs for detecting other viral infections including influenza and HIV. Specialized diagnostic tests utilize specific glycan structures present on viral glycoproteins to identify various viral strains which enables epidemiological research and clinical diagnostics.

Our Solution: Ready-to-Use and Custom Glycopeptides

(1) Catalog Products for Oncology and Virology Panels

Our catalog of ready-to-use GPs is tailored specifically for oncology and virology panels. This catalog is designed to simplify the process of selecting the most appropriate GPs for a given diagnostic or therapeutic application. For oncology panels, the catalog contains GPs that mimic TAAs such as Tn, STn, and sLeX, which are overexpressed in many different cancer types. The catalog GPs for oncology are synthesized to high standards of purity and batch-to-batch consistency, ensuring that they will perform consistently in immunoassays such as ELISA and CLIA. In virology research and diagnostics, the catalog includes GPs that closely resemble viral GPs that are involved in the entry of viruses into host cells. The GPs in this catalog can be applied for the generation of highly specific diagnostic assays for detection of viral infections such as SARS-CoV-2, influenza and HIV. Specific glycan targets on viral glycoproteins can be used for discrimination between viral strains with applications in epidemiological studies and clinical diagnostics. Each product listed in the catalog went through QC tests to confirm uniform identity and quality across different batches. Each batch of GP is fully characterized using MS and HPLC to confirm the identity and purity of the GP before it is offered for sale.

(2) Custom Glycopeptides for Rare or Proprietary Targets

In addition to our catalog offerings, we offer GP synthesis services for custom targets. We have significant expertise in the chemical and enzymatic synthesis of GPs with defined glycan structures and peptide sequences. This enables us to tailor GP synthesis for our customer's needs, as well as work together on new targets and potential new diagnostic and therapeutic uses. We can synthesise GPs with N-linked glycans on asparagine, or O-linked glycans on serine or threonine. Our exact control of glycan structures and peptide sequence allows us to engineer GPs with the required biological activity and specificity. The synthesis platform enables us to create glycan structures featuring multiple branching points and various functional groups. Our synthesis capabilities produce GPs resembling natural antigens and epitopes which enhances diagnostic assay performance. We offer synthesis services for GPs in quantities from milligram to gram scales with purities that exceed 90% of the target product. We provide customized analytical services that produce detailed reports of GP purity and structure.

Peptide Synthesis Services at Creative Peptides

References

1. Image retrieved from Figure 1 " IgG glycans are important biomarkers and functional effectors in health and disease," Krištić J.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Krištić J.; et al. " The importance of IgG glycosylation—What did we learn after analyzing over 100,000 individuals." Immunological Reviews, 2024, 328(1): 143-170.
3. Image retrieved from Figure 1 " A schematic illustration of immunoglobulin G (IgG) and the Fc glycan modification," Gao C.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
4. Gao C.; et al. " Immunomodulation of Antibody Glycosylation through the Placental Transfer." International Journal of Molecular Sciences, 2023, 24(23): 16772.
5. McDonald D M, Byrne S N, Payne R J. Synthetic self-adjuvanting glycopeptide cancer vaccines[J]. Frontiers in chemistry, 2015, 3: 60. https://doi.org/10.3389/fchem.2015.00060.
6. Behren S, Westerlind U. Glycopeptides and-mimetics to detect, monitor and inhibit bacterial and viral infections: recent advances and perspectives[J]. Molecules, 2019, 24(6): 1004. https://doi.org/10.3390/molecules24061004.
7. Yamazaki K, Kawauchi S, Okamoto M, et al. Comprehensive Serum Glycopeptide Spectra Analysis Combined with Machine Learning for Early Detection of Lung Cancer: A Case–Control Study[J]. Cancers, 2025, 17(9): 1474. https://doi.org/10.3390/cancers17091474.