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Why iTAg MHC Tetramers?
Modification of the CD8 Binding Region
iTAg Tetramers have a proprietary mutation in the alpha-3 domain of the MHC molecule that greatly diminishes nonspecific binding but retains specific binding. The iTAg Tetramer alteration significantly improves binding specificity, providing superior tools for staining and sorting CD8+ T cells.
Staining with iTAg Tetramers
Staining with iTAg Tetramers allows precise quantitation of antigen-specific T cells by reducing nonspecific CD8/MHC interactions (A). Native Tetramers nonspecifically stain CD8+ lymphocytes in addition to CD8+ antigen-specific lymphocytes, resulting in substantial nonspecific staining (B).*
Nonspecific CD8/MHC interactions can be blocked by addition of anti-CD8 antibody, confirming the contribution of these interactions to native Tetramer staining (data not shown).
Sorting
iTAg Tetramers are more efficient than native Tetramers in sorting specific T cells. Bodinier. Nat Med. 2000;6:707-710.
- Column A: Unsorted synovial fluid T cells stained with native (top) or mutated (bottom) Tetramer. Column B: Cells sorted with native Tetramer, then stained with native (top) or mutated (bottom) Tetramer. Column C: Cells sorted with mutated Tetramer (top), then stained with mutated Tetramer (bottom).
- The activation marker CD25 is induced in T cells sorted with native (left) or mutated (right) Tetramers after 18-hour exposure to NLV-loaded T2 cells.
Conclusion: T cells sorted with native Tetramer cannot be activated by NLV-loaded T2 cells; thus such T cells represent irrelevant CD8+ cells. In contrast, cells sorted with mutated A2/NLV Tetramers stain brightly with native and mutated Tetramers and express CD25 after incubation with NLV-loaded T2 cells.
Quality Manufacturing
- Manufacturing processes are optimized to ensure stable products and reproducible performance
Stability of A*0201 iTAg Tetramers measured by mean fluorescent intensity (MFI) over 1 year (normalized for variable TCR expression).
- Manufactured with GMP-compliant procedures
- All iTAg Tetramer products undergo quality control analysis
- Verified protein content of each manufactured Tetramer product
- <10% free monomer in all manufactured Tetramer products
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