Custom Glycopeptide Conjugates for Coating, Labeling, and Detection

Applications of Glycopeptide Conjugates in Diagnostic Kits

(1) Glycopeptides Conjugated to BSA, KLH for Immunization or ELISA

Custom glycopeptide (GP) conjugates (GPCs) are often an essential element of a diagnostic kit and can be used for a number of applications including immunization, ELISA, and other detection applications. The specific conjugate used can vary depending on the intended application and the properties of the GP and carrier protein. GPCs are often used for immunization and ELISA assays. The GP is conjugated to a carrier protein such as BSA or KLH in order to increase the immunogenicity of the GP and allow for the generation of antibodies that specifically recognize the glycan structure of interest. GPs conjugated to KLH have been used to generate antibodies against tumor-associated antigens (TAAs) such as Tn and STn. The KLH carrier protein serves as a scaffold for the presentation of the GP to the immune system, which then generates antibodies that specifically recognize the TAA. This can be useful for the development of diagnostic tools for the early detection of cancer. GP-BSA or GP-KLH conjugates can also be used as capture or detection antigens in ELISA assays. The glycan structures on the conjugate provide specific binding sites for antibodies, allowing for sensitive and specific detection of target antigens. For example, GPs conjugated to BSA have been used to detect antibodies against specific glycan structures in patient samples, providing diagnostic information.

Fig. 1 Study workflow.^1,2

(2) Biotinylated or Fluorescent Glycopeptides for Detection

Detection methods use biotinylated GPs (bGPs) and fluorescent GPs (fGPs). The former are amenable to conjugation with streptavidin coated beads or surfaces, offering sensitive and specific detection options. The strong interaction between biotin and streptavidin immobilizes the GPs for detection. fGPs can be employed for real-time detection and imaging applications. These conjugates can be used in flow cytometry, fluorescence microscopy, and other imaging techniques to visualize and quantify glycans in biological samples. For example, fGPs have been used to detect and image the expression of specific glycans on cancer cells, providing insights into disease progression and therapeutic response. A success study with fluorescent glycan-decorated polymers used to probe glycan-lectin interactions and to initiate the detection of pathogens further added to the interest to further develop this concept. Indeed, polymers of various structures have been decorated with glycans for investigation of glycan-lectin interactions, some have also been explored for pathogen detections. A second work provided a system where fluorescence from mannose-decorated fluorescent poly(p-phenyleneethynylene) (PPE) is quenched via the formation of non-fluorescent aggregates with the protein concanavalin A (Con A). This system is of particular interest as the fluorescent quenching is more pronounced with the increase in PPE concentration, which approaches the apparent binding constant of streptavidine and biotin.

Conjugation Strategies and Linker Chemistry That Work

(1) Site-Selective vs Random Conjugation

Site-selective conjugation refers to the attachment of the payload to a single site on a protein or peptide. By using a single conjugation site, a highly uniform product can be prepared. One example of site-selective conjugation is described below. The inclusion of an engineered amino acid or short sequence of amino acids on the protein provides a unique conjugation handle for the modification of the protein with a desired payload. In this example, the "π-clamp" sequence was inserted into the protein, which then promotes selective conjugation via SNAr reaction. Another example of site-selective conjugation shown below takes advantage of non-natural amino acids with the requisite reactive handle. In this example, p-acetylphenylalanine (pAcPhe) can be site-specifically incorporated into proteins, and then conjugated to a desired payload using the principles of bio-orthogonal chemistry. Site-selective methods allow for the conjugation to take place at a predetermined site. Site-selective conjugation strategies have the benefit of being less heterogeneous, but in many cases are more involved in terms of synthetic design and the necessary chemical or biochemical manipulations of the target molecule. As such, the decision to pursue a site-selective approach versus a random conjugation strategy is application-specific. In contrast to site-selective conjugation is random conjugation, which involves the attachment of a payload to multiple sites along a protein or peptide to give a heterogeneous distribution of conjugates. In the majority of instances, the reaction between the protein and the payload occurs at multiple lysine residues or at thiol groups on cysteines. With lysine conjugation, numerous β-lactam linkers are available which will react selectively with a single lysine residue based on subtle steric or chemical differences between the various lysine residues in a protein or peptide. In general, one drawback to this is that a distribution of DARs can result in different pharmacokinetics and efficacy of the final product.

(2) Spacer Optimization for Epitope Accessibility

Spacer design forms a critical element within the overall conjugation strategy. Spacer design forms a critical element within the overall conjugation strategy. Spacer length, flexibility, and composition can all have an impact on the properties of the resulting conjugate. Longer spacers can provide more flexibility and reduce steric hindrance, which can minimize interference with epitope recognition. Researchers can modify spacer chemical compositions by adding functional groups which enable subsequent modification or targeting purposes. For example, spacers with clickable moieties (such as azides or alkynes) can be used in click chemistry reactions to attach payloads in a modular and site-specific manner. One of the most commonly used spacers in bioconjugation is polyethylene glycol (PEG). PEG spacers are highly hydrophilic and can be used to enhance the solubility and stability of the conjugate. PEG's hydrophilic properties assist in minimizing non-specific binding and aggregation. Spacer design plays a critical role in optimizing conjugation strategies because selecting the right spacer substantially influences the conjugate's properties. When creating diagnostic conjugates researchers must ensure epitopes remain accessible to bind specific antibodies or receptors. The spacer requires optimization to ensure both the conformation and accessibility of the epitope and efficient binding capability. The optimization of spacer design requires researchers to combine molecular modeling with experimental validation approaches.

What We Offer?

(1) Flexible Conjugation to Carriers, Beads, or Plates

GPs can be immobilized by conjugating them to different types of beads or plates. The carrier material can be chosen based on the needs of the assay. Common carrier materials include polystyrene, silica, or magnetic nanoparticles. For instance, magnetic beads may be chosen for their ease of separation and compatibility with automated processes, while silica beads have high surface area and good stability. Our conjugation methods can be used to attach the glycopeptides to the carrier while preserving their structure and biological activity. Our bead conjugation services provide a versatile platform for various applications including flow cytometry, magnetic separation, and microarray assays. We can offer a range of bead sizes and materials to suit the specific needs of your assay. For example, we can conjugate glycopeptides to fluorescent beads for multiplexed detection in flow cytometry, enabling the simultaneous measurement of multiple analytes. Our conjugation processes are optimized to ensure high loading efficiency and uniform distribution of GPs on the bead surface, which can improve the sensitivity and specificity of your assays. Glycopeptides can be immobilized on microtiter plates for ELISA and diverse plate-based applications. The immobilization methods we employ enable glycopeptides to spread evenly across plate surfaces thus creating uniform and precise binding locations for antibodies or detection reagents. This uniformity in glycopeptide immobilization is critical for reproducible results in high-throughput screening and diagnostic assays. We can customize conjugation density and conditions to optimize assay performance.

(2) QC Testing of Conjugates for Functional Validation

Quality control (QC) testing of conjugates for functional validation is a critical step in our service to ensure the functionality and reliability of the GP conjugates. This process is designed to confirm that the conjugates perform as expected in their intended applications, providing you with the confidence to use your reagents, knowing they have been quality tested to the highest standards. Our QC testing will include functional validation of the conjugated GPs to ensure that they maintain their biological activity and specificity. This is achieved by testing the conjugates in the relevant assay to confirm that they perform as expected. We will determine the conjugates' binding capability and specificity using target antibodies when glycopeptides are attached to beads for flow cytometry applications. If the GPs are to be used in an ELISA, we will evaluate the sensitivity and specificity of the conjugated glycopeptides in detecting the target analytes to ensure that the conjugation process has not interfered with the functionality. All our QC testing parameters are intended to meet the highest quality assurance criteria. Each batch of conjugated glycopeptides is rigorously tested using techniques like HPLC, MS and ELISA, to confirm its purity, molecular identity and functional performance. We also provide Certificates of Analysis (COA) with every batch that documents the results of our QC tests and certifies that the conjugates meet the required specifications. Diagnostic and research applications require documentation for regulatory compliance and quality assurance.

Peptide Conjugation Services at Creative Peptides

References

1. Image retrieved from Figure 1 " Study workflow," Du J J.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Lohia S.; et al. " Glycosylation analysis of urinary peptidome highlights IGF2 glycopeptides in association with CKD." International Journal of Molecular Sciences, 2023, 24(6): 5402.
3. Stergiou N, Urschbach M, Gabba A, et al. The Development of Vaccines from Synthetic Tumor‐Associated Mucin Glycopeptides and their Glycosylation‐Dependent Immune Response[J]. The Chemical Record, 2021, 21(11): 3313-3331. https://doi.org/10.1002/tcr.202100182.
4. Ji Y, Li G, Wang J, et al. Recent progress in identifying bacteria with fluorescent probes[J]. Molecules, 2022, 27(19): 6440. https://doi.org/10.3390/molecules27196440.