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Introduction

What are MHC tetramers and what can you do with them?

The T-cell mediated immune response is defined by the interaction between antigen presenting cells and T cells, through the Major Histocompatibility Complex (MHC) and the T cell receptor (TCR). MHC molecules present a peptide to antigen-specific T cells that recognize this peptide. Soluble, monomeric MHC molecules bind very weakly to the TCR. However, by making a tetramer through a fluorescently labeled streptavidin conjugate, the complex has greater avidity to the T cell and maintains more stable binding by interacting with several TCRs, making it useful for flow cytometry detection of antigen specific T cells.

What is a Flex-T™?

Flex-T™ is BioLegend's technology to study antigen-specific T cells through their TCRs. It has the unique property of allowing the loading of peptides of interest into the binding site of the MHC groove, by using ultraviolet (UV) light labile, exchangeable peptides. Flex-T™ technology features:

  • Flexible peptide loading
  • Two color combination capability
  • High throughput screening
  • High specificity
  • Ease of use
  • Affordability

How does ultraviolet peptide exchange work?

Flex-T™ is made of MHC monomers loaded with a peptide that can be degraded by the use of a UV light source. This allows for a peptide exchange when the UV irradiation is done in the presence of the peptide of interest (which is not UV-labile). This flexibility permits the screening of virtually any peptide of interest with enough affinity for the MHC allele that it is loaded onto.

 


Representative Data

To increase the resolution when detecting antigen-specific T cells with MHC tetramers, at the same time that improves the specificity of the assay, we recommend a two color staining with the same allele/peptide combination. Furthermore, each allele can be coded with a unique two-color combination, allowing for the detection of multiple antigen-specific T cells at the same time.

A) CD8+ T Cells previously gated on lymphocytes (FSC vs SSC) and 7-AAD negative events, the exclusion cocktail contains Alexa Fluor® 700 anti- CD4, CD19, CD14, and CD16. B) Antigen-specific CD8+ T Cells, gated as described, detected with Flex-T™ tagged with PE and APC. HLA-A*11:01 Flex-T™ is loaded with EBV peptide (IVTDFSVIK).

 

Use our Human Class I Peptide Exchange ELISA kit to qualitatively evaluate the efficiency of the UV peptide exchange when building MHC Tetramers with Flex-T™ reagents prior to cell staining.  Each kit comes with validated, ready-to-use reagents including a pre-coated plate and positive control.

 

 

Flex-T™ HLA-A*02:01 UVX monomer was treated with a 366nm UV in the presence of a Positive or Negative control peptide, a test peptide, or no peptide (UV-only). After peptide exchange, each reaction mixture was then diluted and analyzed by the ELISA. The optical densities of each sample calculated by absorbance at 450 nm subtracted with absorbance at 570 nm are shown in upper bar graph and the percentile (%) of test peptide against Positive control peptide in lower bar graph.