Flex-T™ HLA Class I ELISA Control

Product Details

Verified Reactivity

Human

Formulation

PBS (pH 7.5) containing 0.5% BSA, protease inhibitors, 0.05% sodium azide.

Concentration

0.2 mg/ml

Storage & Handling

Store frozen (-20 or -80°C); shipped in blue ice. Aliquot upon receipt before freezing; avoid repeated freeze-thaw cycles.

Application

ELISA - Quality tested
FC - Verified

Recommended Usage

Recommended final concentrations as positive controls are 2.7nM, 1.4nM, and 0.7nM for HLA Class I ELISA. Use 5 μl to start the dilutions as described in the application notes.

Application Notes

Take 5 µl of the solution and dilute it into 7.88ml of assay dilution buffer. This will result in the High control for the HLA class I ELISA (2.7nM). 2X serial dilutions with assay dilution buffer will result in the Medium (1.4nM) and Low (0.7nM) controls. This amount is sufficient for up to 15 plates. However, it is recommended that ELISA controls be prepared freshly for each assay.

To order custom products please fill out the Custom Flex-T™ Request Form.

Application References

(PubMed link indicates BioLegend citation)

1. Altman JD, et al. 1996. Science 274:94-6.
2. Rodenko B, et al. 2006. Nat. Protoc. 1:1120.
3. Toebes M, et al. 2006. Nat. Med. 12:246.
4. Bakker AH, et al. 2008. Proc. Natl. Acad. Sci. USA 105:3825.

Antigen Details

Biology Area

Immunology

Molecular Family

MHC Antigens

Gene ID

NA

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Surface and Intracellular Cytokine Staining for Flow Cytometry - Video
• Flex-T™ HLA Class I ELISA Protocol
• Cell Surface Flow Cytometry Staining Protocol
• Intracellular Flow Cytometry Staining Protocol
• Flex-T™ Tetramer Preparation and Flow Cytometry Staining Protocol

Related Pages & Pathways

Related FAQs

Is there any peptide length recommended?

There are no special requirements for the peptides. Peptides that naturally bind to MHC molecules will bind to Flex-T™ reagents. For class I molecules, typical length is about 8 – 10 amino acids. Class II molecules accommodate longer peptides, about 14 – 20 amino acids¹.

1) Mohan JF and Unanue ER. Unconventional recognition of peptides by T cells and the implications for autoimmunity. Nat Rev Immunol. 2012 Oct;12(10):721-8.

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

The T-cell mediated innate 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 binds to several TCRs, creating a more stable interaction and making it useful for flow cytometric detection of antigen specific T cells.

I am interested in finding novel peptides instead, are there any resources for this?

There are a number of databases and webpages that can help, these are three of them:

http://www.iedb.org/
http://tools-int-01.liai.org/main/

What are the specifications of the UV source?

Long-wave UV, 366 nm, 8 Watts (We recommend, for example, CAMAG cat# 022.9115, or Ultraviolet Crosslinker CL-1000). The distance from the solution to the light source should be 2 – 5 cm (approximately 0.8 – 3 inches).

What is Flex-T™?

Flex-T™ (Flexible-Tetramers) is BioLegend’s brand name for our Soluble MHC product line. It encompasses monomers, the ultraviolet (UV) peptide exchange technology, and all associated products and applications.

Do I need to know the sequence of the UV-labile peptide?

The sequence of the UV-labile peptide is not needed to use the reagent. MHC molecules are not stable without a peptide, so these peptides are used just for two purposes: stabilize the MHC molecules and serve as a place holder to be substituted by the peptide of interest.

Do you offer mouse monomers or tetramers?

We offer a selection of fixed peptide mouse monomers within our catalog. We also produce mouse monomers and tetramers as Custom Flex-T™ Reagents.

Do you offer custom products and services?

Yes, please contact our Custom Solution Team with your request at cst@biolegend.com, or contact your local BioLegend representative.

How do I evaluate the efficiency of the peptide exchange?

Follow the protocol for HLA class I ELISA. An assay positive control is provided (Cat#280301) that can be diluted to a high, medium, and low concentration. Signal intensity can be correlated to affinity of the peptide.

What is the peptide exchange technology and what's the advantage of using it?

Flex-T™ MHC monomers are 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.

Is it feasible to screen not just peptides, but also several specificities of antigen-specific T cells in one sample?

With the traditional pre-assembled Tetramer approach, this is difficult to do, and not cost-effective. With Flex-T™ technology, as there is more flexibility to assemble the tetramers, it is easy and affordable to screen a sample for several specificities². To facilitate this approach, a combinatorial color coding system has been developed. Please visit the Flex-T™ webpage for a detailed description.

2) Hadrup SR et al. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat Methods. 2009 Jul;6(7):520-6.

Is there any advantage in buying biotinylated monomers?

Yes, the same monomer can be assembled with different Streptavidin conjugates, providing great flexibility for color choices. Additionally, it can be stored frozen and the tetramer assembled shortly before the experiment. This increases the storage time of the reagent.

Certificate of Analysis