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FAQ

【MHC Tetramer】General



  1. What are MHC tetramers?
    MHC tetramers are complexes of four major histocompatibility complex (MHC) molecules, which are associated with a specific peptide and bound to a fluorochrome-conjugated streptavidin (a). Mainly, there are two types of tetramers, class I and class II. Class I tetramers bind to a distinct set of T cell receptors (TCRs) on a subset of CD8+ T cells (b). Class II tetramers bind to a distinct population of CD4+ T cells. Thus, by mixing tetramers with peripheral blood mononuclear cells (PBMCs) or whole blood and using flow cytometry as a detection system, all CD4+ or CD8+ T cells that are specific for one peptide in the context of a particular MHC allele can be detected, regardless of functionality (b, c). Simply stated, MHC tetramer reagents allow measurement of a cellular immune response directed toward single peptide specificity. The heavy chain portion of human and monkey class I tetramers contains a mutation to minimize non-specific binding of tetramers to CD8 on the cell surface, improving the specificity of these reagents to accurately discriminate the rare, antigen-specific T cells from the general CD8+ T cell population.
  2. How are MHC tetramers used for research and immune monitoring?
    MHC tetramers are used for the detection and monitoring of antigen-specific T cells to study the adaptive immune response, including T cell development; disease progression; and evaluation of therapeutics, vaccinations, and other interventions that may have an impact on cellular immune responses. In clinical trials, MHC tetramers are used to monitor T cells that may be related to either the cause of illness (e.g. diabetes or autoimmune disease) or the fight against an ailment (e.g. cancer, infection). In this way, antigen-specific T cells and their phenotype/function may serve as a surrogate or biomarker that correlates to treatment efficacy or disease progression.
  3. What are some examples of diseases and models that have been studied using MHC tetramers?
    Here are just some examples. Now the target research area using tetramer is expanding.
    • Infectious Diseases: HIV, EBV, CMV, HPV, HBV, HCV, Influenza, Measles, Malaria, TB, RSV, Dengue virus, SARS-CoV-2 etc.
    • Cancer: Breast, Prostate, Melanoma, Colon, Lung, Cervical, Ovarian, Leukemia etc.
    • Autoimmune Diseases: Diabetes, Multiple sclerosis, Rheumatoid arthritis, Autoimmune vitiligo, Celiac disease etc.
    • Transplantation: EBV and CMV etc.
    • Animal Models: OVA, E alpha, SIV etc.
    • Cancer Immunotherapy: Peptide vaccine, DC vaccine, TCR therapy etc.
  4. What are the advantages of MHC tetramer analysis in measuring cellular immune response?
    Tetramer analysis offers many potential advantages over some of the more traditional T cell based assays:
    • MHC tetramers allow direct detection of antigen-specific T cells.
    • MHC tetramer staining is quantitative.
    • Cells can be labeled with MHC tetramer and other cell-surface markers at the same time allowing additional characterization of the responding cells.
    • MHC tetramer-labeled cells remain viable and can be sorted by flow cytometry for further study, including functional assays.
    • MHC tetramers can be used in conjunction with intracellular staining (e.g. for cytokines such as IFN-γ) and other flow cytometry techniques to further characterize the antigen-specific T cell functionality and compare them with T cells that are not antigen-specific.
    • Because MHC tetramers are available in different fluorochromes, multiple T cell specificities can be analyzed simultaneously.
  5. How do I choose the peptide and allele for my MHC tetramer studies?
    First of all, please check MHC allele of your samples. We recommend customers do a literature search to determine the best peptide/allele combination to detect the specific T cells of interest.
  6. Which fluorochromes are available for MHC Tetramers?
    Most MHC Tetramers are mainly offered in PE, APC. FITC or BV421 is offered on a subset of Tetramers. If you cannot find the fluorochrome-labeled MHC Tetramer that you want, we can manufacture a custom MHC Tetramer of your interest. Please contact us.
  7. Can tetramers be used on CyTOF?
    Metal conjugated tetramers for use on CyTOF instruments have been produced by individual investigators using MHC Monomers and streptavidin or NeutrAvidin™ reagents that are tagged with rare metals. A poster presented at AAI on CyTOF tetramers made with MBLI (JSR group) MHC Monomers and a NeutrAvidin™ reagent to be commercialized by DVS Sciences can be found on their website.
    https://www.mblintl.com/assets/AAI-Poster-NA-metal-MHC_2015_TC-final.pdf
  8. What is the concentration of tetramer in the vial?
    We do not specify our Tetramer concentration (1 vial = 50 tests), as the fluorochrome contribution can vary in the final formulation. Except for some products, MHC Monomer concentration is basically 100 µg/mL. A more accurate and consistent monomer concentration in the vial can be obtained by asking MBL with your product code and lot number.
  9. What allele/peptide comprises the Negative Tetramer?
    For MHC class I Tetramers, Tetramers with artificially designed peptides that do not exist in nature can be used as negative controls; Code No. TS-M008-P, TS-M151-P, TS-M152-P, TS-M153-P, and the epitope peptide of TB-0029-1, TB-0029-2, TB-0029-4. Because there are few people with HIV, Tetramers loading HIV peptides can also be used as a negative control.
    Because MHC class II molecule loaded with CLIP peptides are formed during the process of MHC class II being presented on the cell surface, CLIP-specific T cells do not exist in nature. Therefore, the Tetramer can be used as a negative control. If you cannot find any Negative Tetramers of your desired allele described above, a tetramer with a peptide unrelated to the experimental system can also be used as a control.
  10. What is the α3 mutation?
    The T cell surface CD8 enhances T cell antigen recognition by binding to HLA class I molecules. Therefore, we produced T-Select HLA class I Tetramers with one point mutation (Ala245Val) at the HLA α3 domain known to reduce the CD8-HLA interaction. These mutated tetramers showed a greatly diminished nonspecific binding but retained specific binding2).

    References:
    1) Gao GF, et al., Nature 387: 630−634 (1997)
    2) Bodinier M, et al., Nat. Med. 6: 707−710 (2000)

  11. How do you purify the MHC complexes?
    Please check “Preparation of Class I MHC Tetramers” on our webpage.
  12. How are the products QC’d?
    Steps in the Tetramer manufacturing process are verified by spectrophotometry and HPLC. Some Tetramers are further tested for functionality.
  13. Do you check for functionality?
    Some Tetramers are tested by flow cytometry and the test data can be found in each product datasheet.
  14. What is the difference between paraformaldehyde and formaldehyde?
    Paraformaldehyde is a form of formaldehyde. The simple chemical formula for formaldehyde is CH2O. Paraformaldehyde is polymerized formaldehyde. When paraformaldehyde is dissolved, it becomes formaldehyde. Only the dissolved formaldehyde form is able to fix tissues.
    When you are making a paraformaldehyde solution, you should make it in a hood. Mix paraformaldehyde with PBS or TBS at 70°C. Use 5N NaOH to make the solution clear. You can then do a quick spin or use a filter syringe to remove any insoluble impurities. It’s always best to use a fresh solution but aliquots can be stored at -20°C and used over a couple months.
  15. Can I use biotinylated antibodies or streptavidin conjugates with MHC Tetramers and Monomers?
    It is not recommended to use biotinylated antibodies and streptavidin conjugates in parallel with MHC Tetramers and particularly with biotinylated Monomers as they could cause non specific staining reactions.

FAQ 【MHC Tetramer】