The MHC class I tetramer facility makes tetramers with the following human and mouse class I alleles: HLA-A2, A3, A11, A24, B7, B8,B27, B35 and H2-Kd, Kb, Db, Ld, M3. We have purified stocks of each of these alleles and also human and murine beta2-microglobulin. We are able to produce two MHC class I tetramers per week. Making a tetramer takes 7 days (if purified heavy chain is available). It is our goal to produce tetramers within 2 - 3 weeks after a request is placed. In general, we will try to make tetramers in the order that they are requested. However, the tetramer facility director or manager may, on occasion, have to change the order due to special circumstances.
The cost of making a Phycoerythrin conjugated tetramer is $800. The most expensive reagents are the biotin ligase (BirA from Avidity, LLC) and the PE-conjugated streptavidin (Prozyme). On occasion, it may be advantageous to use tetramers conjugated with APC, a more expensive fluorophore, which increases the cost of the tetramer to $900. The yield from a tetramer preparation is variable, but typically the product is sufficient for hundreds of stainings, often thousands of stainings. The stability of MHC class I tetramers is variable and is influenced by the affinity of peptide binding by the MHC class I heavy chain. Some tetramers can last over 6 months when properly stored at 4 C and other loose their staining capacity in less than 2 months. Because of this variability, it is important to test the ability of MHC tetramers to stain antigen specific T cells. It is recommended to routinely test new batches of tetramers by staining antigen specific T cells with dilutions of tetramers at concentrations of 1:10, 1:20, 1:40, 1:80 and 1:160. A typical staining dilution is 1:40, but it is critical to determine the optimal working concentration of each tetramer before using it in your experiments.
A standard tetramer preparation requires 12mg of peptide. We recommend ordering peptides from the MSKCC Microchemistry Core Facility (San San Yi x7767) or from a reliable outside vendor (SynPep Inc., BioSyn Inc., Macromolecular Resources) asking them to send the peptide in 12mg aliquots. Peptides cleaved with EDT (ethane dithiol) are substantially better for MHC I refolding, with superior yields and greater stability. Many peptide vendors do not use EDT for cleavage, causing instability of tetramer complexes. Therefore, when ordering peptides for tetramer generation, please request that EDT (ethane dithiol) be used for peptide cleavage. It is not necessary to HPLC purify peptides for tetramer generation, since most crude peptides are generally over 90% pure. Because peptide oxidation during prolonged storage is a significant problem and can decrease or eliminate staining of antigen specific T cells, we recommend ordering fresh peptide for tetramer preparations and storing them under reducing conditions.