Altor BioScience - Products - Principles and Procedures

STAR™ Multimer Technology

The cellular expression of specific peptides in a complex with major histocompatibility complex (MHC) class I molecules plays a major role in stimulating and maintaining immune responses to cancer and infectious diseases. In fact, the presence of disease-related peptide-MHC complexes on the surface of tumor or virus infected cells represents a unique target for vaccine and other immunotherapeutic approaches.

Most MHC class I-presented peptides are derived from the degradation of intracellular proteins by the proteosome complex. The resulting peptide fragments are transported into the lumen of the endoplasmic reticulum where they are bound to the antigen-binding groove of the MHC class I molecule. The peptide-MHC complex is then transported to the cell surface for presentation to the adaptive immune system. In some cases, such as vaccine approaches using dendritic cells, exogenous peptides are incubated with cells to allow direct or indirect loading of MHC class I molecules on the cell surface. Thus, the presence of a peptide antigen on the cell surface can be influenced by the level of endogenous or exogenous protein present, appropriate degradation of these proteins in the intracellular compartments, the ability of the degraded peptides to bind the groove of the particular MHC molecules and the successful transport and stability of the peptide-MHC complex on the cell surface.

STAR™ Multimer reagents allow detection of peptide-MHC complexes based on the ability of a given T-cell receptor (TCR) to recognize a specific peptide presented in the context of a particular MHC molecule. STAR™ Multimers are made up on a soluble single-chain TCR containing a biotin moiety added enzymatically via an AviTag™ sequence (1). The biotinylated TCR molecules are multimerized following addition to streptavidin carrying detectable labels. Although the binding affinities between peptide-MHC complexes and TCRs are generally low, the increased avidity of STAR™ Multimers results in an apparent higher peptide-MHC affinity than that observed for the monomeric TCR. Thus, STAR™ Multimer are able to specifically and stably bind endogenous peptide-MHC complexes under a variety of experimental conditions.

Cells stained with STAR™ Multimers can be analyzed by flow cytometric analysis in order to determine the level of antigen presentation in a given population or the frequency of specific antigen presenting cells in a mixed population. STAR™ Multimers also allow assessment of tumor or viral antigen presentation in normal and disease tissues (formalin fixed or frozen) by immunohistochemistry techniques. To standardize dendritic cell-based research and clinical applications, simple direct assays to measure peptide antigen presentation can be readily developed using STAR™ multimers. Finally these reagents can be employed in a variety of methods to characterize TCR-MHC-peptide interactions and identify novel peptide antigens.

Flow Cytometry Procedure

STAR™ Multimers labeled with phycoerythrin can be used to stain peptide-MHC complexes expressed through endogenous antigen presentation or following addition of exogenous peptide (i.e. peptide-pulsed cells). Altor provides peptide-loaded control cells useful in establishing and standardizing staining methods.
To stain cells, 2 x 10E5 cells in 0.1mL FCM buffer (PBS, 0.5% BSA, 0.1% NaN3) are mixed with STAR™ Multimers labeled with PE and incubated at 4°C for 1 hour.
Samples are washed twice with 1 mL FCM buffer, resuspended in 0.5 mL FCM buffer and analyzed by flow cytometry.

Immunohistochemistry Procedure

STAR™ Multimers labeled with horseradish peroxidase (HRP) are designed to detect peptide-MHC complexes expressed on cells or tissues by standard IHC methods. Altor provides cytospin-prepared slides containing peptide-loaded and non-pulsed control cells to facilitate assay development.
To stain cells or tissue sections, slides are blocked with Blocking buffer (1% BSA in PBS) for 30 minutes at room temperature and incubated with STAR™ Multimers labeled with HRP for 2 hours.
After rinsing twice with TBST (50 mM Tris/HCl pH 7.6, 150 mM NaCl, 0.05% Tween-20), slides are treated with Diaminobenzidine (DAB) reagents to visualize staining.
Slides can then be counterstained with hematoxylin, dehydrated (if required), and mounted for microscopy examination.
(1) AviTag™ technology is covered under U.S. Patent No. 5,723,584, 5,874,239, 5,932,433 and 6,265,552. STAR™ Multimers using this technology are sold under a Limited Use License with Avidity, L.L.C. Please see Terms and Conditions for more details.
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© Altor BioScience Corporation, 2011



	STAR™ Multimer Technology The cellular expression of specific peptides in a complex with major histocompatibility complex (MHC) class I molecules plays a major role in stimulating and maintaining immune responses to cancer and infectious diseases. In fact, the presence of disease-related peptide-MHC complexes on the surface of tumor or virus infected cells represents a unique target for vaccine and other immunotherapeutic approaches. Most MHC class I-presented peptides are derived from the degradation of intracellular proteins by the proteosome complex. The resulting peptide fragments are transported into the lumen of the endoplasmic reticulum where they are bound to the antigen-binding groove of the MHC class I molecule. The peptide-MHC complex is then transported to the cell surface for presentation to the adaptive immune system. In some cases, such as vaccine approaches using dendritic cells, exogenous peptides are incubated with cells to allow direct or indirect loading of MHC class I molecules on the cell surface. Thus, the presence of a peptide antigen on the cell surface can be influenced by the level of endogenous or exogenous protein present, appropriate degradation of these proteins in the intracellular compartments, the ability of the degraded peptides to bind the groove of the particular MHC molecules and the successful transport and stability of the peptide-MHC complex on the cell surface. STAR™ Multimer reagents allow detection of peptide-MHC complexes based on the ability of a given T-cell receptor (TCR) to recognize a specific peptide presented in the context of a particular MHC molecule. STAR™ Multimers are made up on a soluble single-chain TCR containing a biotin moiety added enzymatically via an AviTag™ sequence (1). The biotinylated TCR molecules are multimerized following addition to streptavidin carrying detectable labels. Although the binding affinities between peptide-MHC complexes and TCRs are generally low, the increased avidity of STAR™ Multimers results in an apparent higher peptide-MHC affinity than that observed for the monomeric TCR. Thus, STAR™ Multimer are able to specifically and stably bind endogenous peptide-MHC complexes under a variety of experimental conditions. Cells stained with STAR™ Multimers can be analyzed by flow cytometric analysis in order to determine the level of antigen presentation in a given population or the frequency of specific antigen presenting cells in a mixed population. STAR™ Multimers also allow assessment of tumor or viral antigen presentation in normal and disease tissues (formalin fixed or frozen) by immunohistochemistry techniques. To standardize dendritic cell-based research and clinical applications, simple direct assays to measure peptide antigen presentation can be readily developed using STAR™ multimers. Finally these reagents can be employed in a variety of methods to characterize TCR-MHC-peptide interactions and identify novel peptide antigens. Flow Cytometry Procedure STAR™ Multimers labeled with phycoerythrin can be used to stain peptide-MHC complexes expressed through endogenous antigen presentation or following addition of exogenous peptide (i.e. peptide-pulsed cells). Altor provides peptide-loaded control cells useful in establishing and standardizing staining methods. To stain cells, 2 x 10E5 cells in 0.1mL FCM buffer (PBS, 0.5% BSA, 0.1% NaN3) are mixed with STAR™ Multimers labeled with PE and incubated at 4°C for 1 hour. Samples are washed twice with 1 mL FCM buffer, resuspended in 0.5 mL FCM buffer and analyzed by flow cytometry. Immunohistochemistry Procedure STAR™ Multimers labeled with horseradish peroxidase (HRP) are designed to detect peptide-MHC complexes expressed on cells or tissues by standard IHC methods. Altor provides cytospin-prepared slides containing peptide-loaded and non-pulsed control cells to facilitate assay development. To stain cells or tissue sections, slides are blocked with Blocking buffer (1% BSA in PBS) for 30 minutes at room temperature and incubated with STAR™ Multimers labeled with HRP for 2 hours. After rinsing twice with TBST (50 mM Tris/HCl pH 7.6, 150 mM NaCl, 0.05% Tween-20), slides are treated with Diaminobenzidine (DAB) reagents to visualize staining. Slides can then be counterstained with hematoxylin, dehydrated (if required), and mounted for microscopy examination. (1) AviTag™ technology is covered under U.S. Patent No. 5,723,584, 5,874,239, 5,932,433 and 6,265,552. STAR™ Multimers using this technology are sold under a Limited Use License with Avidity, L.L.C. Please see Terms and Conditions for more details. Back © Altor BioScience Corporation, 2011