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Tools for Comprehensive Evaluation
of the Cellular Immune Response
Immune Monitoring using
Class I and Class II Tetramers
BR-9898A
Today's highly competitive, regulatory
driven environment for drug and vaccine
development demands a standardized
approach to effectively measure the
immune response. Beckman Coulter's
innovative platforms, scientific expertise
and custom product development
knowledge can deliver a comprehensive
immune monitoring strategy.
When you partner with Beckman Coulter,
you benefit from more than 50 years of
experience in life science and diagnostic
cellular assays. From the industry standard
Coulter principle to our revolutionary
iTAg MHC Tetramer technology,
Beckman Coulter has the experience to
design the solution to your immune
monitoring requirements.
B2007-7738 Tetramer Brochv1.qxd:Tetramer Brochure 11/7/07 9:19 AM Page 3
Tools for Comprehensive Evaluation
of the Cellular Immune Response
The iTopia Epitope Discovery System*
High throughput discovery of Class I restricted T cell epitopes
across multiple alleles.
The iTopia system from Beckman Coulter provides high
throughput T cell epitope discovery across eight human alleles. In
contrast to in silico methods, the iTopia system provides
comprehensive eptiope information based on experimental data,
not mathematical predictions, and requires no patient material. A
comprehensive report is provided containing all putative epitopes
in order of probable immunogenicity. Confirmation and
quantitation of the T cell response to each epitope can then be
carried out utilizing MHC Tetramer technology.
The iTopia Epitope Discovery System enables you to:
Identify epitopes from a protein of interest by generating
experimental binding data that mimics in vivo MHC-peptide
binding
Characterize MHC-peptide affinity and off-rate measurements
across a breadth of alleles
Utilize no patient materials
Save time and reduce R&D costs by eliminating peptides
unable to bind to the MHC complexes early in the discovery
process
Detect epitopes within the protein that may potentially be
enhanced or modified to produce a stronger immune response
Identify allele-specific epitopes that can be used in clinical
trials to gather important immune response information
Recognize vaccine candidates not providing the desired
immune response levels earlier in the process, allowing
redesign of clinical trials or elimination of candidate vaccine
from further study
Monitor patient immune response to epitopes identified using
the iTAg MHC Tetramer IFN-y (gamma) assay for combined
quantitative and functional analysis
Automate with 96-well plate format
iTAg
TM
MHC Tetramer Technology*
Prior to the development of Class I and Class II MHC tetramers,
techniques for the isolation and identification of antigen specific
T cells were tedious, imprecise, lacked reproducibility and
inferred the epitope specificity of a T cell based on a functional
test. A complex of 4 MHC-peptide pairs and a streptavidin
flurochrome, MHC Tetramers directly detect antigen specific T
cells at the single cell level based on the structural specificity of
the T cell. Functional analysis of the Tetramer positive cells may
then be performed providing both the structural specificity and
functional profile of the antigen specific T cell response.
Investigation of the antigen specic T cell response begins with
peptide epitopes. Understanding the response requires the
quantitation of T cells of numerous specicities across multiple alleles.
Figure 1: The iTopia System relies on detection of a properly
folded MHC-peptide complex.
CD8 T Cell
CD4 T Cell
Activated CD4 T Cell
B Cell
Plasma Cell
Antibodies
Dendritic Cell Dendritic Cell
Activated CD8 T Cell
Activated CD8 T Cell
CD8 T Cells
CD8 T Cells
CD8 T Cells
Target Cell
Cytokines
Cytokines
Target Cell Death
MHC Class I
MHC Class II
Antigen-Presenting Cell
Antigen-Presenting Cell
Antigen-Presenting Cell
Virus
B2007-7738 Tetramer Brochv1.qxd:Tetramer Brochure 11/7/07 9:20 AM Page 4
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
E1
0.0%
E2
0.0%
E4
100.0%
E3
0.0%
E1
0.0%
E2
0.1%
E4
99.9%
E3
0.0% i
T
A
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M
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C

T
e
t
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a
m
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/
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E
CD4ECD
Class I iTAg
TM
MHC Tetramers*
Enable antigen-specific CD8
+
positive T cells to be
enumerated directly from patient samples without the need
for stimulation
Are the only method to directly detect antigen-specific CD8
+
T cells ex vivo
Reduce the total time for sample preparation and analysis
to hours
Enable both functional characterization and structural
specificity of labeled cells without the need for additional
patient sample
Nonspecific binding is inherent in immunologic assays. To reduce
nonspecific binding, Human Class I iTAg MHC Tetramers contain
a proprietary mutation in the alpha 3 domain of the MHC
(Figure 2).
Class II iTAg
TM
MHC Tetramers*
CD4
+
T cells are critical for the maintenance of long term
immunological memory and play a role in many diseases. Analysis
of antigen-specific CD4
+
T cells has long been hampered by lack
of a suitable assay. As a result of Beckman Coulter's recent
advances in Class II iTAg MHC Tetramer manufacturing process,
high quality reagents for the quantitation of the antigen specific
CD4
+
T cell response are now available.
Class II MHC Tetramers are the only method to directly detect
antigen-specific CD4
+
T cells ex vivo (Figure 3)
Enable functional characterization of the Tetramer labeled
antigen-specific CD4
+
T cell response simultaneously without
the need for additional patient sample
Enumerate antigen-specific CD4
+
T cells from patient samples
following in vitro stimulation (Figure 4)
Figure 4a-f: Flu HA 306-318 307R/DR4 iTAg MHC tetramer: Monitoring
Vaccine Efcacy. Demonstrates immune monitoring and competence
with possible utility of Class II iTAg MHC Tetramers to monitor vaccine
efcacy in patients. Plots show six time points from a single donor.
10
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0
10
0
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C1 C2
C4 C3
10
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C1 C2
C4 C3
i
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CD8FITC
Figure 2a Figure 2b
Figure 2: 3 domain binding modication. Tetramer labeling using
unmutated native Tetramers (2a) vs. mutated Tetramers (2b).
Reduction of non-specic binding resulting from the alpha 3 domain
results in accurate quantitation of antigen specic T cells.
Figure 3a Ii(Clip)/DR1
Irrelevant Tetramer
Figure 3b EBV/DR1
Figure 3: Staining Whole Blood ex vivo. Example of direct whole blood
staining with an EBV Class II iTAg MHC Tetramer. Fugure 3a: Ii(Clip)/DR1
Irrelevant tetramer. Figure 3b: EVB/DR1 Positive tetramer.
10
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C1 C2
C4 C3
i
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F
CD4-ECD
10
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2
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3
C1 C2
C4 C3
i
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F
CD4-ECD
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
C1 C2
C4 C3
i
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M
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e
t
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F
CD4-ECD
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
C1 C2
C4 C3
i
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M
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T
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t
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F
CD4-ECD
4e Day 25 (Post-vaccination)
10
3
10
2
10
1
10
0
10
0
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2
10
3
C1 C2
C4 C3
i
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T
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t
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a
m
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/
P
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F
CD4-ECD
4f Day 80 (Post-vaccination)
4a Day -5 (Pre-vaccination)
0.28%
0.74%
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
C1 C2
C4 C3
i
T
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M
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C

T
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t
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a
m
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/
P
E
F
CD4-ECD
0.06%
4.04%
0.08% 0.70%
4b Day 0 (Day of vaccination)
4c Day 7 (Post-vaccination) 4d Day 14 (Post-vaccination)
*For Research Use Only. Not for use in diagnostic procedures.
B2007-7738 Tetramer Brochv1.qxd:Tetramer Brochure 11/7/07 9:20 AM Page 5
iTAg MHC Tetramer IFN- Kit*
Estimating the number of antigen-specific T cells by inference
based on peptide-stimulated IFN- production may provide
erroneous results. Tetramer analysis combined with intracellular
cytokine staining provides both functional information and
antigenic specificity for a more accurate immune assessment.
For the simplification and reproducibility of this technique,
Beckman Coulter has developed the iTAg MHC Tetramer
IFN- kit.
As demonstrated in Figure 5, performing both assays together
enables differentiation of truly antigen-specific T cells producing
IFN- from cells producing IFN- non-specifically. In this case,
intracellular staining alone overestimated the number of antigen
specific T cells.
Combined quantitative and functional measurements to
provide more accurate data
Requires less sample than if assays were performed
individually
Saves valuable time compared to ELISPOT assays
Flowcytomix: Bead-Based Multiplexing assays for the
detection of cytokines
Bead-based assays have become standard laboratory tools for
the evaluation of cytokines in biological fluids. Flowcytomix
assays from Beckman Coulter are simple, highly flexible systems
that show excellent correlation with standard ELISA assays. The
combination of MHC Tetramers and the Th1/Th2 11plex
Flowcytomix assay is a powerful combination for quantitative
evaluation of the T cell response.
Immunophenotyping Reagents
In addition to the valuable information iTAg MHC Tetramer
analysis provides, Beckman Coulter also offers a wide range of
immunophenotyping reagents, which can be used alone or in
conjunction with Tetramer technology to further characterize the
immune response.
Lineage and the totality of assessment
Acute versus chronic disease classification
Etiology and unexplained leukocytosis investigation
Nave, memory and effector cell detection
Cellular differentiation and activation status
Custom reagent expertise
Beckman Coulter's custom design service provides a wide range
of monoclonal antibodies as conjugates and reagent
configurations that are not commercially available. We provide
customized solutions to meet your unique research needs. Our
scientists partner with you to design reagents such as:
Bulk purified antibodies for research applications
Conjugation service for unique monoclonal antibodies
Alternate conjugates of Beckman Coulter monoclonal
antibodies
Sophisticated multicolor configurations
One of the industry's brightest, highest quality tandem dye
fluorochromes
Quality Matters
Beckman Coulter, the leader in complete immune monitoring
solutions, offers Class I and Class II iTAg MHC Tetramers and
complimentary products that meet your requirements for
consistency. All Beckman Coulter Tetramers are manufactured
under GMP compliant procedures to ensure maximum stability
and to minimize lot-to-lot variation.
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
E1
2.3%
E2
1.1%
E4
0.8%
E3
95.8%
i
T
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M
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t
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E
IFN-FITC
iTAg+/IFN--/CD8+ T cells
CMVpp65 positive
T cells that are
producing IFN-;
iTAg+/IFN-
+/CD8+ T cells
iTAg-/IFN-+/CD8+
T cells, the majority
of which are NK
cells
1.00
0.75
0.50
0.25
1.00
2.00
0.00
3.00
0.00
%

T
e
t
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a
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+
/
C
D
8
+
%

T
e
t
r
a
m
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r

+
/
C
D
8
+
Donor 1 Donor 2 Donor 3 Donor 1 Donor 2 Donor 3
Figure 6: iTAg MHC Tetramer Intra-Assay Precision. These data
represent the mean +/- 1SD of 10 replicate donor samples from two
sites. Each site tested different iTAg Tetramers and different donors.
(Data collected in association with an ACTG validation study.)
Figure 5: iTAg MHC Tetamer IFN- Kit. Demonstrates subdivision of
T cells into quadrants based on function and antigen specicity.
B2007-7738 Tetramer Brochv1.qxd:Tetramer Brochure 11/7/07 9:20 AM Page 6
Class I iTAg MHC Tetramers
Human Class I Allele iTAg MHC Tetramers (in Stock)
Allele Specificity Fluorochrome Category Part#
A*0101 CMV (VTEHDTLLY) PE Premium T01045
APC Select T01050
A*0201 CMV PP65 (NLVPMVATV) PE Premium T01009
APC Select T01023
A*2402 CMV (QYDPVAALF) PE Premium T01043
APC Select T01035
B*0702 CMV (TPRVTGGGAM) PE Premium T01046
APC Select T01051
B*0801 CMV (ELRRKMMYM) PE Premium T01047
APC Select T01052
B*3501 CMV (IPSINVHHY) PE Premium T01048
APC Select T01053
A*0201 EBV (GLCTLVAML) PE Premium T01010
APC Select T01024
A*0201 HBV core (FLPSDFFPSV) PE Select T01018
APC Select T01032
A*2402 HBV core (EYLVSFGVW) PE Select T01038
APC Select T01039
A*2402 HBV pol (KYTSFPWLL) PE Select T01040
APC Select T01041
A*0201 HIV gag (SLYNTVATL) PE Premium T01006
APC RUO T01020
A*0201 HIV pol (ILKEPVHGV) PE Premium T01007
APC Select T01021
A*0201 HPV (YMLDLQPET) PE Select T01016
APC Select T01030
A*0201 Influenza-M1 (GILGFVFTL) PE Premium T01011
APC Select T01025
A*0201 Gp 100 (ITDQVPFSV) PE Select T01013
APC Select T01027
A*0201 Gp100 (IMDQVPFSV) PE Select T01012
APC Select T01026
A*0201 Her-2/neu (KIFGSLAFL) PE Select T01014
APC Select T01028
A*0201 Her-2/neu (RLLQETELV) PE Select T01015
APC Select T01029
Allele Specificity Fluorochrome Category Part#
A*0201 Mart-1 (ELAGIGILTV) PE Premium T01008
APC Select T01022
A*0201 PR-1 (VLQELNVTV) PE Select T01017
APC Select T01031
A*0201 Tyrosinase (YMDGTMSQV) PE Select T01019
APC Select T01033
Negative Tetramer PE Premium T01044
APC Select T01054
Human Class I Allele iTAg MHC Tetramers (Custom)
A*0101 PE CP T02002
APC CP T02046
PE IP T02003
APC IP T02047
A*0201 PE CP T02000
APC CP T02044
PE IP T02001
APC IP T02045
A*0301 PE CP T02004
APC CP T02048
PE IP T02005
APC IP T02049
A*1101 PE CP T02024
APC CP T02026
PE IP T02025
APC IP T02027
A*2402 PE CP T02028
APC CP T02030
PE IP T02029
APC IP T02031
B*0702 PE CP T02006
APC CP T02018
PE IP T02007
APC IP T02019
B*0801 PE CP T02008
APC CP T02020
PE IP T02009
APC IP T02021
B*1501 PE CP T02040
APC CP T02042
PE IP T02041
APC IP T02043
Premium iTAg MHC Tetramers -shipped within 3 days. Select iTAg MHC Tetramers - shipped within 3 to 30 days. All Custom Products are usually shipped within 30 to 60 days.
Use Part Number designated "CP" when peptide is customer-supplied. Use Part Number designated "IP" when Beckman Coulter supplies peptide.
All "IP" products include a peptide acquisition fee, Part Number T05008.
B2007-7738 Tetramer Brochv1.qxd:Tetramer Brochure 11/7/07 9:20 AM Page 1
Eastern Europe, Middle East, North Africa, South West Asia: Switzerland, Nyon (41) 22 365 3707. Australia, Gladesville (61) 2 9844 6000.
Canada, Mississauga (1) 905 819 1234. China, Beijing (86) 10 6515 6028. Czech Republic, Prague (420) 267 00 83 66.
Hong Kong (852) 2814 7431, 2814 0481. France, Villepinte (33) 1 49 90 90 00. Germany, Krefeld (49) 2151 33 35.
India, Mumbai (91) 22 3080 5101. Italy, Cassina de Pecchi (Milan) (39) 02 953921. Japan, Tokyo (81) 3 6745 4704.
Latin America (1) (305) 380 4709. Mexico, Mexico City (52) 55 560 57770. Netherlands, Mijdrecht (31) 297 230630.
Puerto Rico (787) 747 3335. Singapore (65) 6339 3633. South Africa/Sub-Saharan Africa, Johannesburg (27) 11 805 2014.
Sweden, Bromma (46) 8 564 85 900. Switzerland, Nyon 0800 850 810. Taiwan, Taipei (886) 2 2378 3456. Turkey, Istanbul (90) 216 309 1900.
UK, High Wycombe (44) 01494 441181. USA, Brea, CA (1) 800 352 3433, (1) 714 993 5321.
B2007-7738-LC-5K www.beckmancoulter.com 2007 Beckman Coulter, Inc. PRINTED IN U.S.A.
Allele Specificity Fluorochrome Category Part#
B*2705 PE CP T02032
APC CP T02034
PE IP T02033
APC IP T02035
B*3501 PE CP T02010
APC CP T02022
PE IP T02011
APC IP T02023
B*5701 PE CP T02036
APC CP T02038
PE IP T02037
APC IP T02039
Non-human Allele iTAg Tetramers
H-2 Db LCMV (KAVYNFATC) PE Premium T03001
APC Select T03003
H-2 Kb OVA (SIINFEKL) PE Premium T03000
APC Select T03002
Mamu-A*01 SIV gag (CTPYDINQM) APC Select T03005
SIV gag (CTPYDINQM) PE Select T03004
Non-human Allele iTAg Tetramers (Custom)
H-2 Db PE CP T04002
APC CP T04008
PE IP T04003
APC IP T04009
H-2 Kb PE CP T04000
APC CP T04006
PE IP T04001
APC IP T04007
H-2 Kd PE CP T04004
APC CP T04010
PE IP T04005
APC IP T04011
Mamu-A*01 PE CP T04012
APC CP T04014
PE IP T04013
APC IP T04015
Human Class II Allele iTAg MHC Tetramers (Custom)
Allele Fluorochrome Category Part#
DRB1*0101 PE CP T02052
PE CP T02056
PE IP T02053
PE IP T02057
DRB1*0401 PE CP T02050
PE CP T02054
PE IP T02051
PE IP T02055
DRB1*1501 PE CP T02058
PE CP T02060
PE IP T02059
PE IP T02061
DRB1*0301 PE CP T02066
PE CP T02068
PE IP T02067
PE IP T02069
DPB1*0401 PE CP T02062
PE CP T02064
PE IP T02063
PE IP T02065
iTag MHC Tetramer CMV Assay
Multi Allele PE Premium T01049
iTopia Kit
Human Class I
A*0101 T06100
A*0201 T06101
A*0301 T06102
A*1101 T06103
A*2402 T06104
B*0702 T06105
B*0801 T06106
B*1501 T06107
iTAg MHC Tetramer IFN- Kit T08005
iMASC Antibody Gating Kit T09004
iTAg MHC Tetramer Lysing Solution T08002
iTAg MHC Tetramer Fixative Solution T08003
Premium iTAg MHC Tetramers -shipped within 3 days. Select iTAg MHC Tetramers - shipped within 3 to 30 days. All Custom Products are usually shipped within 30 to 60 days.
Use Part Number designated "CP" when peptide is customer-supplied. Use Part Number designated "IP" when Beckman Coulter supplies peptide.
All "IP" products include a peptide acquisition fee, Part Number T05008.
B2007-7738 Tetramer Brochv1.qxd:Tetramer Brochure 11/7/07 9:20 AM Page 2