Method for producing an examination reagent and kit for analysing a T-cell frequency
A method for producing an examination reagent includes adding a helper ligand to a receptor protein which is unfolded in an initial solution so as to provide a pre-solution of the examination reagent with a receptor protein which is folded. The receptor protein which is folded comprises a bonded helper ligand which can be exchanged with an examination peptide.
1. A method for producing an examination reagent, the method comprising:
providing and adding a helper ligand peptide to an unfolded MHC class I receptor protein and β2m in order to produce a folded MHC class I receptor protein, wherein the helper ligand peptide consists of a peptide of 8 to 11 amino acid residues in length and has a lower affinity for a folded MHC class I receptor protein than that of an examination peptide of interest;
adding a glycyl-leucine (GL) dipeptide as a ligand exchanger/ligand releaser to the folded MHC class I receptor protein, wherein the GL dipeptide has a lower affinity for the folded MHC class I receptor protein than the affinity of the helper ligand peptide for the folded MHC class I receptor protein; and
adding an examination peptide having a higher affinity for the folded MHC class I receptor protein than the affinity of the helper ligand peptide for the folded MHC class I receptor protein, so that the examination peptide is exchanged for the helper ligand peptide, thereby providing the examination reagent, and wherein the MHC class I receptor protein is H-2K b .
2. The method as recited in claim 1 , wherein the β2m is human β2m.
3. The method as recited in claim 1 , wherein the helper ligand peptide is FAPGNYPAL (SEQ ID NO: 2) and the β2m is human β2m.
4. The method as recited in claim 1 , wherein the unfolded MHC class I receptor protein is produced in an expression system.
5. The method as recited in claim 1 , further comprising:
attaching a signaling element to the unfolded or folded MHC class I receptor protein,
wherein the signaling element is a fluorescent dye.
6. The method as recited in claim 1 , further comprising:
attaching an anchor element to the unfolded or folded MHC class I receptor protein,
wherein the anchor element is a biotin molecule.
7. The method as recited in claim 1 , further comprising:
providing a plurality of folded MHC class I receptor proteins and assembling the said plurality of folded MHC class I receptor proteins to form a multimer,
wherein at least one of the folded MHC class I receptor proteins comprises the helper ligand.
8. The method as recited in claim 7 , further comprising:
attaching a multimer signaling element to the multimer,
wherein the multimer signaling element is a fluorescent dye.