Preparation empty peptide-receptive MHC class I complex for large-scale detection through photolabile peptide ligands.
Int J Biol Macromol
; 276(Pt 1): 133781, 2024 Jul 09.
Article
in En
| MEDLINE
| ID: mdl-38992528
ABSTRACT
Peptide-major histocompatibility complex (pMHC) multimers are wide recognized as the premier technique for detecting, characterizing, and isolating antigen-specific CD8+ T-cell subsets. These multimers are specifically useful in studying infections, autoimmune conditions, and cancer through single-cell analysis techniques such as flow cytometry and fluorescence microscopy. However, the development of high-throughput assays with commercially available pMHC tetramers can be expensive, while in-house production may pose challenges for most biology research laboratories. In this context, we introduce a cost-friendly and uncomplicated protocol to prepare empty MHC class I tetramers using disulfide-stabilized molecules and photolabile peptide ligands. Our method relies on disulfide bond-stabilized MHC-I molecules, which demonstrated stability when folded into stable monomers in the presence of a photolabile epitope. These monomers, upon ultraviolet irradiation and streptavidin binding, efficiently assemble into tetramers devoid of any peptide. Following a short incubation with the peptide of interest under gentle conditions, the resulting pMHC tetramer effectively detects patient-sourced, neoantigen-specific T cells. Our unique approach streamlines large-scale pMHC generation, thus paving the way for advancements in T cell-based diagnostics and personalized therapies.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Language:
En
Journal:
Int J Biol Macromol
Year:
2024
Document type:
Article
Affiliation country:
China