HUMAN SEPSIS/APOPTOSIS 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 analytes in any combination: soluble VCAM-1 (sVCAM-1), soluble ICAM-1 (sICAM-1), soluble Fas Ligand (sFasL), soluble Fas (sFas), Macrophage Migration Inhibitory Factor (MIF) and Total Plasminogen Activator Inhibitor-1 (tPAI-1). This kit is fully validated for the analysis of the above analytes in tissue extract, cell/tissue culture samples, and diluted human serum or plasma samples. The analytes sVCAM-1, MIF and tPAI-1 show cross-reactivity with monkey serum samples.
This kit is for research purposes only.
A. Antibody Immobilized Beads (20X concentrated): Dilution is required before the assay. The following Antibody-Immobilized Beads are available for your customized kits:
#16-Human sVCAM-1
#24-Human sICAM-1
#29-Human sFas
#33-Human sFasL
#41-Human MIF
#84-Human tPAI-1
Quantity: 0.2 ml per tube
B. Human Sepsis Standard Cocktail
1 vial containing human sepsis/apoptosis standard cocktail, lyophilized
Quantity: 1 vial
C. Human Sepsis Quality Controls
Control I – 1 vial containing mixed sepsis/apoptosis cocktail, lyophilized
Control II – 1 vial containing mixed sepsis/apoptosis cocktail, lyophilized
Quantity: 1 vial each control
D. Human Sepsis/Apoptosis Detection Antibodies
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 bead diluent for bead preparation
Quantity: 3.5 ml/bottle
G. Assay Buffer
50 mM PBS with 25 mM EDTA, 0.08% Sodium Azide, 0.05% Tween 20 and 1% BSA, pH 7.4
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. Serum Matrix, lyophilized (optional-for serum/plasma samples)
Serum containing 0.08% Sodium Azide
Quantity: 5 ml/vial
J. Microtiter Filter Plate
Quantity: 1- 96-Well Filtration Plate
K. Plate Sealers
Quantity: 2 Plate Sealers
L. Mixing Bottle
III. STORAGE CONDITIONS UPON RECEIPT
Recommended storage for kit components is 2 - 8°C. See individual vial for long-term storage recommendations.
Once the standards and controls have been reconstituted, immediately 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, 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 5 m l to 50 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, Inc. Model 4625, or equivalent)
10. Vacuum Filtration Unit (Millipore Vacuum Manifold Catalogue #MAVM0960R, or equivalent)
11. Luminex100 by Luminex Corporation
VI. SPECIMEN COLLECTION AND STORAGE
A. A maximum of 25 m l per well of tissue extract or cell/tissue culture supernatant samples or 1:10 diluted serum, plasma samples can be used.
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:
Allow the blood to clot for at least 30 minutes before centrifugation for 10 minutes at 1000 X g. Remove serum and assay immediately and aliquot and store samples at a temperature £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. It is recommended to centrifuge plasma samples again at 3000 xg for five minutes prior to assay setup.
Serum or plasma samples should be diluted 1:10 or higher using the Serum Matrix provided in the kit as the sample diluent. Additional Serum Matrix is available from LINCO Research Inc. For sFas, sFasL, and MIF, which have relative low concentrations, it is recommended to dilute serum/plasma sample 1:10. For sVCAM-1, sICAM-1 and tPAI-1 which are typically at concentrations much higher the other 3 analytes, it is best to dilute serum/plasma samples at greater than 1:10 (e.g. 1:40). When all six analytes are measured in a single sample, a compromising dilution factor of 1:10 should be used for serum/plasma samples. For 1:10 dilution, add10 µl sample to 90 µl Serum Matrix. For 1:40 dilution, add 2.5 µl sample to 97.5 µl Serum Matrix.
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 ≥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 highest concentration stock 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. Use a fresh tip with each dilution.
J. The plate should be read immediately after the assay is finished. If however the plate cannot be read immediately, 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. Delay in reading a plate may result in decreased signals and sensitivities for some analytes.
K. The microtiter 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 Lincoplex Filter Plate prior to reading an assay.
M. 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 Sepsis/Apoptosis Standard Cocktail
1). Before use, reconstitute the Human Sepsis/Apoptosis Standard Cocktail with 250 ml Deionized Water to give a concentration of 250 ng/ml for sVCAM-1 and sICAM-1 and a concentration of 50 ng/mL for sFasL, sFas, MIF 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 stock standard (Original); the unused portions of this stock may be stored at £ -20° for up to one month.
2). Preparation of Working Standards
Label six polypropylene microfuge tubes 1:4, 1:16, 1:64, 1:256, 1:1024 and 1:4096. Add 150 ml of Assay Buffer to each of the six tubes. Prepare serial dilutions by adding 50 ml of the Original reconstituted standard to the 1:4 tube, mix well and transfer 50 ml of the 1:4 standard to the 1:16 tube, mix well and transfer 50 ml of the 1:16 standard to the 1:64 tube, mix well and transfer 50 ml of the 1:64 standard to 1:256 tube, mix well and transfer 50 ml of the 1:256 standard to the 1:1024 tube and mix well and transfer 50 µl of the 1:1024 standard to the 1:4096 tube and mix well. The 0 standard (Background) will be Assay Buffer.
|
Standard Dilution |
Volume of Deionized Water to Add |
Volume of Standard |
|
Original |
250 ml |
0 |
|
Standard Dilution |
Volume of Assay Buffer to Add |
Volume of Standard |
|
1:4 |
150 ml |
50 µl of Original |
|
1:16 |
150 ml |
50 µl of 1:4 |
|
1:64 |
150 ml |
50 µl of 1:16 |
|
1:256 |
150 ml |
50 µl of 1:64 |
|
1:1024 |
150 ml |
50 µl of 1:256 |
|
1:4096 |
150 ml |
50 µl of 1:1024 |
The serial dilutions result in the following concentrations of standards.
|
Standard Dilution |
sVCAM-1 |
sICAM-1 |
sFas (pg/mL) |
sFasL(pg/mL) |
MIF (pg/mL) |
tPAI-1 (pg/mL) |
|
Original |
250,000 |
250,000 |
50,000 |
50,000 |
50,000 |
50,000 |
|
1:4 |
62,500 |
62,500 |
12,500 |
12,500 |
12,500 |
12,500 |
|
1:16 |
15,625 |
15,625 |
3,125 |
3,125 |
3,125 |
3,125 |
|
1:64 |
3,906 |
3,906 |
781 |
781 |
781 |
781 |
|
1:256 |
977 |
977 |
195 |
195 |
195 |
195 |
|
1:1024 |
244 |
244 |
48.8 |
48.8 |
48.8 |
48.8 |
|
1:4096 |
61 |
61 |
12.2 |
12.2 |
12.2 |
12.2 |
B. Preparation of Controls
Before use, reconstitute Control I and Control II each with 250 ml deionized water. Invert the vials several times to mix and vortex. 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° for up to one month.
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 Serum Matrix
This step is required for serum or plasma samples only.
Add 5.0 ml deionized water to the bottle containing the Serum Matrix, mix well. Allow the bottle to set for at least 10 min for complete reconstitution of serum components. Unused reconstituted serum matrix should be stored at £ -20° for up to one month.
E. Preparation of Antibody-Immobilized Beads
Sonicate each antibody-bead bottle for 30 seconds, and then vortex the bottle for 1 minute. Add 0.15 ml 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: when using 3 human Sepsis/Apoptosis antibody immobilized beads, add 0.15 ml from each of the three bead vials to the mixing bottle. Then add 2.55 ml Bead Diluent.
Example 2: when using 6 human Sepsis/Apoptosis antibody immobilized beads, add 0.15 ml from each of the six bead vials to the mixing bottle. Then add 2.10 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 Standards, 0 (Background), 1:4096, 1:1024, 1:256, 1:64, 1:16, 1:4 and the Original, Controls 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 PLATE). Remove any excess Assay Buffer from the bottom of the plate by blotting on an absorbent pad or paper towels. Residual liquid in the wells will affect the accuracy and precision of the assay.
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. Specifically, when assaying tissue/cell extract or tissue/cell culture medium samples, use identical extraction buffer or control medium as the matrix solution and sample diluent, respectively. When assaying serum or plasma samples, use the Serum Matrix provided in the kit as the matrix solution and sample diluent.
8. Add 25 µL of Sample into the appropriate wells.
9. Vortex Premixed Bead 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 agitation on a plate shaker for 2 hours (for buffer-based or serum-free samples) or overnight (18-20 h, for serum or plasma samples or serum-containing culture medium samples) 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. Residual liquid in the wells will affect the accuracy and precision of the assay.
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 agitation on a plate shaker at room temperature (20-25° C) for 1 hour. DO NOT VACUUM AFTER THIS INCUBATION.
15. Add 25 m L 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. (NOTE: DO NOT INVERT PLATE).
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 Luminex100. Read 50 beads per bead set in 50 uL sample size.
21. Save the data and evaluate the median fluorescence units 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: | 16 for sVCAM-1 |
| 24 for sICAM-1 | |
| 29 for sFas | |
| 33 for sFasL | |
| 41 for MIF | |
| 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 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.
Sensitivity (Minimum Detectable Concentrations, minDC)