HUMAN ADIPOCYTE LINCOplex
By Purchasing this product, which contains fluorescently labeled microsphere beads authorized by Luminex Corporation ("Luminex"), you, the customer, acquire the right under Luminex's patent rights, if any, to use this product or any portion of this product, including without limitation the microsphere beads contained herein, only with Luminex's laser based fluorescent analytical test instrumentation marketed under the name of Luminex100.
This multiplex assay kit manufactured by Linco Research is to be used for the simultaneous quantification of the following 8 human adipokines in any combination, Leptin, Resistin, Adiponectin, IL-6, TNFα, MCP-1, and PAI-1 (total or active).
Note that Active PAI-1 and Total PAI-1 cannot be run together in the same assay.
This kit is for research purposes only.
A. Antibody-Immobilized Beads
The following beads are available for your customized order:
#12-Human IL-6
#22-Human Leptin
#40-Human TNFα
#46-Human MCP-1
#51-Human Adiponectin
#61-Human Resistin
#84-Human PAI-1 (total) or
#86-Human PAI-1 (active)
Mix beads and dilute with Bead Diluent HENDO-65K as described in Section VIII. D.
Quantity: 0.2 ml antibody-immobilized beads/bottle
Note that Active PAI-1 and Total PAI-1 cannot be run together in the same assay.
B. Human Adipokine Standard Cocktail
1 vial human adipokine standard cocktail, lyophilized
Quantity: 1 vial
C. Human Adipokine Controls
Control I – 1 vial containing human adipokine cocktail, lyophilized
Control II – 1 vial containing human adipokine cocktail, lyophilized
Quantity: 1 vial each control
D. Bead Diluent
1 bottle containing Bead Diluent
Quantity: 3.5 ml/bottle
E. Human Adipocyte Detection Antibodies
1 bottle containing a cocktail of biotinylated detection antibodies in Assay Buffer
Quantity: 5.5 ml/bottle
F. Streptavidin-Phycoerythrin
1 bottle containing Streptavidin-Phycoerythrin prepared in Assay Buffer
Quantity: 5.5 ml/bottle
G. Assay Buffer
PBS with 0.08% Sodium Azide, 0.05% Tween 20, Protease Inhibitor and 1% BSA,
pH 6.8
Quantity: 30 ml/bottle
H. 10X Wash Buffer
1:10 dilution required with deionized water to give 10 mM PBS with 0.05% Proclin, and 0.05% Tween 20, pH 7.4
Quantity: 30 ml/bottle
I. Mixing Bottle
Quantity: 1 Bottle
J. Microtiter Filter Plate
Quantity: 1- 96 Well Filtration Plate
K. Plate Sealers
Quantity: 2 Plate Sealers
III. STORAGE CONDITIONS UPON RECEIPT
Recommended storage for kit components is 2 - 8°C. See individual vials for long-term storage recommendations.
Once the standards and controls have been reconstituted, transfer contents into polypropylene vials. DO NOT STORE RECONSTITUTED STANDARDS OR CONTROLS IN GLASS VIALS. For long-term storage, freeze reconstituted standards and controls at £ -20°C. Avoid multiple (>2) freeze/thaw cycles.
DO NOT FREEZE Antibody-Immobilized Beads, Bead Diluent, Detection Antibody, and Streptavidin-Phycoerythrin.
Sodium Azide has been added to some reagents as a preservative. Although the concentrations are low, sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide build up.
V. MATERIALS REQUIRED BUT NOT PROVIDED
A. Reagents
Luminex Sheath Fluid (Luminex Catalogue #40-50000)
B. Instrumentation/Materials
1. Adjustable Pipettes with Tips capable of delivering 10 m l to 1000 m l
2. Multichannel Pipettes capable of delivering 10 m l to 200 m l
3. Reagent Reservoirs
4. Polypropylene Microfuge Tubes
5. Aluminum Foil
6. Absorbent Pads
7. Laboratory Vortex
8. Sonicator (Branson Ultrasonic Cleaner Model #B200 or equivalent)
9. Titer Plate Shaker (Lab-Line Instruments, Model #4625, or equivalent)
10. Vacuum Filtration Unit (Millipore Vacuum Manifold Catalogue #MAVMO960R, or equivalent)
11. Luminex100 by Luminex Corporation
VI. SPECIMEN COLLECTION AND STORAGE
A 25 m l per well of tissue/cell extract or supernatant samples can be used. Appropriate sample dilution with Assay Buffer may be required.
To obtain reliable and reproducible results, the operator should carefully read this entire manual and fully understand all aspects of each assay step before attempting to run the assay.
A. The Antibody-Immobilized Beads are light sensitive, so the assay plate containing beads must be covered with aluminum foil during all incubation steps.
B. The bottom of the Microtiter Filter Plate should not be in contact with any absorbent material during assay set-up or incubation times. Set the plate on a plate cover or any other plate holder to raise the plate from the surface.
C. After the wash steps, dry the bottom of the Microtiter Filter Plate by using paper towels or absorbent pads to prevent any leakage due to capillary action.
D. Keep the vacuum suction on the plate as low as possible. It is recommended to have a vacuum setting that will remove 200 ml of buffer in ≥ 5seconds (equivalent to < 100 mmHg).
E. The standards prepared by serial dilution must be used within 1 hour of preparation. Use a fresh tip with each dilution. Discard any unused standards except the highest standard (standard 7) which may be stored at £ -20° C for 1 month and at £ -80° C for greater than one month.
F. Any unused mixed Antibody-Immobilized Beads may be stored in the bead mix bottle at 2-8° C for up to one month.
G. During the preparation of the standard curve, make certain to vortex the higher concentration well before making the next dilution.
H. If the plate cannot be read on the same day as the assay was finished, do the final bead suspension in 100 ml Sheath Fluid, seal the plate, cover with aluminum foil, and store at 2-8° C for up to 24 hours. Prior to reading, agitate the plate on the plate shaker at room temperature for 10 minutes.
I. The titer plate shaker should be set at a speed to provide maximum agitation without splashing of liquid outside the wells. For the recommended plate shaker, this would be a setting of 5-7 which is approximately 500-800 rpm in a 0.3 cm orbit.
J. Ensure the needle probe is clean. This may be achieved by sonication and/or alcohol flushes. Adjust probe height to the Lincoplex Filter Plate prior to reading an assay.
K. For cell culture supernatants or tissue extraction, use the culture or extraction medium as matrix in blank, standard curve and controls. If samples are diluted in Assay Buffer, use the Assay Buffer as matrix.
VIII. PREPARATION OF REAGENTS FOR IMMUNOASSAY
A. Preparation of Human Adipokine Standard Cocktail
Reconstitute the Human Adipokine Standard Cocktail with 250 μl deionized water and allow the vial to set for about 5 minutes, invert the vial several times to mix gently. Vortex and mix well. Transfer the standards to a microfuge tube labeled "Standard 7". Label six polypropylene microfuge tubes "Standard 6", "Standard 5", "Standard 4", "Standard 3", "Standard 2", and "Standard 1". Add 200 ml of Assay Buffer to each of the six tubes. Perform 5 times serial dilutions by adding 50 ml of the "Standard 7" to the "Standard 6" tube, mix well and transfer 50 ml of the "Standard 6" to the "Standard 5" tube, mix well and transfer 50 ml of the "Standard 5" to "Standard 4" tube, mix well and transfer 50 ml of the "Standard 4" pg/mlto the "Standard 3", mix well and transfer 50 ml of the "Standard 3"pg/mlto the "Standard 2" tube, mix well and transfer 50 ml of the "Standard 2" pg/mlto the "Standard 1". The 0 standard (Background) will be Assay Buffer.
|
Standard tube |
Volume of Assay Buffer to Add |
Volume of Standard to Add |
|
Standard 6 |
200 ml |
50 ml of Standard 7 |
|
Standard 5 |
200 ml |
50 ml of Standard 6 |
|
Standard 4 |
200 ml |
50 ml of Standard 5 |
|
Standard 3 |
200 ml |
50 ml of Standard 4 |
|
Standard 2 |
200 ml |
50 ml of Standard 3 |
|
Standard 1 |
200 ml |
50 ml of Standard 2 |
Standard Concentrations
|
Standard tube |
IL-6, TNFα, MCP-1 |
Resistin, PAI-1(pg/ml) |
Leptin, Adiponectin(pg/ml) |
|
Standard 1 |
0.64 |
3.2 |
16 |
|
Standard 2 |
3.2 |
16pg/m |
80 |
|
Standard 3 |
16 |
80 |
400 |
|
Standard 4 |
80 |
400 |
2,000 |
|
Standard 5 |
400 |
2,000 |
10,000 |
|
Standard 6 |
2,000 |
10,000 |
50,000 |
|
Standard 7 |
10, 000 |
50,000 |
250,000 |
B. Preparation of Controls
Reconstitute Human Adipokine Control I and Human Adipokine Control II with 250 µl deionized water. Allow the vials to set for about 5 minutes, invert the vial several times to mix and vortex. Transfer the Controls into a microfuge tube.
C. Preparation of Wash Buffer
Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. Dilute 30 ml of 10X Wash Buffer with 270 ml deionized water. Store unused portions at 2-8° C for up to one month.
D. Preparation of Antibody-Immobilized Beads
Sonicate each antibody-bead bottle for 30 seconds, vortex for 1 minute. Add 0.15 ml from each antibody bead bottle to the Mixing Bottle and bring final volume to 3 ml with Bead Diluent. Unused portions may be stored at 2-8° C for up to one month.
Example 1: when using 7 antibody-immobilized beads, add 0.15 ml from each of the seven bead sets to the mixing bottle and 1.95 ml Bead Diluent.
Example 2: when using 4 antibody-immobilized beads, add 0.15 ml from each of the four bead sets to the mixing bottle. Add 2.4 ml Bead Diluent.
Note that Active PAI-1 and Total PAI-1 cannot be run together in the same assay.
Prior to beginning this assay, it is imperative to read this protocol completely and to thoroughly understand the Technical Guidelines outlined in Section VII.
1. Diagram placement of Standards, 0 (Background) 1, 2, 3, 4, 5, 6, 7, Control I and II, and samples on Well Map Worksheet in a vertical configuration. (Note: the instrument will only read the 96-well plate vertically). It is recommended to run the assay in duplicate.
2. Block the filter plate by pipetting 200 µL of Assay Buffer into each well of the microtiter plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25° C).
3. Remove Assay Buffer by vacuum (NOTE: DO NOT INVERT PLATES) and dry the bottom of the plate by using paper towels.
4. Add 25 µL of Assay Buffer to the 0 Standard (Background).
5. Add 25 µL of Assay Buffer to the Sample wells.
6. Add 25 µL of each Standard or Control into the appropriate wells.
7. Add 25 µL of appropriate adipokine-free media to the Background, Standard, and Control wells.HENDO-65K
8. Add 25 µL of Samples into the appropriate wells.
9. Add 25 μL of Mixed Beads to each well. (Note: during addition of Mixed Beads, shake bead mix intermittently to avoid settling)
10. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker overnight (16-18 hours) at 2-8oC. After overnight incubation, it is important to allow the plate and reagents to warm to room temperature (20-25° C) before continuing with the assay.
11. Gently remove fluid by vacuum.
12. Wash plate 3 times with 200 µL/well of Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Dry the bottom of the plate by using paper towels.
13. Pipette 50 µL of Detection Antibody Cocktail into each well. (Note: allow the Detection Antibody to warm to room temperature, 20-25° C, prior to addition.)
14. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 30 minutes at room temperature. DO NOT VACUUM AFTER INCUBATION.
15. Add 50 m L Streptavidin-Phycoerythrin (SAPE) to each well containing the 50 m L of Detection Antibody Cocktail. (Note: allow the SAPE to warm to room temperature prior to addition.)
16. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 30 minutes at room temperature.
17. Gently remove all contents by vacuum.
18. Wash plate 3 times with 200 µL/well Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Wipe any excess buffer on the bottom of plate with a tissue.
19. Add 100 µL of Sheath Fluid to all wells. Cover with aluminum foil and resuspend the beads on a plate shaker for 5 minutes.
20. Read plate on Luminex100.
21. Save and evaluate the median data using a 5-parameter or spline fit data reduction.
Select the following equipment settings:
| Events: | 50, per bead | |
| Sample Size: | 50 ml | |
| Bead Set: | 012 for IL-6 | |
| 022 for Leptin | ||
| 040 for TNFα | ||
| 046 for MCP-1 | ||
| 051 for Adiponectin | ||
| 061 for Resistin | ||
| 084 for PAI-1 (total) | ||
| 086 for PAI-1 (active) | ||
| *Gate (for IS System): | 8,000 to 15,000 | |
| *Gate (for 1.7 System): | 8,060 to 13,000 | |
*These specifications are for the Luminex100v.1.7 or Luminex100IS v2.1/2.2. Luminex instruments with other software (e.g. Masterplex, Starstation, LiquiChip, Bioplex, LABScan100) would need to follow instrument instructions for gate settings and additional specifications.
The ranges for each analyte in Quality Control I and II are provided on the card insert or can be located at the Linco Research website www.lincoresearch.com.
A. Accuracy (recovery in buffer)
IL-6 95.7%
Leptin 95.7%
TNFα 93.3%
MCP-1 92.0%
Adiponectin 95.5%
Resistin 90.2%
PAI-1 (total) 93.1%
PAI-1 (active) 93.1%
B. Sensitivity (Minimum Detectable Concentrations, MinDC)
IL-6 0.3 pg/ml
Leptin 27.4 pg/ml
TNFα 0.1 pg/ml
MCP-1 0.5 pg/ml
Adiponectin 80.3 pg/ml
Resistin 4.5 pg/ml
PAI-1 (total) 0.5 pg/ml
PAI-1 (active) 0.5 pg/ml
C. Precision
Intra-assay variation (%CV) 1.4 - 7.9%
Inter-assay variation (% CV) < 21%
D. Linearity of Dilution
90 – 116%
E. Cross-Reactivity
The antibody pairs used in this assay are specific and no significant cross-reactivity
within this panel was observed.
Human Adipokine Standard Cocktail LHA-8061
Human Adipokine Quality Control I and II LHA-6061
Human Adipocyte Detection Antibodies LHAC-1061
Streptavidin–Phycoerythrin L-SAPE7
Bead Diluent LHE-BD
Assay Buffer LE-ABGLP
Set of two 96-Well Filter Plates with sealers L-PLATE
10X Wash Buffer L-WB
12-Human IL-6 Beads HIL-6
22-Human Leptin Beads HE-LPTN
40-Human TNF alpha Beads HTNF-A
46-Human MCP-1 Beads HMCP-1
51-Human Adiponectin Beads HA-ADPN
61-Human Resistin Beads HA-RES
84-Human total PAI1 Beads HASP-PAI1
86-Human active PAI1 Beads HA-PAI1