Fluorescent Label
MHC tetramers are typically prepared with most of the commonly used fluorescent labels used for flow cytometry. One major advantage of the technique is that the label is already attached to the streptavidin molecule, eliminating the need for further chemical modification of the MHC / peptide complex. The choice of label is based upon the signal-to-noise ratio of the label, the capabilities of the flow cytometer that will be used for the assay, and the availability of other fluorescent antibodies used in conjunction with the tetramer.
Tetramers provided by our core are labeled with either streptavidin-phycoerythrin (PE) or streptavidin-allophycocyanin (APC), which usually give the brightest signals and have been used in most experiments.
b2-microglobulin
Although we can provide MHC class I tetramers with either human or murine b2-microglobulin, we typically fold all tetramers of every species with human b2-microglobulin. Use of murine b2-microglobulin may reduce the stability of the reagent. Typically staining of murine CD8+ T-cells with tetramer is NOT affected by the use of human b2-microglobulin.
Risk of Failed Productions
One of the factors that determine the success of the tetramer production is the binding affinity of the peptide for the MHC class I molecule. Unfortunately, we cannot accurately predict the binding properties of a peptide according to its sequence. We also do not have specific numbers that would represent a binding threshold for the peptide. Therefore, the failure of tetramer production happens occasionally. In that case, a second attempt for the MHC/peptide folding and chromatographic purification will be tried with no additional charge. If the 2nd attempt also fails, we will terminate the preparation and the order will be partially charged.
Some online databases can help to predict the binding affinity between MHC molecules and peptides. Below are some examples. The positive result returned from searching these databases may not guarantee a successful tetramer production but can be used as a good reference.
- NetMHC: Binding of peptides to MHC class I molecules (DTU Health Tech)
- Epitope prediction: This page allows you to find out the ligation strength to a defined HLA type for a sequence of aminoacids. (syfpeithi)
- IEDB Analysis Resource
- Rankpep: prediction of binding peptides to Class I and Class II MHC molecules (Immunomedicine Group)
Quality Control
Quality control of tetramer reagents produced includes the following:
- All tetramers will be purified by size exclusion and ion exchange chromatography. FPLC chromatograms of the reagent demonstrating proper folding and multimerization will be recorded.
- Each tetramer is tested for the level of biotinylation.
- ELISA using anti b2-microglobulin antibodies specific for properly folded MHC I.
Please direct ordering status inquiries or technical questions to Lily Wang at (713) 798-3918 or lxwang@bcm.edu.
