Tetramer assay
Encyclopedia
A tetramer assay is used to detect the presence of antigen
specific T-cells. In order for a T-cell to detect the peptide to which it is specific, it must both recognize the peptide AND the MHC complex at the surface of a cell it comes into contact with. Because the binding affinity of a T-cell receptor to MHC complexed with a peptide is so low, this has historically been a challenging problem. A solution was realized by creating a tetramer of MHC molecules each presenting an identical peptide. This increases the avidity of the binding.
The main component of the assay is an MHC
tetramer. This reagent has to be custom made for each antigen specific t-cell that one desires to detect. The MHC tetramer consists of four units of the MHC molecule each of which is tagged with a biotin
molecule. A peptide
from the antigen is attached to the MHC molecules. The MHC/peptides are made into a complex by the addition (typically fluorescently labeled) streptavidin
, which has an extremely high affinity to biotin. The MHC with its bound peptide will only bind the specific T-cells that respond to that peptide. The tetramer can be detected by flow cytometry
via the fluorescent label.
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
specific T-cells. In order for a T-cell to detect the peptide to which it is specific, it must both recognize the peptide AND the MHC complex at the surface of a cell it comes into contact with. Because the binding affinity of a T-cell receptor to MHC complexed with a peptide is so low, this has historically been a challenging problem. A solution was realized by creating a tetramer of MHC molecules each presenting an identical peptide. This increases the avidity of the binding.
The main component of the assay is an MHC
Major histocompatibility complex
Major histocompatibility complex is a cell surface molecule encoded by a large gene family in all vertebrates. MHC molecules mediate interactions of leukocytes, also called white blood cells , which are immune cells, with other leukocytes or body cells...
tetramer. This reagent has to be custom made for each antigen specific t-cell that one desires to detect. The MHC tetramer consists of four units of the MHC molecule each of which is tagged with a biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
molecule. A peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
from the antigen is attached to the MHC molecules. The MHC/peptides are made into a complex by the addition (typically fluorescently labeled) streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
, which has an extremely high affinity to biotin. The MHC with its bound peptide will only bind the specific T-cells that respond to that peptide. The tetramer can be detected by flow cytometry
Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
via the fluorescent label.