Reversible MHC multimer staining for functional purification of antigen-specific T cells

A method for the reversible staining of a target cell, said target cell comprising a receptor molecule on its surface, with a detectable label, the method comprising:

• A) contacting a sample of cells comprising said target cell with
• (i) a receptor molecule binding reagent comprising (a) a ligand which specifically binds to said receptor molecule to form a first binding pair and (b) a first partner of a second binding pair, wherein said ligand and said receptor molecule exhibit a binding interaction having a dissociation constant (Kd) in the range of 10−2 to 10−7 M, and wherein said first partner binds to a second partner to form a first partner:
• second partner second binding pair,
• (ii) a second partner which forms part of said first partner:
• second partner second binding pair, wherein said second partner has at least two binding sites for said first partner, each binding site capable of forming a reversible bond with said first partner, wherein the dissociation constant (Kd) of said reversible bond is in the range of 10−2 to 10−13 M, and
• wherein multivalent complexes that bind to said target cell are formed by the binding of at least two said ligand-containing receptor molecule binding reagents to one said second partner, said multivalent complexes providing increased avidity, relative to the binding of a single ligand to said target cell, due to the presence on each multivalent complex of at least two of said ligand-containing receptor molecule binding reagents;
• and
• (iii) said detectable label, wherein said detectable label is bound to or binds to said ligand-containing receptor molecule binding reagent and/or said second partner,
• wherein said target cell is stained by binding of said multivalent complexes to said target cell, and wherein staining of said target cell is reversible upon disruption of the reversible bond between said first partner and said second partner.