Streptamer

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The Streptamer technology allows the reversible isolation and staining of antigen-specific T cells. This technology combines a current T cell isolation method with the Strep-tag technology. In principle, the T cells are separated by establishing a specific interaction between the T cell of interest and a molecule that is conjugated to a marker, which enables the isolation. The reversibility of this interaction and the fact that it is performed at low temperatures is the reason for the successful isolation and characterization of functional T cells. Because T cells remain phenotypically and functionally indistinguishable from untreated cells, this method offers new strategies in clinical and basic T cell research.^[1]

Classic methods in T cell research[]

T cells play an important role in the adaptive immune system. They are capable of orchestrating, regulating and coordinating complex immune responses. A wide array of clinically relevant aspects are associated with the function or malfunction of T-cells: Autoimmune diseases, control of viral or bacterial pathogens, development of cancer or graft versus host responses. Over the past years, various methods (ELISpot Assay, , secretion assay) have been developed for the identification of T cells, but only major histocompatibility complex (MHC) procedures allow identification and purification of antigen-specific T cells independent of their functional status.

In principle, MHC procedures are using the T cell receptor (TCR) ligand, which is the MHC-peptide complex, as a staining probe. The MHC interacts with the TCR, which in turn is expressed on the T cells. Because TCR-MHC interactions have only a very weak affinity towards each other, monomeric MHC-epitope complexes cannot provide stable binding. This problem can be solved by using multimerized MHC-epitopes, which increases the binding avidity and therefore allows stable binding. Fluorochromes conjugated to the MHC-multimers then can be used for identification of T cells by flow cytometry. Nowadays, MHC molecules can be produced recombinantly together with the antigenic peptides which are known for a fast-growing number of diseases.

The Streptamer technology[]

The Streptamer backbone[]

The Streptamer staining principle combines the classic method of T cell isolation by MHC-multimers with the Strep-tag/ technology. The Strep-tag is a short peptide sequence that displays moderate binding affinity for the biotin-binding site of a mutated streptavidin molecule, called Strep-Tactin. For the Streptamer technology, the Strep-Tactin molecules are multimerized and form the "backbone", thus creating a platform for binding to strep-tagged proteins. Further, the Strep-Tactin backbone has a fluorescent label to allow flow cytometry analysis. Incubation of MHC-Strep-tag fusion proteins with the Strep-Tactin backbone results in the formation of a MHC-multimer, which is capable for antigen-specific staining of T cells.

Reversible staining[]

Because the molecule d-biotin has a much higher affinity to Strep-Tactin than Strep-tag, it can effectively compete for the binding site.^[2][3] Therefore, a MHC multimer based on the interaction of Strep-tag with Strep-Tactin is easily disrupted in the presence of relatively low concentrations of d-biotin. Without the Strep-Tactin backbone, the single MHC-Strep-tag fusion proteins spontaneously detach from the TCR of the T cell, because of weak binding affinities (monomeric MHC-epitope complexes cannot provide stable binding, see above).

References[]