HUMAN CARDIOVASCULAR DISEASE (CVD)
By purchasing this product, which contains fluorescent-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 Luminex*.
*Luminex instrumentation refers to Luminex®100, Luminex 200 and other Luminex instruments from MiraiBio® (MasterPlex CT™), ACS® (STarStation™), Bio-Rad Laboratories, Inc.® (Bio-Plex™) and Qiagen® (LiquiChip™). For technical assistance on running LINCOplex Kits, contact our technical service department Toll Free U.S. at 866-441-8400 or 636-441-8400 or email us at info@lincoresearch.com.
This multiplex assay kit manufactured by LINCO Research, Inc. is to be used for the simultaneous quantification of the following analytes in any combination: Adiponectin (Acrp30), soluble E-Selectin (sE-Selectin), soluble VCAM-1 (sVCAM-1), soluble ICAM-1 (sICAM-1), Matrix Metalloproteinase-9 (MMP-9), Myeloperoxidase (MPO), and Plasminogen Activator Inhibitor-1 (tPAI-1, total). This kit can be used for the analysis of the above analytes in tissue extract, cell/tissue culture samples, and diluted human serum or plasma samples. Adiponectin, sVCAM-1, MMP-9, MPO, and tPAI-1 have strong detectable signals in non-human primate serum or plasma samples (not fully validated). This kit is for research purposes only.
A. Human CVD Panel 1 Beads (20X concentrated): Dilution is required before the assay. The following Antibody-Immobilized Beads are available for your customized kits:
#02-sE-Selectin
#16- sVCAM-1
#24- sICAM-1
#27- MMP-9
#31- MPO
#51- Adiponectin
#84- tPAI-1 (total)
Quantity: 200 µl per tube
B. Human CVD Panel 1 Standard
1 vial containing mixed analytes in a cocktail, lyophilized
Quantity: 1 vial
C. Human CVD Panel 1 Controls
Control 1 – 1 vial containing mixed analytes, lyophilized
Control 2 – 1 vial containing mixed analytes, lyophilized
Quantity: 1 vial each control
D. Human CVD Panel 1 Detection Antibody
1 bottle containing a cocktail of biotinylated detection antibodies in assay buffer
Quantity: 3.2 ml/bottle
E. Streptavidin-Phycoerythrin
1 bottle containing Streptavidin-Phycoerythrin prepared in assay buffer
Quantity: 3.2 ml/bottle
F. Bead Diluent
1 bottle containing diluent for bead preparation
Quantity: 3.5 ml/bottle
G. LINCOplex Assay Buffer
PBS with 0.08% Sodium Azide and 1% BSA, pH 7.4
Quantity: Two bottles containing 30 ml/bottle
H. LINCOplex 10X Wash Buffer
1:10 dilution required with deionized water to give 10 mM PBS with 0.05% Proclin 300, and 0.05% Tween 20, pH 7.4.
Quantity: 30 ml/bottle
I. Microtiter Filter Plate
Quantity: 1- 96-Well Filtration Plate
J. Plate Sealers
Quantity: 2 Plate Sealers
K. Mixing Bottle
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, immediately transfer contents into polypropylene vials. DO NOT STORE STANDARDS OR CONTROLS IN GLASS VIALS. Freeze reconstituted standards and controls at £ -20°C. Avoid multiple (>2) freeze/thaw cycles.
DO NOT FREEZE Antibody-Immobilized Beads, 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. 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 Pipettors with Tips capable of delivering 25 m l to 1000 m l
2. Multichannel Pipettor capable of delivering 25 m l to 200 m l
3. Reagent Reservoirs
4. Polypropylene Microfuge Tubes
5. Aluminum Foil
6. Absorbent Pads
7. Laboratory Vortex
8. Sonicator
9. Titer Plate Shaker (Lab-Line Instruments, Inc. Model 4625, or equivalent)
10. Vacuum Filtration Unit (Millipore Vacuum Manifold Catalogue #MAVM0960R, or equivalent)
11. Luminex Instrument
VI. SPECIMEN COLLECTION AND STORAGE
A. A maximum of 25 m l of tissue extract or cell/tissue culture supernatant samples or diluted serum, plasma samples can be used per well.
B. Preparation of Tissue Culture Supernatant:
Centrifuge the sample to remove cell debris and run the assays immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. Tissue culture supernatant may require a dilution with an appropriate control medium prior to assay. Users need to provide the control medium as the sample diluent.
C. Preparation of Serum or Plasma Samples:
For serum, allow the blood to clot for at least 30 minutes. For plasma, use appropriate anti-coagulant. Blood should be centrifuged at 1000xg for 10 min. After centrifugation, remove serum or plasma and assay immediately or aliquot and store samples at a temperature £ -20ºC. Avoid multiple (>2) freeze/thaw cycles.
A dilution of 1:100 is recommended for this assay when using serum or plasma samples (eg. 5 µl sample and 495 µl Assay Buffer). NOTE: This dilution factor may result in below detectable levels for sE-Selectin in some samples. 1:50 is a preferred dilution factor for such samples.
D. All samples must be stored in polypropylene tubes. DO NOT STORE SAMPLES IN GLASS.
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. The following notes should be reviewed and understood before the assay is set-up.
A. The Antibody-Immobilized Beads are light sensitive and must be covered with aluminum foil at all times. Cover the assay plate containing beads with aluminum foil during all incubation steps.
B. It is critical to allow all reagents to warm to room temperature (20-25° C) before use in the assay.
C. The bottom of the Microtiter Filter Plate should not be in direct contact with any absorbent material during assay set-up or incubation times. The plate can be set on the non-flat side of the plate cover or any other plate holder to raise the plate from the surface.
D. After the wash steps, keep the bottom of the Microtiter Filter Plate clean by blotting on paper towels or absorbent pads to prevent any leakage during assay set-up and incubation steps due to capillary action.
E. 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 4-5 seconds (equivalent to < 100 mmHg).
F. After hydration, all standards and controls must be transferred to polypropylene tubes.
G. The standards prepared by serial dilution must be used within 1 hour of preparation. Discard any unused standards except the original hydrated standard, which may be stored at £ -20° C for 1 month and at £ -80° C for greater than one month.
H. Any unused mixed Antibody-Immobilized Beads may be stored in the bead mix bottle at 2-8° C for up to one month.
I. During the preparation of the standard curve, make certain to vortex the higher concentration well before making the next dilution.
J. The plate should be read immediately after the assay is finished. Prior to reading, agitate the plate on the plate shaker for 2 to 10 minutes. Delay in reading the plate may result in decreased signals and sensitivities for some analytes.
K. 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.
L. Ensure the needle probe is clean. This may be achieved by sonication and/or Alcohol Flushes. Adjust probe height to the filter plate prior to reading an assay.
M. For cell culture supernatant or tissue extract samples, use the culture or extraction medium as matrix in blank, standard points and controls and as sample diluent for sample dilutions.
VIII. PREPARATION OF REAGENTS FOR IMMUNOASSAY
A. Preparation of Standard Cocktail
1). Prior to use, reconstitute the Standard Cocktail with 250 ml Deionized Water to give a 250 ng/ml final concentration for Adiponectin, sE-Selectin, sVCAM-1, sICAM-1, and 50 ng/mL final concentrations for MMP-9, MPO, and tPAI-1. Invert the vial several times to mix. Allow the vial to set for 5-10 minutes to make sure that the standards are completely reconstituted, and then transfer the standard to an appropriately labeled polypropylene microfuge tube. This will be used as the Original Standard; the unused portions of this standard may be stored at £ -20° for up to one month.
2). Preparation of Working Standards
Label five polypropylene microfuge tubes Standard 1, Standard 2, Standard 3, Standard 4, and Standard 5. Add 200 ml of Assay Buffer to each of the five tubes. Prepare serial dilutions by adding 50 ml of the Original reconstituted standard to the Standard 5 tube, mix well and transfer 50 ml of the Standard 5 tube to the Standard 4 tube, mix well and transfer 50 ml of the Standard 4 tube to the Standard 3 tube, mix well and transfer 50 ml of the Standard 3 tube to the Standard 2 tube, mix well and transfer 50 ml of the Standard 2 tube to the Standard 1 tube and mix well. The 0 standard (Background) will be the Assay Buffer.
|
Standard Dilution |
Volume of Deionized Water to Add |
Volume of Standard |
|
Original Standard |
250 ml water |
0 |
|
Standard Dilution |
Volume of Assay Buffer to Add |
Volume of Standard |
|
Standard 5 |
200 µl |
50 µl of Original |
|
Standard 4 |
200 ml |
50 µl of Standard 5 |
|
Standard 3 |
200 ml |
50 µl of Standard 4 |
|
Standard 2 |
200 ml |
50 µl of Standard 3 |
|
Standard 1 |
200 ml |
50 µl of Standard 2 |
The serial dilutions result in the following concentrations of standards.
|
Standard Dilution |
Adiponectin(ng/ml) |
sE-Selectin(ng/ml) |
sVCAM-1(ng/mL) |
sICAM-1(ng/mL) |
MMP-9(pg/mL) |
MPO(pg/mL) |
tPAI-1(pg/mL) |
|
Original |
250.0 |
250.0 |
250.0 |
250.0 |
50,000 |
50,000 |
50,000 |
|
1:5 |
50.0 |
50.0 |
50.0 |
50.0 |
10,000 |
10,000 |
10,000 |
|
1:25 |
10.0 |
10.0 |
10.0 |
10.0 |
2,000 |
2,000 |
2,000 |
|
1:125 |
2.0 |
2.0 |
2.0 |
2.0 |
400 |
400 |
400 |
|
1:625 |
0.4 |
0.4 |
0.4 |
0.4 |
80 |
80 |
80 |
|
1:3,125 |
0.08 |
0.08 |
0.08 |
0.08 |
16 |
16 |
16 |
B. Preparation of Controls
Before use, reconstitute Control 1 and Control 2 each with 250 ml deionized water. Invert the vial several times to mix. Allow the vial to set for 5-10 minutes, then transfer each Control to an appropriately-labeled polypropylene microfuge tube. Unused portions may be stored at £ -20° C for up to one month.
C. Preparation of Wash Buffer
Bring the 10X Wash Buffer to room temperature and mix well 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.
E. Preparation of Antibody-Immobilized Beads
Sonicate each antibody-bead bottle for 30 seconds, and then vigorously vortex the bottle for 1 minute. Add 150 µl from each antibody-bead bottle to the Mixing Bottle and bring final volume to 3.0 ml with Bead Diluent. Vortex well. Unused portions may be stored at 2-8° C for up to one month.
Example 1: For a 3 plex assay, add 150 µl of each of the 3 beads to the Mixing Bottle, add 2.55 ml Bead Diluent.
Example 2: For a 7 plex assay, add 150 µl of each of the 7 beads to the Mixing Bottle, add 1.95 ml Bead Diluent.
Prior to beginning this assay, it is imperative to read this protocol completely and to thoroughly understand the Technical Guidelines outlined in Section VII.
Allow All Reagents To Warm To Room Temperature (20-25° C) Before Use In The Assay.
1. Diagram placement of background, standards, controls 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. See recommended well map at end of protocol.
2. Pre-wet the filter plate by pipetting 200 µL of 1X Wash Buffer into each well of the microtiter plate. Seal plate with a plate sealer and agitate on a plate shaker for 10 minutes at room temperature (20-25° C).
3. Remove Wash Buffer by vacuum. (NOTE: DO NOT INVERT PLATE). Remove any excess Wash Buffer from the bottom of the plate by blotting on an absorbent pad or paper towels. Repeat if necessary.
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 an appropriate matrix solution to the Background, Standards, and Control wells. If diluted plasma or serum samples are being assayed, use Assay Buffer as matrix solution. If cell culture supernates or tissue extracts are being assayed, use culture or extraction medium as matrix solution.
8. Add 25 µL of Sample into the appropriate wells.
9. Vortex Bead Mix Bottle and add 25 µL of Mixed Beads to each well. (Note: during addition of Mixed Beads, shake the bead suspension intermittently to avoid settling).
10. Seal, cover with aluminum foil, and incubate with vigorous agitation on a plate shaker overnight (16-18 h) at 2-8° C.
11. Gently remove fluid by vacuum filtration.
12. Wash plate 2 times with 200 µL/well of Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Remove any excess Wash Buffer from the bottom of the plate using an absorbent pad or paper towels.
13. Add 25 µL of Detection Antibody Cocktail into each well. (Note: It is important to allow the Detection Antibody to warm to room temperature, 20-25° C, before adding to wells.)
14. Seal, cover with aluminum foil, and incubate with vigorous agitation on a plate shaker at room temperature (20-25° C) for 1 hour. DO NOT VACUUM AFTER THIS INCUBATION.
15. Add 25 µL Streptavidin-Phycoerythrin to each well containing the 25 µL of Detection Antibody Cocktail.
16. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25° C).
17. Gently remove all contents by vacuum.
18. Wash plate 2 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 paper towel or tissue.
19. Add 100 µL of Sheath Fluid to all wells. Cover with aluminum foil and resuspend the beads by shaking on a plate shaker for 5 minutes.
20. Run plate on Luminex Instrument. Read 50 beads per bead set in 50 µL sample size.
21. Save the data and evaluate the Median Fluorescence Intensity using appropriate curve-fitting software. A 5-parameter logistic method with weighting or cubic spline method is recommended. Remember to multiply the results by the sample dilution factor.
Select the following equipment settings:
| Events: | 50, per bead |
| Sample Size: | 50 ml |
| Bead Set: | 02 for sE-Selectin |
| 16 for sVCAM-1 | |
| 24 for sICAM-1 | |
| 27 for MMP-9 | |
| 31 for MPO | |
| 51 for Adiponectin | |
| 84 for tPAI-1 | |
| **Gate (for IS System): | 8,061 to 13,091 |
| **Gate (for 1.7 System): | 8,061 to 13,091 |
**These specifications are for the Luminex 100 or Luminex 200 with software v 1.7 or IS. Luminex instruments with other software (e.g. Masterplex, Starstation, LiquiChip, Bioplex, LABScan100, etc.) would need to follow instrument instructions for gate settings and additional specifications.
The ranges for each analyte in Quality Control 1 and 2 are provided on the card insert or can be located at the Linco Research website www.lincoresearch.com.
A. Sensitivity (Minimum Detectable Concentrations, minDC)
|
Analyte |
MinDC (pg/mL) |
|
sE-Selectin |
79.0 |
|
sVCAM-1 |
16.0 |
|
sICAM-1 |
9.0 |
|
MMP-9 |
1.0 |
|
MPO |
7.0 |
|
Adiponectin |
56.0 |
|
tPAI-1 |
1.0 |
B. Precision:
|
Analyte |
Intra-Assay |
Inter-Assay (CV%) |
|
sE-Selectin |
11.2 |
13.4 |
|
sVCAM-1 |
4.5 |
8.5 |
|
sICAM-1 |
7.9 |
9.7 |
|
MMP-9 |
6.8 |
11.7 |
|
MPO |
12.3 |
16.3 |
|
Adiponectin |
9.2 |
15.9 |
|
tPAI-1 |
11.8 |
12.5 |
Intra-assay precision is generated from the mean of the %CV’s from 12 reportable results across two different concentrations of analytes represented by serum samples in a single assay. Interassay precision is generated from the mean of the %CV’s from 2 reportable results across 2 different concentrations of analytes across 5 different assays.
C. Accuracy in Serum (% Spiked):
|
Analyte |
Serum |
|
sE-Selectin |
104.1 |
|
sVCAM-1 |
103.9 |
|
sICAM-1 |
83.0 |
|
MMP-9 |
91.1 |
|
MPO |
91.7 |
|
Adiponectin |
96.4 |
|
tPAI-1 |
84.6 |
Accuracy, defined as percentage of measured analyte concentration in serum samples spiked with known concentrations of analyte, was determined by taking the average recovery of 3 levels of analytes in serum samples (2, 10, and 50 ng/ml for Adiponectin, sE-Selectin, sVCAM-1 and sICAM-1; 0.4, 2.0 and 10 ng/ml for MMP-9, MPO and tPAI-1).
D. Dilution Linearity (% Recovery):
|
Analyte |
Dilution 1:100 |
Dilution 1:200 |
Dilution 1:400 |
|
sE-Selectin |
109.9 |
117.5 |
109.2 |
|
sVCAM-1 |
113.7 |
114.6 |
118.1 |
|
sICAM-1 |
98.7 |
107.9 |
126.8 |
|
MMP-9 |
102.7 |
115.2 |
125.8 |
|
MPO |
96.9 |
102.2 |
89.6 |
|
Adiponectin |
70.0 |
84.0 |
81.9 |
|
tPAI-1 |
95.1 |
100.0 |
102.6 |
Dilution Linearity, defined as the analyte levels measured in diluted samples as a percentage of the 1:50 diluted samples, was determined by measuring analyte concentrations in 6 independent serum samples. The samples were diluted 1:50, 1:100, 1:200 and 1:400 in the kit Assay Buffer, and then analyzed in duplicate. The average percent recovery was reported.
E. Crossreactivity: There is no detectable cross reactivity within the panel.
Note: Adiponectin, sVCAM-1, MMP-9, MPO, and tPAI-1 have strong detectable signals in non-human primate serum or plasma samples (not fully validated).
| REAGENTS | Cat # |
| Human CVD Panel 1Standard | HCVD1-8067-1 |
| Human CVD Panel 1 Quality Controls | HCVD1-6067-1 |
| Human CVD Panel 1 Detection Antibody | HCVD1-1067-1 |
| Streptavidin-Phycoerythrin | L-SAPE6 |
| LINCOplex Assay Buffer | L-AB |
| Bead Diluent | LBD |
| 10X Wash Buffer | L-WB |
| Set of two 96-Well Filter Plates with sealers | L-PLATE |
| Antibody-Immobilized Beads: | |
| 02-Human soluble E-Selectin Beads | HESEL |
| 16-Human soluble VCAM-1 Beads | HSP-SVCM1 |
| 24-Human soluble ICAM-1 Beads | HSP-SICM1 |
| 27-Human MMP-9 Beads | HMMP-9 |
| 31-Human MPO Beads | HMPO |
| 51-Human Adiponectin Beads | HA-ADPN |
| 84-Human total PAI-1 Beads | HASP-PAI1 |