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Immune Monitoring Solutions

Immune Monitoring Cellular Immune Response
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. Use the on-line wizard below to order or request a quote. Click on any of the 3 icons for more information, documentation and promotions on immune monitoring products. All Tetramers are sold as 50 tests vials. Suggested use is a standard 10µl per test. Therefore a 500µl volume is provided in each vial.
Tetramers iTopia™ Streptamer™
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 qualified candidates quickly through the development process.
The Streptamer technology is a novel method to isolate and characterize functional antigen-specific T-cells.

Tetramers  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.  (1 vial = 50 tests).
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Tetramer documentation and white papers

Author YearTitle

 
Jacques Fellay 2007 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"  
 

Gratama JW, Brooimans R, Lowenberg B, Sullivan L, Gasior G, Boyce C, Southwick P, Cornelissen J. Immune monitoring with Brooimans RA, Gratama JW, Lowenberg B, Sullivan LA, Gasior GH, Boyce CS, Southwick PC, Cornelissen JJ. Prediction of recurrent CMV reactivation in allogeneic stem cell transplant recipients. Measurement of Antigen-Specific Immune Responses (MASIR), Annual Meeting, Courmayeur, Italy, January, 2005.

 
 

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

 


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Frequently asked Questions

1. What are iTAg MHC Tetramers?

MHC Tetramers are complexes of four Major Histocompatibility Complex (MHC) 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. 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+).

Phycoerythrin: (PE) Fluorochrome CD8+ Human T Cell Fluorescence: CD8

2. For what diseases has research been conducted?
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
Tetramers can be customized for unique applications on our website www.coulterflow.com/tetramer.html.

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 analysis, namely to identify individual T cells on the basis of their specific 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 sequence identified and isolated. However, Tetramer analysis offers many potential advantages over some of the more traditional T cell based assays.
·  Tetramer staining is quantitative.
·  Tetramer staining does not require the use of radioactive isotopes.
·  Staining is rapid, allowing fresh blood (or tissue-derived) samples to be analyzed. Specific T cells can be analyzed from blood samples without the prerequisite of in vitro culture.
·  Large numbers of samples can be processed using a flow cytometer.
·  Cells can be labeled with Tetramer and other cell-surface molecules at the same time allowing additional characterization of the responding cells.
·  Tetramer-labeled cells remain viable and can be sorted into uniform populations by flow cytometry to be placed into additional assays to study functionality.
·  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 detect 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 specific 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 Select and Custom iTAg MHC Tetramers.

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 µL of iTAg MHC Tetramer and any additional antibodies (e.g. anti-CD8). Add 200 µL 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 MHC Tetramer Lysing Solution supplemented with 50 µL iTAg MHC Tetramer Fixative Solution per tube. Vortex for 5 seconds immediately after the addition of the iTAg MHC 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 µL 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 106cells/mL. 200 µL of sample is required for each iTAg Tetramer determination. To each 12x75 mm test tube add 10 µL of iTAg MHC Tetramer and any additional antibodies (e.g. anti-CD8). Add 200 µL PBMC into each test 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 µL 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 µL of iTAg MHC Tetramer for 200 µL of whole blood or 200 µL of PBMC). However, it is important to evaluate the amount of background staining using a suitable staining reagent such as the iTAg MHC Negative Tetramer. It is also recommended that custom MHC Tetramer reagents be titrated to determine the optimum concentration for use.

8. How is Tetramer staining affected by binding to CD8?
Beckman Coulter has obtained an exclusive license to utilize a mutated Class I heavy chain in the manufacture of human and rhesus macaque iTAg MHC Tetramers. This mutation greatly diminishes nonspecific MHC binding to CD8, but peptide-specific/MHC binding to TCR is retained.
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 cells displaying the specific antigen of interest should be used. As a negative control for Class I Tetramer staining, a cell sample can be labeled with iTAg MHC Class I Negative Tetramer.

11. 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

12. General ordering
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.

How do I order tetramer X (or allele X with peptide Y)?
Contact your BCI sales representative, customer service (1-866-485-1270) or order directly online at our new website: www.coulterflow.com.

How much does tetramer X cost?
Contact your BCI sales representative, customer service (1-866-485-1270) or view prices online at our new website: www.coulterflow.com

How do I order peptide? You can request we order the peptide directly from one of our approved vendors when you place your order. There is a peptide acquisition fee. Contact your BCI sales representative or customer service (1-866-485-1270) for details.

How much is the peptide acquisition fee? Contact your BCI sales representative or customer service (1-866-485-1270) for details.

Can I provide my own peptide? Yes, if it meets our requirements for purity and quantity (see below).

How much peptide is needed? What purity is needed? We require a minimum of 20 mgs for HLA-“A” alleles or 50 mgs for HLA-“B” alleles. Peptide purity must be ≥ 90%.  The Certificate of Analysis for that peptide must be shipped with the materials to Beckman Coulter in order to prevent delays to the manufacturing process.

Can I get the leftover peptide back? Yes, but this request must be made at the time you place your order. The remaining quantity of peptide may be only 0-2 mgs.

How long will it take to make the tetramer and ship it to me? Once your order is booked and a purchase order is received, you will be quoted a ship date by customer service. The ship date is contingent on the type of tetramer you request (premium; select; custom).

I would like to order custom peptide Y with allele X. Is it available? Have you made it before? We will check our inventory to see if it is available. If not, we will make an evaluation of the peptide / allele combination and its suitability for manufacture. If we believe the tetramer can be made, we will then process your order.

Do you have allele Y? (e.g. African alleles) We only offer the alleles listed in our catalog / on our website. We cannot prepare custom alleles. If you are interested in an allele that is not listed, please contact your local sales representative or send us an email with your inquiry.

Do you have literature references citing use of your tetramers? For Class I and Class II? Yes, most are CMV related studies. A selection of references is listed under “Documents” on our new website: www.coulterflow.com. To submit your publication for posting, please email a copy of your publication to Beckman Coulter for review.

Do you offer fluorochrome X labeled tetramer? (e.g. FITC; Alexa dyes) For Class I custom tetramers, we offer PE and APC as fluorochromes. For Class II, we only offer PE. We cannot prepare tetramers using other fluorochromes.

Why are the part numbers and lot numbers on the tetramer box and the tetramer vial different? The lot number on the tetramer vial label is the tetramer lot number. The lot number on the box label is the kit lot number which encompasses all the components of the kit: the tetramer, the box, the foam insert, the box label, etc. The part numbers are specific for either a particular tetramer vial or a particular tetramer kit.

What is the concentration of tetramer? We do not specify a tetramer concentration ( 1 vial = 50 tests). The concentration of monomer in the vial is 0.1 mg/ml.

13. Technical/Problems

Can I stain with tetramer and antibodies simultaneously? Yes. For simple 2, 3 or 4 color assays using antibodies conjugated to FITC, PE, APC, PC5, or PC7, you should have no problems. More complex staining experiments (ex. 6, 8, 10 colors) may require titration of reagents and/or reagent order of addition studies to optimize staining results.

What tetramer should I use as a negative control? For Class I? For Class II? For Class I HLA-A*0201 tetramers, our Negative Tetramer is the perfect choice. The Negative Tetramer may also be used as a control for other human alleles but a tetramer of the same allele as the tetramer of interest, but with a non-specific peptide, would be best. This applies to non-human alleles especially.

For Class II DR alleles, tetramers made using the HuClip peptide or a non-specific peptide as described above are good negative controls.

What is the Negative Tetramer? (i.e. What allele/ peptide?) The Negative Tetramer is prepared with the HLA-A*0201 allele and a proprietary peptide whose sequence does not occur in nature. We offer the Negative Tetramer conjugated to either PE (PN T01044) or APC (PN T01054)

What is the “population coverage” for the 5 tetramer CMV kit (PN T01049)? The CMV multi-tetramer kit covers 80-100% of the population, depending on the literature source. It includes the following alleles:

iTAgTetramer/PE-HLA-A*0201CMVPP65(NLVPMVATV)
iTAgTetramer/PE-HLA-A*2402CMV(QYDPVAALF)
iTAgTetramer/PE-HLA-A*0101CMV(VTEHDTLLY)
iTAgTetramer/PE-HLA-B*0702CMV(TPRVTGGGAM)
iTAgTetramer/PE-HLA-B*0801CMV(ELRRKMMYM)
iTAgTetramer/PE-HLA-B*3501CMV(IPSINVHHY)
iTAgTetramer/PE-NegativeTetramer

Which peptide should I chose to make a tetramer for doing experiment X? We recommend customers do a literature search to determine the best peptide / allele for their application. Tech Ops can conduct a limited search to try to assist the customer.

Which CD8-FITC should I use? We recommend BCI’s T8-FITC (PN 6603861).

Which murine allele should be used with mouse X? For C57BL6 mice – Kb and Db alleles For BALB/c mice – Kd, Dd and Ld alleles (Note: we only offer Kd)

Which blood collection tubes are compatible with tetramer use? EDTA, Li Heparin and Na Heparin tubes are all compatible with the tetramer staining protocol.

Can I use a biotinlyated Ab as part of my staining protocol? Yes, but note that there is a small quantity of free biotinylated monomer in the tetramer preparation. This should be considered when planning your staining protocol.

What is the formulation of the fixative reagent, the lyse reagent, and the various buffers? The formulation of the fixative reagent is detailed in IFU No. T10300. Other reagent formulations may be proprietary and are therefore not detailed in the IFUs.

Can tetramers be used with paraffin sectioned tissue? Yes, work has been published on using tetramers for this purpose.

How do I correct the high background I see when I stain T cells with the Kb OVA tetramer and an anti-CD8 antibody? You should perform a cross titration series with the tetramer and the CD8 antibody being used. A good reference is: Current Protocols in Immunology, MHC-Peptide Tetramers to Visualize Antigen-Specific T Cells, Section 17.3.23-24, Supplement 53.


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New Tetramer Products
Part # Category Description                           Buy  To purchase these items enter the part number in the wizard below. Price  
T01064 Select

iTAg Tetramer/PE - HLA-A*0101 (SLLMWITQC)

$1800.00  
T01065 Select

iTAg Tetramer/APC - HLA-A*0101 (SLLMWITQC)

$1800.00  
T01066 Select

iTAg Tetramer/PE - HLA-A*0201(RMFPNAPYL)

$1800.00  
T01067 Select

iTAg Tetramer/APC - HLA-A*0201(RMFPNAPYL)

$1800.00  
T01068 Select

iTAg Tetramer/PE - HLA-B*5701 (KAFSPEVIPMF)

$1800.00  
T01069 Select

iTAg Tetramer/APC - HLA-B*5701 (KAFSPEVIPMF)

$1800.00  
T03006 Select

iTAg Tetramer/PE - H-2 Kd (AMQMLKETI)

$1500.00  
T03007 Select

iTAg Tetramer/APC - H-2 Kd (AMQMLKETI)

$1500.00  
T03008 Select

iTAg Tetramer/PE - H-2 Kd (SYIGSINNI)

$1500.00  
T03009 Select

iTAg Tetramer/APC - H-2 Kd (SYIGSINNI)

$1500.00  
T03010 Select

iTAg Tetramer/PE - H-2 Db (KVPRNQDWL)

$1500.00  
T03011 Select

iTAg Tetramer/APC - H-2 Db (KVPRNQDWL)

$1500.00  
T03012 Select

iTAg Tetramer/PE - H-2 Db (RAHYNIVTF)

$1500.00  
T03013 Select

iTAg Tetramer/APC - H-2 Db (RAHYNIVTF)

$1500.00  
T03014 Select

iTAg Tetramer/PE - H-2 Kb (SVYDFFVWL)

$1500.00  
T03015 Select

iTAg Tetramer/APC - H-2 Kb (SVYDFFVWL)

$1500.00  
T03016 Premium

iTAg Tetramer/PE - H-2 Kb LCMV (AVYNFATC)

$1800.00  
T03017 Premium

iTAg Tetramer/APC - H-2 Kb LCMV (AVYNFATC)

$1800.00  
T03018 Premium

iTAg Tetramer/PE - H-2 Kb LCMV (AVYNFATCGI)

$1800.00  
T03019 Premium

iTAg Tetramer/APC - H-2 Kb LCMV (AVYNFATCGI)

$1800.00  
T03020 Premium

iTAg Tetramer/PE - H-2 Kb LCMV (YTVKYPNL)

$1800.00  
T03021 Premium

iTAg Tetramer/APC - H-2 Kb LCMV (YTVKYPNL)

$1800.00  
T03022 Premium

iTAg Tetramer/PE - H-2 Kb LCMV (ISHNFCNL)

$1800.00  
T03023 Premium

iTAg Tetramer/APC - H-2 Kb LCMV (ISHNFCNL)

$1800.00  
T03024 Premium

iTAg Tetramer/PE - H-2 Kb LCMV (LEYDFNKL)

$1800.00  
T03025 Premium

iTAg Tetramer/APC - H-2 Kb LCMV (LEYDFNKL)

$1800.00  
T03026 Premium

iTAg Tetramer/PE - H-2 Db LCMV (SGVENPGGYCL)

$1800.00  
T03027 Premium

iTAg Tetramer/APC - H-2 Db LCMV (SGVENPGGYCL)

$1800.00  
T03028 Premium

iTAg Tetramer/PE - H-2 Db LCMV (FQPQNGQFI)

$1800.00  
T03029 Premium

iTAg Tetramer/APC - H-2 Db LCMV (FQPQNGQFI)

$1800.00  

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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.
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iTopia  iTopia™
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.
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iTopia documentation and white papers

Author YearTitle

 
William H. Shingler 2008 Identification and Functional Validation of MHC Class I Epitopes in the Tumor-associated Antigen 5T4
Bianca D. Santomasso 2007 A T Cell Receptor Associated with Naturally Ocurring Human Tumor Immunity
Margaret M. Bachinsky 2004 Mapping and Binding Analysis of Peptides Derived from the Tumor-associated antigens Surviving for Eight HLA Alleles
FF Weichold 2007 Impact of MHC Class I Alleles on the M. Tuberculosis Antigen-Specific CD8+ T-Cell Response in Patients with Pulmonary Tuberculosis

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iTopia Frequently Asked Questions

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 or request a quote for 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.

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New iTopia products          To purchase these items enter the part number in the wizard below or click on the  icon.

#

Part No.

Name

Regulatory Status

UNSPSCShop

1

T06000

iTopia Start up Kit RUO

41116100

2

T06001

iTopia™ Epitope Discovery System (Pre-requisite & customer guide)RUO

41116100

3

T06100

iTopia Assay Kit - HLA-A*0101RUO

41116100

4

T06101

iTopia Assay Kit - HLA-A*0201RUO

41116100

5

T06102

iTopia Assay Kit - HLA-A*0301RUO

41116100

6

T06103

iTopia Assay Kit - HLA-A*1101RUO

41116100

T06104

iTopia Assay Kit - HLA-A*2402RUO

41116100

8

T06105

iTopia Assay Kit - HLA-A*0702RUO

41116100

9

T06106

iTopia Assay Kit - HLA-A*0801RUO

41116100

10

T06107

iTopia Assay Kit - HLA-A*1501RUO

41116100

11

T06210

iTopia™ System Software CDRUO

41116100

12

T06300

iTopia Peptides <=10mer (IP)RUO

41116100

13

T20304

iTopia Wash SolutionRUO

41116100


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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.
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cell signaling  Streptamers
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.
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Streptamer Manual

DOC   Year

 

Antigen-specific staining and functional isolation of T-cells

 

 

2009

 

Material Safety Data Sheets

MSDS    

 

MSDS fin 6-7xxx Streptamer MHC class I

 

 

 

 

#

Instructions for Use

IFU   # Instructions for Use IFU

1

6-7005-XXX MHC A02 HIV pol ILKEPVHGV

 

17

6-7021-XXX MHC A02 Muc-1 STAPPVHNV

2

6-7006-XXX MHC A02 HBV core FLPSDFFPSV

 

18

6-7022-XXX MHC B07 Muc-1 VPGWGIALL

3

6-7007-XXX MHC A02 MART1 ELAGIGILTV

 

19

6-7023-XXX MHC A02 Proteinase-3 VLQELNVTV

4

6-7008-XXX MHC A02 Her-2-neu KIFGSLAFL

 

20

6-7024-XXX MHC A01 CMV pp50 VTEHDTLLY

5

6-7009-XXX MHC A02 Her-2-neu RLLQETELV

 

21

6-7025-XXX MHC A02 gp100 YLEPGPVTA

6

6-7010-XXX MHC A02 Gp100 IMDQVPFSV

 

22

6-7026-XXX MHC A24 WT-1 CMTWNQMNL

6-7011-XXX MHC A02 Gp100 ITDQVPFSV

 

23

6-7027-XXX MHC B07 CMV TPRVTGGGAM

8

6-7012-XXX MHC A02 Tyrosinase YMDGTMSQV

 

24

6-7028-XXX MHC A24 CMV pp65 QYDPVAALF

9

6-7013-XXX MHC A02 NY-ESO-1 SLLMWITQV

 

25

6-5000-XXX StrepTactin PE conjugate

10

6-7014-XXX MHC H2Kd LLO91-99 GYKDGNEYI

 

26

6-5010-XXX StrepTactin APC conjugate

11

6-7015-XXX MHC H2Kb Oval SIINFEKL

 

27

6-5500-005 Streptamer Magnetic Beads human

12

6-7016-XXX MHC H2Db gp33-LCMV KAVYNFATM

 

28

6-5600-005 Solution set for Streptamer magnetic beads

13

6-7017-XXX MHC B08 CMV IE QIKVRVDMV

 

29

6-7001-XXX MHC A02 CMV NLVPMVATV

14

6-7018-XXX MHC A02 HA-1H VLHDDLLEA

 

30

6-7002-XXX MHC A02 EBV GLCTLVAML

15

6-7019-XXX MHC A02 WT-1 RMFPNAPYL

 

31

6-7003-XXX MHC A02 InfluenzaM GILGFVFTL

16

6-7020-XXX MHC A02 BST-2 KLQDASAEV

 

32

6-7004-XXX MHC A02 HIV gag SLYNTVATL


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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.

t-Cell 

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®

Fluorescence Assisted Cell Sorting 

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

 

Beads   

Magnetic Beads

11. How do I order Streptamer?
The easiest way to order or request a quote for 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.

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New Streptamer products
"Available in select markets. Click here to inquire."
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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.
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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

On-line order and request quote wizard



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