Beckman Coulter's Immune Monitoring tools for the comprehensive evaluation of the Cellular Immune Response include: the iTopia™ Epitope Discovery System and the iTAg™ MHC Tetramer portfolio for Class I and Class II molecules. For Research Use Only. Not for diagnostic procedures.
With the iTAg™ MHC Tetramer portfolio for Class I and Class II molecules, antigen specific T cells can be accurately detected at a single cell level.
The iTopia Epitope Discovery System helps researchers advance important candidates quickly through the development process.
The Streptamer technology is a novel method to isolate and characterize functional antigen-specific T-cells.
Tetramers
Beckman Coulter's iTag™ MHC Tetramers are complexes of four MHC (Major Histocompatibility Complex) molecules, which are associated with a specific peptide and bound to a fluorochrome. There are two types of Tetramers, Class I and Class II. Class I Tetramers bind to a distinct set of T cell receptors (TCRs) on a subset of CD8+ T cells, and Class II Tetramers bind to a distinct population of CD4+ T cells. Beckman Coulter's patented MHC iTag tetramers reduce non-specific bindings to allow unique detection and enumeration of Ag specific T cells. For Research Use only. Not for use in diagnostic Procedures.
A Whole-Genome Association Study of Major Determinants for Host Controls of HIV-1
Rick A. Brooimans
2008
Analytical
Performance of a Standarized Single-Platform MHC Tetramer Assay for the
Identification and Enumeration of CMV-Specific CD8+ T Lymphocytes
Citations
Transplant research applications
Cytometry Part A, Vol 73A, November 2008 Special Issue:
Measurement of Antigen-Specific Immune Responses. Author: Mike Keeney,
"Analytical Performance of a Standardized Single-Platform MHC Tetramer for the
Identification and Enumeration of CMV-Specific CD8+ T Lymphocytes"
Le Boulaire C, Bossy D, Brinkmann A, Grohal S, Webner S, Klingebiel
T, Koehl U, Krueger L, Lofaro L. Detection and enumeration of CMV
antigen-specific T lymphocytes to assess the immune status after stem
cell transplantation. 3rd International Symposium on the Clinical
Use of Cellular Products, Cellular Therapy, Regensburg, Germany, March,
2005.
Gratama JW, Brooimans RA, Lowenberg B, Sullivan LA, Gasior GH, Boyce
CS, Southwick PC, Cornelissen JJ. Immune monitoring with iTAg MHC
tetramers: Prediction of recurrent CMV infection in stem cell
transplant (SCT) recipients. 10th International CMV/Betaherpesvirus
Workshop, Williamsburg, Virginia, April, 2005
Lofaro L, Broyles D, Chamberlin J, Johnson C, Boyce C, Faye D, Bossy
D, Gautherot E, Bray K. Analytical performance and preliminary
clinical assessment of iTAg
™ MHC CMV Tetramers in measurement of
CMV-specific T cells in whole blood.
5th Euroconference on Clinical Cell Analysis and 8th European Working
Group on Clinical Cell Analysis (EWGCCA) Workshop: Standardization of
Novel Assays for Clinical Cell Analysis, Athens, Greece, September, 2005
1. What are iTAg MHC Tetramers? There are two types of Tetramers, Class I and Class II. MHC Tetramers are complexes of four MHC (Major Histocompatibility Complex) molecules,
which are associated with a specific peptide and bound to a fluorochrome. Class I Tetramers bind to a distinct set of T cell receptors (TCRs)on a subset of CD8+ T cells, and Class II Tetramers bind to a distinct population of CD4+ T cells. Thus, by mixing Tetramers with PBMCs or whole blood and using flow cytometry as a detection system, a count of all
CD4+ or CD8+ T cells that are specific for one peptide and its matched MHC allele is provided, regardless of functionality. Simply stated, iTAg
reagents will allow us to measure the cellular response directed toward single peptide specificity. The Class I Tetramers, (human and rhesus
macaque) have been mutated to minimize binding of the MHC molecule to CD8 cell surface. These reagents show diminished CD8-mediated binding to the general CD8 positive lymphocyte population, but retain
MHC peptide-specific binding to TCR thus facilitating accurate discrimination of rare, specific T cells (less than 1% of CD8+).
2. For what diseases has research been conducted? Examples of MHC Tetramer Research Applications:
Infectious Diseases: HIV-AIDS, EBV, CMV,HPV, HBV, HCV, Influenza, Measles, Malaria, TB and RSV Cancer: Breast, Prostate, Melanoma, Colon, Lung, Cervical, Ovarian and Leukemia Autoimmune Diseases: Diabetes, Lyme disease, Multiple sclerosis, Rheumatoid arthritis, Autoimmune vitiligo Transplantation: EBV and CMV
Relevant peptides have been identified for each of these applications and can be customized for unique applications. For more information on the application of iTAg MHC Tetramers in many of these diseases please see our list of Publications
3. What are the advantages of iTAg MHC Tetramer analysis in measuring cellular immune response? Recent technological advances have now made possible one of the long-term goals of T-cell assays, namely to identify individual T cells on the basis of the specificity of the binding to the MHC-peptide complex.
The Tetramer-analysis method has already been used to study CD8+ T-cell responses in humans with acute viral infections, such as HIV. Interestingly, in these cases, Tetramer analysis has revealed that the increase of antigen-specific CD8+ T cells during the acute phase of the response is far greater than previously thought.
Due to the specificity of Tetramer technology, the MHC type of the patient must be known and the specific peptide identified and isolated. However, Tetramer analysis offers many potential advantages over some of the more traditional T cells assays.
This method is quantitative.
It does not require the use of radioactive isotopes.
It is rapid, allowing fresh blood (or tissue-derived) samples to be analyzed, and large numbers of samples to be processed.
Because the analysis is performed using a flow cytometer, cells can be labeled with the Tetramer and other cell-surface molecules at the same time. This allows additional characterization of the responding cells.
Tetramer staining does not kill the labeled cells; therefore, the cells can be sorted into uniform populations by flow cytometry and placed into additional assays to study functionality.
Specific T cells can be analyzed from blood samples without the prerequisite of in vitro culture. Some populations of CTLs (Cytotoxic T Lymphocytes) that have been expanded in vivo might have limited growth potential in vitro and, therefore, might not be detected by other technologies with any degree of accuracy.
All specific CTLs are detected, regardless of their functional status. iTAg MHC Tetramers can be used in conjunction with the iTAg MHC Tetramer IFN gamma kit to simultaneously quantitate antigen specific T cells and IFN gamma production.
4. What is the shelf life of iTAg reagents? The shelf life of Premium iTAg Tetramers such as HIV pol, HIV gag,
Melanoma MART-1, CMV, EBV, and influenza Tetramers is a minimum 12 months from the date of manufacture when stored at 2-8°C. Opened vials
of iTAg Tetramer are stable for 90 days when stored at 2-8°C.
Stability data is not available for Custom iTAg MHC Tetramer reagents.
5. How should the Tetramers be stored? Tetramers should be stored at 2-8°C. They should not be frozen
and exposure to light should be minimized. Vials should be refrigerated
at 2-8°C immediately after use.
6. What is the recommended labeling procedure? Whole Blood
Collect blood by venipuncture into a blood collection tube containing
an appropriate anti-coagulant.
To each 12x75 mm test tube add 10 uL of iTAg MHC Tetramer
and any additional antibodies (e.g. anti-CD8).
Add 200 uL of whole blood into each test tube.
Vortex gently.
Incubate for 30 minutes at room temperature protected from light.
Lyse red blood cells using 2 mL of iTAg HMC Tetramer Lysing Solution supplemented
with 50 uL iTAg HMC Tetramer Fixative Solution per tube.
Vortex for 5 second immediately after the addition of the iTAg HMC Tetramer
Fixative solution.
Incubate for a minimum of 10 minutes at room temperature protected
from light.
Centrifuge tubes at 150 x g for 5 minutes.
Aspirate or decant the supernatant.
Add 3 mL of PBS.
Centrifuge tubes at 150 x g for 5 minutes.
Aspirate or decant the supernatant.
Resuspend the pellet in 500 uL of PBS with 0.5% paraformaldehyde
or formalin.
Store at 4°C in the dark and analyze by flow cytometry within
24 hours.
Mononuclear Cell Preparation
Prepare peripheral blood mononuclear cells (PBMC) according
to established procedures. Cells should be resuspended at a concentration
of 5 x 10E6 cells/mL. 200 µL of sample is required
for each iTAg Tetramer determination.
To each 12x75 mm test tube add 10 uL of iTAg MHC Tetramer
and any additional antibodies (e.g. anti-CD8).
Add 200 uL PBMC into each test tube(e.g. 1 x 10E6 cells
per tube).
Vortex gently.
Incubate for 30 minutes at room temperature protected from light.
Add 3 mL of PBS.
Centrifuge tubes at 400 x g for 5 minutes.
Aspirate or decant the supernatant.
Resuspend the pellet in 500 uL of PBS with 0.5% paraformaldehyde
or formalin.
Store at 4°C in the dark and analyze by flow cytometry within
24 hours.
7. What can be done about high background staining? Premium iTAg MHC Tetramer labeling has been optimized using the cell
preparation procedure outlined above (i.e. 10 uL of iTAg MHC Tetramer
for 200 mL of whole blood or 200 mL of PBMC). However, it is important
to evaluate the amount of background labeling. This may be accomplished
using the iTAg MHC Negative Tetramer. It is also recommended that Custom
MHC Tetramer reagents be titrated to determine the optimum concentration.
8. How is Tetramer staining affected by binding to CD8? Beckman Coulter has obtained an exclusive license to utilize a mutated
Class I in human and rhesus macaque iTAg MHC Tetramer manufacturing.
This mutation greatly diminishes nonspecific MHC binding to CD8, but
retains peptide specific MHC binding to TCR. For additional information,
a reprint of the article Efficient detection and immunomagnetic sorting
of specific T cells using multimers of MHC Class I and peptide with reduced
CD8 binding by Bodinier, et al, published in Nature Medicine,
Vol. 6, Number 6, June 2000, is available.
9. Do CD8 antibodies affect the binding of Tetramer to
specific CD8 T cells? The mutation that greatly reduces the binding of iTAg Tetramer to human
and rhesus macaque CD8 also minimizes the aberrant effect that some CD8
antibodies have on the specific binding of MHC Class I Tetramer.
10. What are the recommendations for positive and negative
controls? For positive controls, cell lines or frozen cell samples displaying
the antigen specificity of interest should be used. For negative controls
for Class I Tetramer, a cell sample can be labeled with iTAg MHC Class
I Negative Tetramer.
11. How do I order iTAg MHC Tetramer reagents? The easiest way to order iTAg MHC Tetramers is online by clicking
here
This is a secure section of the website and we will initiate your order
immediately.
To initiate the Custom Tetramer Solution process for iTAg Custom MHC
Tetramers, click here and select the CUSTOM category to send an inquiry
or call toll free at 1-866-485-1270. Outside the US, call 33 49 1 1727273520.
12. What Class I and Class II alleles are available? At present we are able to provide:
Class II Tetramers
HLA-DRB1*0101
HLA-DRB1*0401
HLA-DRB1*1501
HLA-DRB1*0301
HLA-DPB1*0401 Human Class I Alleles
HLA-A*0101
HLA-A*0201
HLA-A*0301
HLA-A*1101
HLA-A*2402
HLA-B*0702
HLA-B*0801
HLA-B*1501
HLA-B*2705
HLA-B*3501
HLA-B*5701 Murine Class I Alleles
H2-Kb
H2-Kd
H2-Db Rhesus Macaque Alleles Mamu-A*01
Get 10% off on your first Tetramer, iTopia or Streptamer web order.
Enter the promotional code WEB026710 in the coupon field of our checkout page which appears when you click the SUBMIT ORDER button on the order wizard below.
The science behind the iTopia Epitope Discovery System, which mimics in vivo peptide binding reactions, help researchers advance important candidates more quickly through the development process. It does so not only by facilitating peptide identification and ranking based on binding characterization, but also through its ability to assist the selection of individuals populations most likely to respond. This may improve the productivity of clinical trials and can form the basis for an immune monitoring system to follow individuals. For Research Use only. Not for use in diagnostic Procedures.
1. What are the Peptide Specifications for the Epitope Mapping System Assay?
The assay was designed to use peptides with a purity of > 80%. It is very important to consider the real peptide content. This means measuring the net peptide content or considering the contribution to the mass of the counter ions derived from the buffer used for the peptide purification. The most common counter ions include trifluoroacetic acid (TFA), sodium (Na), phosphate (PO3), and acetic acid (AcOH).
2. How many alleles are available and what percentage of the world wide population is covered?
We offer eight different Class I HLA alleles at this time. They are HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-A*1101, HLA-A*2402, HLA-B*0702, HLA-B*0801, HLA-B*1501. 85&43;90% of the world population will express one or more of these alleles according to the MORI database and The HLA Facts Book (Marsh, S.G.E., Parham, P., Barber, L.D. The HLA Facts Book, Academic Press, 2000).
3. Can one peptide bind different alleles?
Yes, it has been reported that there can be cross reactivity of peptides on more than one allele. However the peptides ability to form a stable peptide-MHC complex can vary between alleles.
4. What is the optimum peptide length?
MHC Class I molecules bind peptides that are between 8 to 12 amino acids in length. The most common size is a 9. The length of the peptides will be allele dependent.
5. Can one peptide have good affinity but poor stability?
Yes. During feasibility testing we found peptides that have comparable affinities, being relatively high, but the ability to form a stable peptide-MHC complex varies between the peptides, with some being very low. In other words, a peptide may have high affinity for the MHC and binds relatively quickly, but has a relatively rapid rate of dissociation. The reverse can also be true. A certain peptide may take longer to bind initially, but once it does bind, the rate of dissociation it has is relatively slow.
6. What is the iScore?
The iScore is a proprietary multi-parametric calculation that enables you to easily visualize the Off-rate and Affinity values. By ranking your results in descending order, you can easily see the peptides that have high affinity and slow rates of dissociation from the MHC. These will be the peptides you may want to consider immediately for functional studies.
7. How is the iScore calculated?
iScore is a calculation (formula) that produces a value that combines the binding, affinity (ED50) and off-rate (t1/2) resulting in a single number.
8. How were the control peptides selected?
The control peptides were selected from the scientific literature taking in to consideration their biological relevance and their affinity and stability.
9. How comparable is the assay with the T2 assay?
The assay is not comparable to the T2 assay. The variability linked to the differences in the expressed molecules on the cell surface has been eliminated.
10. How can I test the biological relevance of the selected positive peptides?
The biological relevance of peptides can be tested using iTAg™ MHC Tetramers and iTAg MHC Tetramer IFN - assays, using patient samples.
11. Do you plan to develop this assay for Class II?
Yes. The feasibility testing is in process.
12. How do I order iTopia products?
The easiest way to order iTopia products is online by using the on-line order wizard below This is a secure section of the website and we will initiate your order immediately. You can also click here to send an inquiry or call toll free at 1-866-485-1270. Outside the US, call 33 49 1 1727273520.
Get 10% off on your first Tetramer, iTopia or Streptamer web order.
Enter the promotional code WEB026710 when checking out using the online order wizard below.
The Streptamer technology is a novel method to isolate and characterize functional antigen-specific T-cells. Streptamer enable for the first time a reversible, antigen-specific staining of T-cells, leaving the isolated T-cells phenotypically and functionally indistinguishable from untreated cells. Streptamer-isolated T-cells may offer new strategies for treating uncurable diseases including viral infections and cancer by adoptive T-cell transfer. In addition, the technology is likely to advance basic T-cell research as an essential prerequisite for novel immunological medicines. For Research Use only. Not for use in diagnostic Procedures.
A recent publication by Dirk H. Busch (et al.), the inventor of the Streptamer technology, was featured in the June 2002 issue of Nature Medicine. It introduced the Streptamer technology as a method to isolate, for the first time, fully functional antigen-specific T-cells. Experiments in mice demonstrate that the Streptamer technology is capable to provide antigen-specific T-cells, which efficiently can be applied in adoptive transfer protocols. Reference: Knabel M et. al., 2002, Nature Medicine 6: 631-637.
Streptamer - a break-through in T-cell research
T-cells and immune response T-cells represent probably the most important arm of the adaptive immune system. They are capable to orchestrate, regulate, and coordinate complex immune responses. Thus, T-cells are in the centre of the body's acquired immune system, which can specifically attack foreign antigens, while still being able to discriminate self from non-self. 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.
The major histocompatibility complex (MHC) T-cell reactivity depends on unique cell surface molecules called "major histocompatibility complexes" (MHC), which present antigen fragments to the T-cell receptor. Just at this complex interface the specific link is made for the host to recognize invading pathogens or to control cancer cells. This specific interaction can be exploited to stain and even to isolate specific T-cell clones by the following proceeding.
MHC multimer staining This method includes the advantages of conventional MHC multimer staining procedures, but it further allows removal of the staining reagents at 4° C before negative effects of ligand-binding can begin to take place. The field of development of T-cell-based therapeutics and diagnostics is constantly growing, and we are currently evaluating several promising clinical applications using Streptamer technologies. For example, adoptive transfer therapies based on directly ex vivo sorted or more efficiently ex vivo expanded T-cells would benefit from the Streptamer technology. Furthermore, expanded T-cell lines or clones can be easily purified from contaminating stimulator cells or cell debris before transfer to clinical applications. Thus, Streptamer-sorted T-cells comprise a highly defined population reducing the probability of unwanted side effects.
The Streptamer® approach
Using IBA's Strep-tag technology to tag MHCs The Streptamer approach is based on a reversible staining procedure that utilizes IBA's core Strep-tag® technology to tag MHCs which are non-covalently attached to fluorescently labeled Strep-Tactin or Strep-Tactin coupled to magnetic beads. Highly stable streptavidin derivatives are optimized for binding Strep-tag, a short peptide sequence with affinity for the biotin-binding site of Strep-Tactin. MHC/peptide complexes fused with a specifically engineered Strep-tag sequence are generated and multimerized in the presence of Strep-Tactin. This complex - called "Streptamer" - is ideally suited to bind and isolate cognate T-cells by fluorescence activated cell sorting (FACS) or magnetic assisted cell sorting.
Truly reversible staining and fully functional T-cells A key aspect of the Streptamer approach is the ability to rapidly remove the staining reagent after the isolation step by disassembly of the MHC/Strep-Tactin multimer in the presence of low concentrations of d-biotin at 4° C, which competes with high affinity for the binding of Strep-tag to Strep-Tactin. The weak binding monovalent MHCs dissociate spontaneously and rapidly from the T-cell surface. The realization of a truly reversible staining enables for the first time the collection of fully functional T-cells that are indistinguishable from untreated cells. This is in contrast to the unfavorable effects of conventional MHC multimer staining techniques, especially regarding the consequences for the outcome of adoptive T-cell transfers.
Several factors are important for a reversible T-cell staining technique:
Rapid and complete monomerization at low temperatures
The extreme affinity differences between Strep-tag / Strep-Tactin and d-biotin /
Strep-Tactin allow complete and very fast binding of d-biotin, even at very low temperatures (preferably at 4°C).
Procedure is non-toxic to T-cells d-biotin (vitamin H) in low concentrations, which are sufficient for Streptamer disassembly, is non-toxic for T-cells.
Substances must be harmless for (clinical) in vivo applications
The amounts of d-biotin that might be transferred with reversibly stained T-cells are far lower than d-biotin concentrations found in conventional vitamin supplementations and therefore unlikely to be harmful.
Thus, the Streptamer technology provides an optimal solution for reversible, antigen-specific T-cell staining and isolation, and it represents the essential gateway for new innovative clinical applications.
T-cell isolation by Fluorescence Assisted Cell Sorting (FACS)
Fluorescently labeled Streptamers can be used for T-cell staining or T-cell isolation using Fluorescence Assisted Cell Sorting (FACS).
Advantages:
· Reversible, antigen-specific staining of T-cells.
· Isolation and characterization of functional T-cells
· High staining intensities
· Mild and rapid dissociation of staining reagents using biotin
· Improved viability of T-cells after isolation
· FACS analysis with Phycoerythrin (PE) or Allophycocyanin (APC)
Fluorescent Streptamer®
T-cell isolation by magnetic cell separation
Streptamer® Magnetic Beads provide a rapid and economic alternative for T-cell isolation at 4°C under sterile conditions by Magnet Assisted Cell Sorting.
We are pleased to announce the new Streptamer Magnetic Beads for T-cell isolation by magnetic cell separation. Due to our unique Streptamer technology positively selected antigen-specific T-cells are phenotypically and functionally indistinguishable from untreated cells. In addition to IBA's fluorescent conjugates allowing for sensitive T-cell detection and T-cell isolation by FACS, the paramagnetic beads enable a rapid and economic T-cell isolation at 4°C under sterile** conditions.
Advantages:
· Easy-to-handle T-cell purification with magnets
· Fast and sterile
· Reversible T-cell labelling
· Functional isolation and characterization of T-cells
· Mild, rapid and complete removal of Streptamer Beads using biotin
· Improved viability of T-cells after isolation
· Bead isolates can be ideally detected with fluorescent conjugates
11. How do I order
Streptamer? The easiest way to order Streptamer products is online by using the on-line order wizard below
This is a secure section of the website and we will initiate your order
immediately. You can also click here to send an inquiry
or call toll free at 1-866-485-1270. Outside the US, call 33 49 1 1727273520.
Get 10% off on your first Tetramer, iTopia or Streptamer web order.
Enter the promotional code WEB026710 when checking out using the online order wizard below.
To order, use our online order wizard below or click on these links to view our Immune Monitoring
Solutions as a list.
TETRAMERS - iTOPIA -
STREPTAMER. For custom products we recommend using the on-line wizard
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