Frequently asked questions

immunAware provides reagents and services related to major histocompatibility complexes (MHC) class I and II molecules, including:

• HLA-class I easYmer® – the fastest and most flexible way to obtain peptide-MHC (pMHC)
• MHC-class I catalog – ready to use pMHC class I mono- or tetramers with known epitope
• MHC-class II catalog – ready to use pMHC class II mono- or tetramers with known epitope
• MHC-class I custom – generation of pMHC class I complexes with your choice of peptide
• MHC-class II custom – generation of pMHC class II complexes with your choice of peptide
• Affinity and stability measurements of pMHC interactions – also on unknown peptides

Please click here for further information.

After placing your order you will receive an email with an order summary. All normal orders are
shipped under incoterms DAP, and the delivery time depends on the product in order.

• easYmers® the delivery time is estimated 1-2 weeks
• catalog pMHC the delivery time is estimated 2-3 weeks
• custom pMHC the delivery time is estimated 3-6 weeks

Please contact us here for inquiries and further information.

All our reagents are distributed directly from the facilities of immunAware Aps in Hørsholm, Denmark. Our subsidiary company immunAware Inc. is the distributor for customers in the US through Eagle Biosciences, Inc. Our easYmer® reagents are also distributed by Immudex and SelleckChemKorea.

Read more about our partners here.

MHC is short for major histocompatibility complex and defines a large, highly polymorphic genetic locus encoding MHC-molecules – the locus was first discovered through the study of transplanted tissue compatibility, hence the name. MHC-molecules are presented on cell surfaces throughout the body in nearly all vertebrates and display epitopes for immune cells. In humans, MHC molecules are also known as human leucocyte antigens (HLA).

Thus, MHC-molecules are genetically determined membrane structures that are important in the regulation of immunological reactions, i.e. important in relation to infectious diseases, vaccination, autoimmunity, immune-oncology, organ transplantation and graft rejection. Discover our range of MHC reagents here.

MHC class I and II molecules are structurally different, applying for differences in their extracellular peptide-binding clefts and ability to bind to different T cell receptors.

MHC class I molecules are heterodimers of a heavy α-chain and β2-microglobulin. They are found on all cells and display epitopes from internally digested proteins (typically 8-10 amino acids in length), i.e. presenting a “piece of self”, which is recognized by CD8+ T cells – also called cytotoxic T cells. If the epitope is recognized by the T cell as foreign, e.g. in case of infection, it will be destroyed.

MHC class II molecules are also heterodimers, but unlike MHC class I molecules, they exist of two homogenous peptides, an α and β chain. They are found on antigen presenting cells (APCs) and display epitopes (typically 10-30 amino acids in length) from extracellular proteins. They are recognized by CD4+ T cells, also called T helper cells, which stimulate B cells to produce antigen specific antibodies. See our range of MHC reagents here.

A tetramer is an oligomer of four identical monomers. Thus, a MHC tetramer is four identical peptide-MHC monomer molecules assembled by a (fluorochrome-conjugated) streptavidin molecule.

MHC class I tetramers can be used to label CD8+ T cell subsets expressing a cognate T cell Receptor (TcR) which will interact with a specific peptide-MHC complex (pMHC). Similarly, MHC class II tetramers interact with the CD4+ T cell subsets expressing a TcR specific to the peptide-MHC class II complex.

The pMHC:TcR interaction is weak and fast dissociating, which prevents a distinct labelling of T cells. Tetramerization of pMHC monomers increases the avidity resulting in a stable interaction between the tetrameric pMHC and specific TcRs which can be detected in a flow cytometer. Identification of antigen-specific T cell was first published by Altman et al. at Standford in 1996.

Get more information about MHC monomers and tetramers here.

MHC tetramers allows:

• Direct detection and monitoring of antigen-specific T cells.
• Different fluorochrome-conjugations allows simultaneously detection of multiple T cell specificities.
• Combined staining with other cell surface markers or intracellular cytokine or chemokine staining.
• Staining of PBMCs from blood samples, tissue-derived cell samples or in vitro cell cultures
• High-throughput of large number of cell samples.
• FACS sorting of MHC tetramer labelled T cells for downstream functional assays and analysis.

Get more information about MHC monomers and tetramers here.

A peptide MHC tetramer staining is a functional assay where antigen-specific T cells are stained with e.g. a fluorochrome-labelled MHC tetramer loaded with the corresponding peptide epitope. A flow cytometry analysis allows you to detect and monitor antigen-specific T cells — i.e. T cells expressing the cognate T cell receptor to the peptide-MHC complex in question in a population of T cells with multiple specificities. Co-staining with cell surface markers or intracellular staining for cytokines and chemokines can provide you with a detailed view and profile of the antigen-specific T cells of interest.

Consult our protocols on HLA class I TMR staining of CD8+ T cells and HLA class II TMR staining of CD4+ T cells.

No, to get optimal tetramer staining of your antigen-specific T cells you should work with live cells. You can however, fixate the tetramer stained cells in PBS suppl. with 1% formaldehyde if you wish to analyze your cells at a later time.

Consult our protocols on HLA class I TMR staining of CD8+ T cells and HLA class II TMR staining of CD4+ T cells.

Peptide-MHC class I / class II monomers are stable in the freezer (-20°C) for years — however, we recommend to avoid repeated freeze-thaw cycles. All our molecules are biotinylated, so it is possible to store the monomer for years and just assemble the tetramers at will. Tetramers (four peptide-MHC monomers assembled by streptavidin) should be stored in the fridge (4°C) where it is stable for months. The long-term stability of tetramers may depend on the peptide, thus some tetramers are stable for longer periods of time than others.

Read more about MHC monomers and tetramers here.

We do not recommend to store a fluorescence-conjugated tetramer frozen (below 0°C). If you need to store a tetramer for a longer period of time, we recommend storing it in the fridge (4°C).

Read more about MHC monomers and tetramers here.

All our catalog and custom HLA monomers are being functional tested through beads assay and flow-cytometry (FACS) before tetramerization and shipping.

Read more about our MHC monomers and tetramers here.

easYmers® are basically “empty” HLA class I molecules. Our proprietary formulation renders highly active peptide receptive HLA class I molecules which allows any non-expert user to, seamlessly, produce HLA monomers and tetramers just by adding peptides of interest. Consult our protocols on HLA class I TMR staining of CD8+ T cells and HLA class II TMR staining of CD4+ T cells.

Included in the easYmer® kit is an allotype relevant positive control peptide, which allows you to validate the quality of your own peptide-HLA complexes in a simple flow cytometry based assay – read more in our protocol Assesment of HLA-compxlex folding (FACS assay).

For more information on easYmers®, click here

The HLA easYmers® provide maximum flexibility for your T cell research compared to premade or even custom HLA tetramers and have several advantages:

• Save time and money – the easYmers® allow you to maintain a repository of “empty” HLA molecules in your own lab allowing you to make your own custom mono- or tetramers from day-to-day, similar to what you would only find in a tetramer core facility service.
• The easYmers® are stable for >18 months at -20°C in its “empty” peptide receptive conformation.
• All easYmers® are 100% biotinylated providing maximum flexibility in terms of choosing flow colours or metal-chelated streptavidin.
• Being natural MHC molecules, there are no UV-exchange or artificial thiol-bonds with easYmers®.
• Use the easYmers® to screen a large number of different epitopes or make a large amount of a single HLA class I tetramer and screen multiple donors.
• You are no longer limited to work on the most common available allotypes – the easYmers® are available in more than 30 allotypes (HLA-A, HLA-B and HLA-C). See our full range of available allotypes here.
• You can use our flow cytometry based protocols to assess the folding efficiency of your custom peptide HLA complexes.
• Keep your epitope sequence proprietary as there is no need to disclose the sequence to a custom tetramer manufacturer.

Read more about easYmers® here

easYmers® can be used to:

• detect and monitor antigen-specific T cells
• isolate and enrich antigen-specific T cells
• charge artificial APCs (antigen specific cells) with specific peptide-MHC complexes to stimulate T cells
• engineer TcRs for improved stability or affinity
• screen for TcR-like antibodies/reagents

Read more about easYmers® here

No, there are no special equipment requirements to set up an easYmer® folding. Our easYmer® kit provides you with all reagents needed for you to generate specific peptide-HLA class I monomers of your choice in your own laboratory – simply add your peptide and incubate.

Each lot of our easYmers® undergoes functionally testing with positive control peptides to validate the folding efficiency. Additionally, a control peptide is provided with the easYmer® kit, allowing you to validate your peptide-HLA complexes in your lab. Assessment of peptide loading.

MHC class I molecules typically binds nonamer (9-mer) peptides but other peptide lengths may be relevant. Especially for MHC class I it is important to identify the optimal peptide you wish to bind to the MHC molecule of interest. We recommend using a MHC binding prediction tool before making tetramers with easYmers® or ordering a tetramer at: NetMHCPan 4.1 server

We offer to measure the stability of your peptides in complex with MHC class I molecules. Most of our MHC class I molecules (a total of 86 molecules covering HLA-A | HLA-B | HLA-C and H-2) that are available for custom class I tetramer production are also available for stability assessments. Read more about MHC class I stability.

Yes, we offer to measure the stability and report the results on unknown peptides sequences – hence you can keep the peptide sequences confidential if so desired. The results will be delivered in a report, briefly describing the experimental setup and the stability data reported as half-lives accompanied by dissociation curves for each measured peptide-MHC combination.

Follow the links below to find protocols or Material Safety Data Sheet:

• Protocols
• MSDS Europe