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MHC Tetramers Frequently Asked Questions
iTopia™ Frequently Asked Questions
MHC Tetramers Frequently Asked Questions
Q. 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 receptor. 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+).1
Q. 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. Please click on the links for details.
For more information on the application of iTAg MHC Tetramers in many of these
diseases please see our list of Publications.
Q. 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-γ kit to simultaneously quantitate antigen specific T cells and IFN-γ
production.
Q. What is the shelf life of iTAg MHC Tetramer
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蚓. Opened vials of iTAg Tetramer are stable
for 90 days when stored at 2-8蚓.
Stability data is not available for Custom iTAg MHC Tetramer reagents.
Q. How should the Tetramers be stored?
Tetramers should be stored at 2-8蚓. They should not be frozen and exposure
to light should be minimized. Vials should be refrigerated at 2-8蚓 immediately
after use.
Q. What is the recommended labeling procedure?
SAMPLE PREPARATION FOR LABELING WITH CLASS I iTAg TETRAMERS
Whole Blood
- Collect blood by venipuncture into a blood collection tube containing an
appropriate anti-coagulant.
- To each 12x75 mm test tube add 10 無 of iTAg MHC Tetramer and any additional
苔ntibodies (e.g. anti-CD8).
- Add 200 無 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 無 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 無 of PBS with 0.5% paraformaldehyde or formalin.
- Store at 4蚓 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 106 cells/mL.
200 無 of sample is required for each iTAg Tetramer determination.
- To each 12x75 mm test tube add 10 無 of iTAg MHC Tetramer and any additional
antibodies (e.g. anti-CD8).
- Add 200 無 PBMC into each test tube (e.g. 1 x 106 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 無 of PBS with 0.5% paraformaldehyde or formalin.
- Store at 4蚓 in the dark and analyze by flow cytometry within 24 hours.
Q. 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 無 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.
Q. 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.
Q. Do CD8 antibodies affect the binding of iTAg
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.
Q. 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.
Q. 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 to send an inquiry or call toll free at 1-866-485-1270. Outside the
US, call 33 49 1 1727273520.
Q. 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 |
|---|
| H2-Kb |
| H2-Kd |
| H2-Db |
|
| Rhesus Macaque Alleles |
|---|
| Mamu-A*01 |
|
iTopia Frequently Asked Questions
Q. 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).
Q. 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 – 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).
Q. 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.
Q. 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’mer. The length of the peptides will be
allele dependent.
Q. 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.
Q. 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.
Q. 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.
Q. 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.
Q. 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.
Q. 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-γ assays, using patient samples.
Q. Do you plan to develop this assay for Class
II?
Yes. The feasibility testing is in process.
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