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Catalog No. PN9210-0101
| 7-PLEX CYTOKINE REAGENT KIT (100 REACTIONS; STORE AT 4°C): |
7-PLEX CYTOKINE CALIBRATOR
KIT (STORE AT -20°C): |
| 7-plex Human Cytokine Antibody Reagent (2 vials, 1.2 ml) | 7-plex Cytokine HI Calibrator (1 Vials, 300 µL) |
| 7-plex Scissors Reagent A (1 vial, 1.2 ml) | Negative Calibrator (2 vial, 1.5 mL) |
| 20x Exchange Buffer 1 (1 vial, 1.1 ml) | Cell Lysis Buffer P (2 vial, 1.5 ml) |
| 10x CES Reagent 1 (1vial, 0.6 ml) | |
| 10x Assay Buffer H(1 vial, 1 ml) | |
| eTagTM Reporter Mobility Control 0203 (1 vial, 250 µl) | |
| 96-well Filter plate (1) |
For Research Use Only. Not for Use in Diagnostic Procedures. Not for Resale.
PRECAUTIONS
- Reagents and calibrators contain non-sterile bovine serum albumin.
- Antibody Reagent, CES Reagent, and Mobility Control are light sensitive.
- Store reagents at the specified storage condition. Do not use the kit after the expiration date.
- Reagents and calibrators contain materials that may cause sensitivity on contact with skin.
FOR FURTHER TECHNICAL INFORMATION OR TO ORDER, CONTACT:
ACLARA BioSciences, Inc.
1288 Pear Avenue
Mountain View, California 94043
Technical Support: 800.257.7121
Customer Support: 800.257.7121
Web Site: http://www.aclara.com/
GENERAL LICENSE AND WARRANTY FOR PROTEIN PRODUCTS
Notice of Limited License
The recipient of this product from ACLARA BioSciences, Inc (ACLARA), or its authorized distributor, is granted a limited, non-exclusive license under intellectual property rights* held by ACLARA as part of an eTagTM Expert Access Agreement or Strategic Collaborator Agreement. In the event of a conflict between what is stated here and the eTag Expert Access Program Agreement, the eTag Expert Access Program Agreement shall control. Such license is solely for the research use of this product to perform the analytical assay for which it is intended. The limited license accompanying this product does not include a license or right to use the materials, which may accompany the product as controls, for any purposes other than those recommended for performing the assay. The materials shall not be reverse-engineered, manipulated or used for any purposes other than the performance of the assay consistent with the instructions provided in the package insert by ACLARA. The foregoing license does not include rights to use this product for diagnostic, agricultural, veterinary, medical, or any other commercial applications. Except as expressly provided in this paragraph, no other license is granted either expressly, impliedly, or by estoppel. No license to make, use or sell any components and/or products covered by any third party patents is conveyed expressly or by implication to the customer by the purchase of this product. Nothing in the foregoing grants customer any license or rights in ACLARA's eTag InformerTM software. A limited non-exclusive license to use ACLARA's eTag Informer software in connection with this product is provided separately. For information concerning the availability of additional licenses to practice eTag technology for any purpose other than that stated above, please contact the Vice President of Business Development, ACLARA BioSciences, Inc., 1288 Pear Avenue, Mountain View, CA 94043 (phone. 650-210-1200).
* The eTagTM technology is covered by U.S. patents 6,322,980; 6,514,700; and other pending patents in the U.S. and other countries.
ACLARA warrants that this product meets the specifications stated on the product information sheet. If any component of this product does not conform to these specifications, ACLARA will, at its sole discretion, replace the product at no charge to the customer or refund the cost of the product to the customer; provided that notice of non-conformance is given to ACLARA within thirty (30) days of receipt of the product. This warranty limits ACLARA's liability to the replacement of this product or refund of the cost of the product. NO OTHER WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING WITHOUT LIMITATION IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE OR NON-INFRINGMENT, ARE PROVIDED BY ACLARA. ACLARA shall have no liability for any direct, indirect, consequential or incidental damages arising out of the use, the results of use, or the inability to use this product or its components.
© Copyright 2003 ACLARA BioSciences, Inc.
All Rights Reserved. ACLARA BioSciencesTM, ACLARATM, eTagTM and eTag
InformerTM are trademarks of ACLARA BioSciences, Inc. Invader® and
Cleavase® are registered trademarks of Third Wave Technologies.
* All other names and marks are the property of their respective
owners.
Cytokines are critical protein mediators in the development of cellular immune responses, induction of inflammation and control of cellular differentiation. Investigating these complex phenomena requires an assay technology that can profile a variety of cytokines simultaneously. Cytokines are measured by immunoassays or immunoblots a single cytokine at a time. Establishing a profile by the conventional methods is time consuming and requires large amount of samples. Incorporating the eTagTM Multiplex Protein Proximity Assay, the Human Cytokine Protein 7-plex Kit measures multiple analytes simultaneously using a small, single sample and results a profile for every test. Furthermore, the eTagTM Multiplex Invader® Gene Expression Kit for human cytokine genes (sold separately) is available to investigate the concurrent mRNA profile of a biological specimen.
TECHNOLOGY OVERVIEW
The eTag Assay System includes a family of eTag assays and chemistry methods developed at ACLARA Biosciences®. The system is an innovative approach that combines multiple target analysis in a single microwell with the high sensitivity of quantification by capillary array electrophoresis (CE).
The eTag reporters are fluorescent, small, biology compatible molecules each of which has unique migration property on CE analysis. When each reporter is attached to a distinct probe, a set of eTag reporters can be used to monitor multiple reactions involving binding of the probes to the targets followed by the chemical or enzymatic release of eTag reporters. The released eTag reporters are then separated using commercially available capillary electrophoresis instruments. Peaks representing the eTag reporters are identified and quantified using eTag InformerTM software, which is developed by ACLARATM for a variety of eTag assay applications.
ASSAY PRINCIPLE
The Human Cytokine Protein 7-Plex Kit is based on specific recognition of a cytokine by a pair of antibodies. The cytokines are IL-1 , IL-2, IL-4, IL-6, IL-8, IFN and TNF
In the Human Cytokine Protein 7-Plex Kit, the eTag reporters are linked to antibodies and the eTag reporters are released chemically. As shown in the figure at the right, the first antibody is labeled with specific eTag reporters. The second antibody is attached to a proprietary molecular scissor.
During incubation with a sample containing target cytokines, the antibodies recognize and bind specifically to the respective cytokines. Illumination of the reaction mixture by visible light using the eTagTM Illuminator device activates the "scissor" molecules. These activated scissor molecules subsequently cause release of the eTag reporters from the bound antibodies.
eTag MULTIPLEX PROTEIN ASSAY
The eTag Multiplex Protein Assay requires reagents, the eTag Illuminator device and eTag Informer data analysis software. A Reagent Kit provides all reagents necessary to perform the eTagTM assay; some assays also require a calibrator kit (including the Human Cytokine Protein 7-plex Kit). Mobility controls for eTag reporters are included for capillary electrophoresis (CE) instrument validation. The eTag Illuminator device is required to activate the detection signals, and eTag InformerTM software identifies the specific eTag reporter signals, and determines the level of protein expression.
REQUIRED, BUT NOT INCLUDED
- eTag InformerTM Software Version 2.0 (ACLARA BioSciences Inc., Cat. No. PN1500-0010)
- eTag Illuminator Device (ACLARA BioSciences Inc., Cat. No. PN9500-0001
RECOMMENDED REQUIRED EQUIPMENT AND ACCESSORIES
- Multiscreen Filter plate, pore size 0.22 m (Millipore cat #MAGV N22 50)
- Microplate sealer
- Vacuum pump (Millipore cat #XX55 000 00) (115 V, 60 Hz), operating at (5 in Hg)
- Vacuum manifold (Millipore cat #MAVM 096 0R)
- Flat bed Shaker (Titer Plate Shaker from Lab-Line Instrument, Inc., speed setting @ 2.5)
- Aluminum foil
- 96-well polypropylene microplate
- 0.2ml thin-wall tubes
- Multichannel pipets (0.5-10 µL, 2.5-20 µL, 20-200 µL)
- Capillary Electrophoresis Instrument
REAGENTS REQUIRED, BUT NOT INCLUDED
- DI water
OPTIONAL EQUIPMENT, NOT INCLUDED
- Electronic repeat pipette (250 µL)
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ASSAY WORKFLOW
| A. Sample and Control Preparation Depend upon sample type (cell lysate, tissue culture supernatant) |
B. eTag Multiplex Protein Proximity Assay:
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| C. Preparation for Capillary Electrophoresis (CE): Analyze samples using specified CE run parameters. (Instrument specific CE protocol provided in separate documentation) |
CALIBRATION AND SAMPLE PREPARATION
For a calibration curve, dilute the high calibrator to 3000pM,
then serially using one part of 7-plex calibrator and two parts
of Negative calibrator.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8(Neg) |
| 3000 pM | 1000 pM | 333 pM | 111 pM | 37 pM | 12 pM | 4 pM | 0 pM |
Samples : Assay cell free supernatants or cell lysates immediately or store samples at <= -20°C. Avoid repeated freeze-thaw cycles. Dilute the samples with negative calibrator, if necessary.
CELL LYSATE SAMPLE PREPARATION: 96-WELL PLATE FORMAT
NOTE: This cell lysate detection format is used for both suspension and adherent cells. Cells are typically seeded at 10,000-40,000 cells per well and cultured in the 96-well tissue culture plates. Different seeding densities may be required depending on cell type
- Thaw sufficient ready-to-use Cell Lysis Buffer P.
- For adherent cells, carefully remove the culture medium from the wells using a multichannel pipet. For suspension cells, spin down the cells and remove the culture medium.
- Add 30 µL Cell Lysis Buffer P per well. Lyse cells at room temperature for 5-10 minutes.
- Using a multichannel pipette, carefully transfer each lysate sample into the reaction microplate.
- Assay Buffer:
Dilute the 10x Assay Buffer to 1x working reagent with DI water for use. Each 96-well plate requires 5mL of assay buffer to prime the filter plate - Exchange Buffer:
Dilute the 20x Exchange buffer to 1x working buffer with DI water for use. Each 96-well plate requires 20mL of Exchange buffer - CE Standard (CES) working Reagent:
Dilute the 10x CES reagent to 1x working reagent with DI water for use. Each 96-well plate requires 5mL of CES Reagent. Bound eTag reporters are released into CES reagent for analysis - Calibrators:
Thaw both High and Negative Calibrators for calibrator and sample preparations - Cell Lysis Buffer P:
Thaw sufficient amount for lysate preparation. Each 96-well requires 30 µL of Lysis buffer.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | Sample 1 | Sample 1 | Sample 9 | Sample 9 | Sample 17 | Sample 17 | Sample 25 | Sample 25 | Sample 33 | Sample 33 | Calibrator Level 1 | Calibrator Level 1 |
| B | Sample 2 | Sample 2 | Sample 10 | Sample 10 | Sample 18 | Sample 18 | Sample 26 | Sample 26 | Sample 34 | Sample 34 | Calibrator Level 2 | Calibrator Level 2 |
| C | Sample 3 | Sample 3 | Sample 11 | Sample 11 | Sample 19 | Sample 19 | Sample 27 | Sample 27 | Sample 35 | Sample 35 | Calibrator Level 3 | Calibrator Level 3 |
| D | Sample 4 | Sample 4 | Sample 12 | Sample 12 | Sample 20 | Sample 20 | Sample 28 | Sample 28 | Sample 36 | Sample 36 | Calibrator Level 4 | Calibrator Level 4 |
| E | Sample 5 | Sample 5 | Sample 13 | Sample 13 | Sample 21 | Sample 21 | Sample 29 | Sample 29 | Sample 37 | Sample 37 | Calibrator Level 5 | Calibrator Level 5 |
| F | Sample 6 | Sample 6 | Sample 14 | Sample 14 | Sample 22 | Sample 22 | Sample 30 | Sample 30 | Sample 38 | Sample 38 | Calibrator Level 6 | Calibrator Level 6 |
| G | Sample 7 | Sample 7 | Sample 15 | Sample 15 | Sample 23 | Sample 23 | Sample 31 | Sample 31 | Sample 39 | Sample 39 | Calibrator Level 7 | Calibrator Level 7 |
| H | Sample 8 | Sample 8 | Sample 16 | Sample 16 | Sample 24 | Sample 24 | Sample 32 | Sample 32 | Sample 40 | Sample 40 | Calibrator Level 8 | Calibrator Level 8 |
Figure 3. Example of microplate layout
- Plan the 96-well microplate layout for each experimental run. An example layout includes serially diluted Calibrators in duplicate, and 32 samples, in duplicate. When analyzed by the eTag Informer software, the calibrators can be in the same row or column and the number of replicates is flexible.
- Label the 96-well filter plate as the reaction plate
- Set up the vacuum manifold and the vacuum pump at 5 in Hg
- Prime the filter plate by wetting the filter with 50 µL of the assay buffer per well, place on the vacuum block, and apply vacuum for approximately 10 seconds to remove all liquid. Excessive vacuum may break the membrane. If partial plate is used, cover the unused wells with the rubber seal while applying vacuum.
- Add 20 µL of Antibody Reagent to each well of the reaction plate.
- Add 10 µL of each calibrator and sample to the corresponding well, place the plate seal, and cover the plate with foil. Place the covered plate on a shaker for 30 minutes at room temperature. Set shaker to low speed (200-300 rpm)
- Add 10 µL of Scissors Reagent to each well and mix by pipetting up and down 1-2 times. Cover the plate and incubate on the shaker for 30 minutes at room temperature.
- Remove the cover, place the filter plate on the vacuum manifold, and and apply vacuum to remove all liquid
- Add 100 µL of Exchange buffer to each well, and then apply vacuum to remove all liquid.
- Repeat step 9
- Add 50 µL of CE standard (CES) Reagent to each well, place the eTag Illuminator on the plate, and irradiate for 5 minutes to release eTag reportors. To ensure proper irradiation, match the cut coner of the Illuminator with the filter plate and place the Illuminator flat on the filter plate.
- Place the plate on the shaker and mix for 5 minutes .
- Label a 96-well microplate for CE analysis and transfer a minimum of 10 µL eTag reporter solution to the corresponding well.
- Run capillary electrophoresis using the assay specific CE protocol.
- Analyze data using the eTag Informer Software version 2.0
The eTag reporter assignment for each cytokine in the 7-plex Cytokine Kit is as follows:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
| Analyte | IL1a | IL2 | IL4 | IL6 | IL8 | TNFa | IFNy | US-1 |
| eTag Reporter | Pro-11 | Pro-12 | Pro-1 | Pro-10 | Pro-14 | Pro-2 | Pro-13 | Pro-8 |
A sample electropherogram is displayed below:
click for larger detail view
CAPILLARY ELECTROPHORESIS
A CE instrument equiped with a laser-induced fluorescence (LIF)
detector is required to separate and detect eTag reporter
molecules. The instrument should be configured optically to detect
fluorescein (488 nm excitiation and 525 nm emission wavelengths).
Either the ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems,
Foster City, CA) or the Amersham MegaBACETM 1000
DNA Analysis System (Molecular Dynamics, Sunnyvale, CA) are recommended.
For successful integration with the eTag InformerTM software, these instruments need to be configured
correctly and run with the kit specific separation protocols
(Appendix I). For detailed information on instrument set-up and
operation please refer to the eTag Informer software manual
(Instrumentation Parameters) or the ACLARA BioSciences website
(www.aclara.com).
QC
An eTag reporter mobility control is provided for validation
of CE instrument performance. The control contains 8 eTag reporters
and 2 electrophoresis markers. It is a ready-to-use reagent, add
10 µL per well and perform CE after installation of parameters
and the protocol. We recommend to test one well per capillary.
The resulting electropherogram should apear as displayed in the
previous section.
DATA ANALYSIS
Analysis of CE separations of eTag reporters is carried
out using eTag Infomer software (catalog # PN1500-0010).
The kit specific peak ID database is provided. The software identifies
all eTag reporter peaks in the electropherogram, labels
peaks with the appropriate target protein name and provides quantitative
results based on peak area and calibration curves. Processed data
can be exported into Microsoft* Excel or other data analysis program
for further manipulation. For details on software installation
and instructions for use, please refer to the eTag Informer Software
User Manual.
The CES reagent contains two CE internal standards: M1 and M2. Peak areas are normalized against an universal assay standard (US-1), which minimizes well-to-well variation. The software uses the normalized peak area to establish the calibration curves and calculates the concentration results of samples.
TYPICAL CALIBRATION CURVES
The eTag Informer software establishes the fitted calibration
curves based on the choozen curve-fitting type. It is reconnended
to use the point-to-point fitting for the humna cytokine assay.
These calibration curves are provided for demonstration only. Calibration
curves should be generated for each set of samples assayed. The
ACLARA cytokine assay is calibrated with purified recombinant human
cytokines.
PRECISION
Samples over 2.5-fold dynamic range were tested on one plate. Within-run
precision is summarized as %CV.
| Conc. pM | IL4 | IL1a | IL8 | IL6 | TNFa | IL2 | IFNg |
| 3000 | 5 | 3 | 5 | 3 | 3 | 9 | 8 |
| 333 | 3 | 4 | 5 | 3 | 4 | 12 | 7 |
| 111 | 4 | 14 | 6 | 5 | 10 | 16 | 8 |
| 37 | 5 | 10 | 14 | 3 | 14 | 14 | 6 |
| 4 | 4 | 24 | 14 | 21 | 13 | 16 | 14 |
SPECIFICITY
Individual cytokine samples were assayed using the 7-plex assay.
When cross-reactivities occur, peak(s) would be detected in multiple
assays. The percent cross-reactivities were calculated and summarized.
As shown below, the ACLARA 7-plex assay is highly specific.
Appendix 1: CE Protocol for 7-plex Cytokine Protein Assay
a. For ABI PRISM* 3100: use the module:
| PRO_80secj_1200sec_36cm | |
| Run Temperature | 45°C |
| Cap Fill Volume | 200 steps |
| Current Tolerance | 100 µAmps |
| Run Current | 100 µAmps |
| Voltage Tolerance | 0.6 kVolts |
| Pre Run Voltage | 15 kVolts |
| Pre Run Time | 180 sec |
| Injection Voltage | 3 kVolts |
| Injection Time | 80 sec |
| Run Voltage | 15 kVolts |
| Number of Steps | 10nk |
| Voltage Step Interval | 1 sec |
| Data Delay Time | 1 sec |
| Run Time | 1200 sec |
| Sampling Rate | 160 msec |
b. For MegaBACE* 1000, use the following parameter:
| MegaBace | ||
| Run Temperature | 30°C | |
| Matrix flush time (1) | 22 sec | |
| Matrix flush time (2) | 7 sec | |
| Matrix fill/High pressure time | 40 sec | |
| Matrix fill/Relaxation time | 1 min | |
| Pre Run Voltage | 9 kV | |
| Pre Run Time | 5 min | |
| Sample Injection Voltage | 15kV | |
| Sample Injection Time | 100sec | |
| Run Voltage | 15kV | |
| Pre injection Voltage | 10 kV | |
| Pre Injection Time | 15 sec | |
| Chemistry Name | ET Terminators | |
| Run Time | 30 min | |
| PMT 1 | 750 | |
