Preparation Protocol - MHC Tetramer Laboratory

Introduction

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 labeled with phycobiliproteins such as phycoerythrin (PE) or allophycocyanin (APC) usually give the brightest signals and have been used in most experiments.

Please note that many commonly used flow cytometers do not have the capacity to measure APC (e.g. the FACScan, the Coulter EPICS XL). When using these instruments, PE is the recommended choice for the tetramer label.

Method

Choose the appropriate fluorescent label.
Calculate the total moles of biotinylated MHC in the purified stock. Our biotinylated MHC is typically stored in 100 µl aliquots at a concentration of 2 mg/ml.
µMoles of MHC = (100 µl x 2mg/ml) / (MW of MHC heavy chain + light chain + peptide)

Calculate the concentration of biotin binding sites (BS) in the streptavidin stock.

To perform this calculation, it is necessary to know the concentration of the streptavidin conjugate, whether the concentration is expressed in terms of streptavidin content alone or in terms of the total mass of streptavidin conjugate, and the ratio of label to streptavidin. As an example, Molecular Probes provides streptavidin-PE conjugates at a concentration of 1 mg/ml (equivalent to 1 g/liter). The concentration refers to the mass of the total conjugate, not just the streptavidin moiety. In addition, the material from Molecular Probes is size selected to have a 1:1 ratio of phycoerythrin:streptavidin. The average molecular weight of a 1:1 streptavidin-PE conjugate is 300,000 Da. Therefore, the concentration of biotin binding sites is:

(1 g/L) / (300,000 g/mol) x (4 Biotin BS/mol) = 1.33x10^-5 M
This is equivalent to 1.33x10^-11 moles per µl.

A table of the average number of biotin binding sites for additional streptavidin conjugates is included below.

Label Molecular	Weight	mg/ml	Moles Biotin BS/ µl
Phycoerythrin	300,000	1	1.33 x 10^-11
Allophycocyanin	164,000	1	2.44 x 10^-11
FITC	60,000	1	6.67 x 10^-11

Calculate the volume of streptavidin conjugate that must be added to give a 1:1 ratio of biotinylated MHC to biotin binding sites.
For example, when adding streptavidin-APC to 200 µg biotinylated MHC, the proper total volume of streptavidin-APC is 174 µl.
Add the streptavidin slowly - in 1/10 volume aliquots - waiting 10-15 minutes between each addition. This process performed at room temperature, and the tube containing the MHC and streptavidin is stored in the dark (e.g., in a drawer).
If the volume of streptavidin required has been overestimated, this procedure will produce the maximum concentration of tetramers because all biotin binding sites will be saturated after the early additions.
Store the tetramer at 4°C in an amber polypropylene tube in a covered box. Tetramer stocks prepared in this way have been used for 2-3 months.
As with most phycobiliprotein conjugates, freezing of the tetramers is not generally recommended, although reagents that have been frozen may retain some activity.

Final Words

Because of the different molecular weight of each MHC allele and the peptide, the amount of streptavidin conjugates needed in each tetramer preparation varies. However, the variation is quite small. To avoid the pain of all the calculations, you can follow a general rule:

Streptavidin APC: 174 µl total per 200 µg of MHC monomer
Streptavidin PE: 318 µl total per 200 µg of MHC monomer