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SIAT® In Vitro Class II HLA Binding Assay

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Although various in silico models have been developed to predict the binding affinity of peptides in biotherapeutic drug candidates to class II human leukocyte antigen (HLA, also called major histocompatibility complex, MHC) alleles, the results of these prediction algorithms is not always in accordance with physical binding data. Therefore, after having defined the epitopes of interest in silico, further assays are needed to validate these predictions, among which class II HLA/MHC binding assay is most widely used.

Creative Biolabs offers SIAT® in vitro class II HLA/MHC binding assay service that directly measures the affinity of predicted epitope peptide sequences to HLA alleles. The false positive and negative results generated by in silico models can therefore be eliminated. The information of immunogenic peptides identified by this assay is of great value in the development of novel biotherapeutic drugs. Based on the results of this assay, protein engineering strategies to deplete these T cell epitopes can be used to avoid side effects and the generation of anti-drug antibodies (ADAs).

Typically, an overlapping peptide library from a known protein of interest is generated by chemical synthesis. Afterward, a number of different HLA/MHC binding assay methods, including competition binding assay, direct binding assay, and real-time kinetic binding assay are used to determine the binding affinity of each peptide to one or more class II HLA/MHC alleles.

Competition binding assay
Peptides of interest are tested for their ability to compete against labeled high-affinity control peptides for binding to HLA/MHC molecules. IC50 data is calculated by analyzing the dose-response curve. The binding assays can be performed for a broad representation of class II HLA/MHC molecules.

SIAT® In Vitro Class II HLA Binding Assay

Direct binding assay
This assay is based on measuring of the ability of each peptide to stabilize the HLA/MHC-peptide complex, which will keep the native conformation if the binding affinity of tested peptide is high enough. A known T cell epitope is used as a positive control, and each peptide will be given a score by testing versus the positive control peptide. Currently, up to 60 DR, DQ and DP alleles and several mouse alleles are available for this assay.

SIAT® In Vitro Class II HLA Binding Assay

Real-time kinetic binding assay
This assay can give kinetic information about the on- and off-rate at which each peptide interacts with HLA/MHC molecules in real-time. It can provide a complete information of whether a peptide could be presented long enough to be a good T cell epitope. For example, peptides with fast on- and off-rate may not be suitable candidate T cell epitopes. Protocols based on fluorescence polarization or surface plasmon resonance (SPR) are employed.

SIAT® In Vitro Class II HLA Binding Assay


More SIAT® Immunogenicity Related Services at Creative Biolabs
In Silico Immunogenicity Assessment
In Vitro Class II HLA Binding Assay
Ex Vivo Immunogenicity Assessment
    DC-T Cell Proliferation Assay
    Antigen Presentation Assay
In Vivo Immunogenicity Assessment
Anti-drug Antibodies (ADA) Assays
De-immunization Service




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