T and B Cell-based X-ray Crystallography Assay

Cancer epitope analysis is the research that determines the specific cancer peptides directly bind to cell receptors or antibodies and elicit immune responses. Creative Biolabs provides the advanced and standard X-ray crystallography assay for the inspection of specific interactions between tumor antigen epitopes and immune cell receptors and antibodies.

Using X-ray Crystallography for Epitope Mapping

Protein antigen epitopes classify into two kinds, linear and conformational epitopes. Linear epitopes consist of several continuous amino acid sequences, and conformational epitopes are not only amino acid sequences decided but also associated with the 3D structure of these peptides. In T cell immunity, the T cell receptor is responsible for the recognition of HLA-restricted antigen peptides, but most TCRs bind sub-optimally to tumor antigens. Moreover, most epitopes directly in contact with antibodies are complex, discontinuous conformational epitopes and thus should be screened by structural methods. X-ray crystallography is a gold standard of epitope mapping for identifying specific conformational epitope-receptor interactions.

Fig.1 Protein structure determination using X-ray crystallography.¹

X-ray Crystallography Assay Services at Creative Biolabs

Creative Biolabs provides the X-ray crystallography assay for cancer epitope analysis and structure determination to accelerate your research and development. We have established a high-resolution platform to reveal the exact identity of a specific protein.

Workflow

Key Features of X-ray Crystallography Assay

✔ Co-crystallization of the antibody-antigen complex.

✔ Visualizing the antigen peptide-immune cell interactions in atomic resolution.

✔ Facilitates the inspection of complex conformational epitopes.

✔ Identifies the interaction domain of antigens against antibodies and receptors.

Identify Conformational Epitope for Multiple Types of Receptors

Any antibody format can be used for corresponding epitope analysis, including:

Antibody Format
Fab	scFv	sdAbbody VHH
Soluble TCR	Tailored Format

Description of Specific Steps for Cancer Epitope Determination

Protein Expression

Custom protein expression vector design and construction.
Protein expression in E. coli or insect expression systems allows proper disulfide bond formation.
Purified proteins with high homogeneity.

Proteins Complex Purification

Mix soluble TCRs with HLA-restricted-peptide, or antibody segment with antigen peptide with a proper ratio for hours.
The protein complex is purified on the column and verified by SDS-PAGE.

Protein Crystallization

Crystal screening: screen more than 3,600 crystallization reagents.
Automated operation: allows precise handling of small volume samples and imaging.
Crystal optimization: optimize crystallization conditions as commercial kits recommended.

3D Structure Determination and Epitope Analysis

Collect X-ray data at a synchrotron with high resolution.
Process data with suitable analysis software.
Examine the interface and interactions between the antibody/TCR and the antigen peptide.

Fig.2 X-ray crystal structure of the Cetuximab binding domain on soluble EGFR.²

Creative Biolabs possess a leading technique in protein structure analysis and focuses on cancer epitope-receptor interactions using X-ray structure determination for years. Creative Biolabs works closely with our clients to provide tailored strategies for the structure and interaction determination and provide detailed and accurate data and information on the complex. No more looking around and contact us for a detailed quote.

References

Valavanidis, Athanasios. Predicting 3D Protein Structure by Computational Approach from Amino Acid Sequence. A leap forward in solving the classic problem of biochemistry. 2020.
Li, S.; et al. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer cell. 2005, 7(4): 301–311.

For Research Use Only | Not For Clinical Use