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 The Strep-tag system is a method which allows the purification and detection of proteins by affinity chromatography. The Strep-tag II is a synthetic peptide consisting of eight amino acids (Tryptophan, Trp-Serine, Ser-Histidine, His-Proline, Pro-G ...

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 low temperatures at which it is performed allows for the isolation and characterization of functional T cells. Because T cells remain phenotypically and functionally indistinguishable from untreated cells, this method offers modern strategies in clinical and basic T cell research.

Classic methods in T cell research

T cells play an important role in the adaptive

immune system The immune system is a network of biological processes that protects an organism from diseases. It detects and responds to a wide variety of pathogens, from viruses to parasitic worms, as well as cancer cells and objects such as wood splinte ...

. 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 An autoimmune disease is a condition arising from an abnormal immune response to a functioning body part. At least 80 types of autoimmune diseases have been identified, with some evidence suggesting that there may be more than 100 types. Nearly a ...

, control of

viral Viral means "relating to viruses" (small infectious agents). Viral may also refer to: Viral behavior, or virality Memetic behavior likened that of a virus, for example: * Viral marketing, the use of existing social networks to spread a marke ...

bacterial Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...

pathogens, development of

cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...

or graft versus host responses. Over the past years, various methods ( ELISpot Assay, intracellular cytokine staining,

secretion assay Secretion assay is a process used in cell biology to identify cells that are secreting a particular protein (usually a cytokine). It was first developed by ''Manz et al.'' in 1995. Usually, a cell that is secreting the protein of interest is isol ...

) have been developed for the identification of T cells, but only

major histocompatibility complex The major histocompatibility complex (MHC) is a large locus on vertebrate DNA containing a set of closely linked polymorphic genes that code for cell surface proteins essential for the adaptive immune system. These cell surface proteins are calle ...

(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 The T-cell receptor (TCR) is a protein complex found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The binding b ...

(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 Affinity may refer to: Commerce, finance and law * Affinity (law), kinship by marriage * Affinity analysis, a market research and business management technique * Affinity Credit Union, a Saskatchewan-based credit union * Affinity Equity Partn ...

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 A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescence, fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromaticity, aromatic groups, or planar o ...

conjugated to the MHC-multimers then can be used for identification of T cells by

flow cytometry Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...

. 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-Tactin technology. The ''Strep''-tag is a short peptide sequence that displays moderate binding

for the

biotin Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bor ...

-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. Additionally, 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.{{cite web , url=http://www.strep-tag.com/ , title=Home , website=strep-tag.com 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

Protein methods Biotechnology Molecular biology techniques Biochemistry Laboratory techniques