As a leading services provider of life science, Creative Biolabs has extensive experience in the immunologic analysis field. We offer high-quality CreMap™ antigen processing services which provide valuable information for epitopes from antibody drug candidates and other proteins of interest to help understand and manage their potential immunogenicity.

Overview of Antigen Processing

In order to be capable of engaging the key elements of adaptive immunity (specificity, memory, diversity, self/nonself discrimination), antigens have to be processed and presented to immune cells. Antigen presentation is mediated by Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules found on the surface of antigen-presenting cells (APCs) and certain other cells. Antigen processing and presentation refer to the processes occurring within cells that result in fragmentation (proteolysis) of proteins, binding of fragments to MHC, and expression of the peptide-MHC molecules at the cell surface where they can be recognized by the TCR (T Cell Receptor) on T cell. TCR can only recognize antigens in the form of peptides that bind to MHC molecules on the surface of human cells. The antigens recognized by T cells are peptides produced by cleavage of macromolecular antigen into short fragments by antigen processing.

• Endogenous/intracellular antigens

Endogenous/intracellular antigens are processed by the proteolytic system and presented by MHC molecules. Intracellular proteins are cut into short peptides in the cell's multifunctional protease complex, the proteasome. The proteasome subunit induces proteolytic complex to produce short peptides that specifically bind to the MHC-I molecule. The short peptide is transported into the ER (endoplasmic reticulum) by TAP (Transporters Associated with Antigen Processing) for subsequent assembly with MHC-I molecules. The peptide is then loaded onto the MHC-I molecule and transported to the cell surface for recognition by CD8⁺ T cells.

• Exogenous/extracellular antigen

The exogenous protein is mainly presented by MHC-II molecules. Antigens are internalized by several pathways, including phagocytosis, macropinocytosis, and endocytosis. Internalized antigens are processed in three increasingly acidic endosomal environments: early endosomes (pH 6.0-6.5), late endosomes (pH 5.0-6.0) and lysosomes (pH 4.5-5.0), and finally traffic to a mature endosomal compartment where they are processed and loaded onto MHC-II molecules. At the cell surface, the antigen is presented to CD4⁺ T-cells.

Fig.1 Trafficking of antigens for processing and presentation with MHC molecules. (Cresswell, 1991)

CreMap™ Antigen Processing Assay at Creative Biolabs

The CreMap™ antigen processing assays can be used to measure antigen processing and presentation, which directly identify epitopes of protein antigens that are bound to MHC molecules and displayed to T cells via APC. The processing activity of the antigen may affect epitope production and presentation. Thus antigen processing assays provide valuable and accurate information for epitopes from biotherapeutic drug candidates and other proteins of interest to help understand and manage their potential immunogenicity. During antigen processing, the antigen is degraded into peptides by protease, and thus the processing activity of the antigen can be evaluated by measuring the degradation activity or proteolytic activity. At Creative Biolabs, our CreMap™ antigen processing analysis process are as follows:

We are dedicated to providing the best services to help our clients accelerate their basic research, preclinical or clinical development. Please feel free to contact us for more details about our CreMap™ antigen processing services.

Other optional CreMap™ T cell epitope discovery services:

• CreMap™ MHC-Peptide Binding Assay

FAQ

1. What is an antigen processing assay?

   An antigen processing assay is used to examine how antigen-presenting cells (APCs) process and present parts of antigens on their surfaces using Major Histocompatibility Complex (MHC) molecules. This assay helps researchers understand how the immune system recognizes and reacts to foreign proteins, which is essential for vaccine development and immunotherapy.

2. How are antigen processing assays vital for vaccine development?

   These assays are crucial for identifying which peptide fragments of a pathogen will effectively interact with MHC molecules and subsequently stimulate an immune response. This information helps scientists design vaccines that can elicit strong and specific immune responses, targeting the most effective epitopes of pathogens.

3. What are the main components required for an antigen processing assay?

   The main components include antigen-presenting cells such as dendritic cells or macrophages, the antigen to be tested, and reagents to detect peptide-MHC complexes on the cell surface. Techniques such as flow cytometry, ELISA, or mass spectrometry might be used to analyze the peptide presentation.

4. What types of antigens can be used in these assays?

   Both synthetic peptides and whole proteins can be used as antigens in processing assays. Whole proteins allow examination of the processing pathway, while synthetic peptides, typically representing known epitopes, can be used to study the specificity and efficacy of presentation.

5. How does the choice of antigen-presenting cell affect the assay results?

   The choice of APC can significantly influence the results as different cells may express different sets and levels of MHC molecules, possess varying capacities for antigen uptake, and processing pathways. Typically, professional APCs like dendritic cells are used for their efficient processing and presentation capabilities.

6. What is the role of MHC molecules in antigen processing assays?

   MHC molecules play a critical role by binding peptide fragments of processed antigens and displaying them on the cell surface for recognition by T cells. The stability and affinity of peptide-MHC binding are crucial factors studied in these assays to determine how effectively an antigen can be presented.

7. How do researchers measure the effectiveness of antigen presentation in these assays?

   Effectiveness is often measured by the ability of the peptide-MHC complexes to activate T cells, which can be assessed by detecting cytokine release, T-cell proliferation, or the expression of activation markers on T cells. The more robust the T-cell response, the more effective the antigen presentation.

Resources

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All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.