HUMAN APOLIPOPROTEIN
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 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 is to be used for the simultaneous quantification of the following six apolipoproteins in any combinations: Apo AI, Apo AII, Apo B, Apo CII, Apo CIII, and Apo E. This kit may be used for the analysis of the above apolipoproteins in serum, plasma, tissue extract, other biological fluids, or tissue culture supernatant samples.
This kit is for research purposes only.
A. Antibody-Immobilized Beads
The following antibody-immobilized beads are available as individual bead sets at 20X concentrations. The user needs to mix selected antibody beads and dilute the beads with Bead Diluent to achieve a final 1X concentration for each bead in the mixture.
#08-Human Apolipoprotein AI
#04-Human Apolipoprotein AII
#06-Human Apolipoprotein B
#09-Human Apolipoprotein CII
#11-Human Apolipoprotein CIII
#14-Human Apolipoprotein E
Individual bead bottles require mixing and dilution with Bead Diluent as described in Section VIII. D.
Quantity: 0.2ml antibody-immobilized beads/bottle at 20X concentration
B. Human Apolipoprotein Calibrator Cocktail
1 vial containing human Apolipoprotein Calibrator Cocktail, lyophilized
Quantity: 1 vial
C. Human Apolipoprotein Controls
Control I – 1 vial containing human Apolipoprotein Cocktail, lyophilized
Control II – 1 vial containing human Apolipoprotein Cocktail, lyophilized
Quantity: 1 vial/Control
D. Human Apolipoprotein Detection Antibodies
1 bottle containing a cocktail of biotinylated antibodies in Assay Buffer
Quantity: 5.5 ml/bottle
E. Streptavidin-Phycoerythrin
1 bottle containing Streptavidin-Phycoerythrin prepared in Assay Buffer
Quantity: 5.5 ml/bottle
F. Assay Buffer
10 mM PBS with 0.08% Sodium Azide and 1% BSA, pH 7.4
Quantity: 2 bottles; 30 ml/bottle
G. 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.
H. Bead Diluent
1 bottle containing a buffered solution for stabilizing beads
Quantity: 4.0 ml/bottle
I. Microtiter Filter Plate
Quantity: 1- 96-Well Filtration Plate
J. Plate Sealers
Quantity: 2 Plate Sealers
K. Mixing Bottle
Quantity: 1 Bottle (not provided in kits containing premixed beads)
III. STORAGE CONDITIONS UPON RECEIPT
Recommended storage for kit components is 2 - 8°C. See individual vials for long-term storage recommendations.
Once the calibrators and controls have been reconstituted, transfer contents into polypropylene vials. DO NOT STORE RECONSTITUTED CALIBRATORS OR CONTROLS IN GLASS VIALS. For long-term storage, freeze reconstituted calibrators and controls at £ -20°C. Avoid multiple (>2) freeze-thaw cycles.
DO NOT FREEZE Antibody-Immobilized Beads, Detection Antibody, and Streptavidin-Phycoerythrin. Any unused mixed Antibody-Immobilized Beads must be discarded.
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 25 m l to 1000 m l
2. Multichannel Pipettes 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 (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 #9;Catalogue #MAVM0960R, or equivalent)
11. Luminex Instrument
VI. SPECIMEN COLLECTION, STORAGE AND PREPARATION
A. A maximum of 10 m l of appropriate culture medium or diluted tissue human serum or plasma sample can be used per well.
B. Preparation of Tissue Culture Supernatant:
Centrifuge the sample to remove cell debris and run assay immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. Tissue Culture Supernatant may require a dilution with the appropriate control medium prior to assay.
C. Preparation of Human Serum Samples:
Allow the blood to clot for at least 30 minutes. Centrifuge samples for 10 minutes at 1000 x g. Remove serum and run the assay immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. We recommend that human serum and plasma samples be diluted 10,000-fold using Assay Buffer as the diluent. Samples can be diluted using a two-step protocol. Step 1, add 4 µl serum or plasma to 396 µl Assay Buffer (i.e. 100-fold). Step 2, add 4 µl of the 100-fold diluted sample from Step 1 to another microfuge tube containing 396 µl Assay Buffer. Users may make similar dilutions using less sample volume to conserve Assay Buffer. For diluted samples, multiply the final concentration of each analyte by the dilution factor. For plasma samples is it recommended to centrifuge samples again at 3000 xg for five minutes prior to assay setup.
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 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 calibrators and controls must be transferred to polypropylene microfuge tubes.
G. The calibrators prepared by serial dilution must be used within 1 hour of preparation. Discard any unused calibrators except the highest stock calibrator, which may be stored at £ -20° C for 1 month and at £ -80° C for greater than one month.
H. Mix only required amount of beads prior to assay setup. Discard any unused premixed beads.
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 the plate may result in decreased signals and sensitivities for some apolipoproteins.
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 in a 0.3 cm orbit.
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 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 Apolipoprotein Calibrator Cocktail
1.) Before use, reconstitute the Human Apolipoprotein Calibrator Cocktail with 250 ml of Deionized Water. Invert the vial several times to mix. Vortex the vial for 10 seconds. Allow the vial to set for 5-10 minutes and then transfer the calibrator to an appropriately labeled polypropylene microfuge tube. This will be used as the highest concentration calibrator (i.e. Original); the unused portions of this calibrator solution may be stored at £ -20° for up to one month.
2). Preparation of Working Calibrators
Label eight polypropylene microfuge tubes, i.e. BG (Background), 1:5, 1:25, 1:125, 1:625, 1:3125, 1:15625 and 1:78125 to indicate number of dilution fold. Add 160 ml of Assay Buffer to each of the eight tubes. Prepare serial dilutions by adding 40 ml of the original (highest concentration) reconstituted calibrator cocktail to the 1:5 tube, mix well and transfer 40 ml of the 1:5 diluted calibrator cocktail to the 1:25 tube, mix well and transfer 40 ml of the 1:25 diluted calibrator cocktail to the 1:125 tube, mix well and transfer 40 ml of the 1:125 diluted calibrator cocktail to the 1:625 tube, mix well and transfer 40 ml of the 1:625 diluted calibrator cocktail to the 1:3125 tube, mix well and transfer 40 µl of the 1:3125 diluted calibrator to the 1:15625 tube, mix well and transfer 40 µl of the 1:15625 diluted calibrator to the 1:78125 tube and mix well. The BG (Background) will be Assay Buffer.
|
Calibrator Dilution |
Volume of Deionized Water to Add |
Volume of Calibrator |
|
Original |
250 ml |
0 |
|
Calibrator Dilution |
Volume of Assay Buffer |
Volume of Calibrator to Add |
|
1:5 |
160 ml |
40 µl of Original Calibrator |
|
1:25 |
160 ml |
40 µl of 1:5 Calibrator |
|
1:125 |
160 ml |
40 µl of 1:25 Calibrator |
|
1:625 |
160 ml |
40 µl of 1:125 Calibrator |
|
1:3125 |
160 ml |
40 µl of 1:625 Calibrator |
|
1:15625 |
160 ml |
40 µl of 1:3125 Calibrator |
|
1:78125 |
160 ml |
40 µl of 1:15625 Calibrator |
After dilutions, the calibrators will have following concentrations of apolipoproteins.
The Apolipoprotein Calibrator Concentrations (ng/mL)
|
Calibrator Dilution |
Apo AI |
Apo AII |
Apo B |
Apo CII |
Apo CIII |
Apo E |
|
Background |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
1:78125 |
0.88 |
0.22 |
0.57 |
0.03 |
0.08 |
0.04 |
|
1:15625 |
4.42 |
1.10 |
2.84 |
0.15 |
0.42 |
0.21 |
|
1:3125 |
22.08 |
5.50 |
14.18 |
0.74 |
2.11 |
1.06 |
|
1:625 |
110.40 |
27.52 |
70.88 |
3.68 |
10.56 |
5.28 |
|
1:125 |
552.00 |
137.60 |
354.40 |
18.40 |
52.80 |
26.40 |
|
1:25 |
2760.00 |
688.00 |
1772.00 |
92.00 |
264.00 |
132.00 |
|
1:5 |
13800.00 |
3440.00 |
8860.00 |
460.00 |
1320.00 |
660.00 |
|
Original |
69000.00 |
17200.00 |
44300.00 |
2300.00 |
6600.00 |
3300.00 |
B. Preparation of Controls
Before use, reconstitute Human Apolipoprotein Control I and Control II each with 250 ml Deionized Water. Invert the vial several times to mix and vortex. Make certain that each control is completely dissolved. Allow the vial to set for 5-10 min and then transfer each control to an appropriately labeled polypropylene microfuge tube. Vortex well. 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 Antibody-Immobilized Beads
Vortex each bead bottle vigorously for 1 minute. Pipette 0.15ml of the 20x concentrated beads from each bottle and mix them in the mixing bottle provided. Add appropriate volume of Bead Diluent to bring the final volume to 3.0 ml.
Example 1: when using 6 antibody-immobilized beads, add 0.15 ml from each of the six bead sets to the mixing bottle. Add 2.1 ml Bead Diluent.
Example 2: when using 3 antibody-immobilized beads, add 0.15 ml from each of the three bead sets to the mixing bottle. Add 2.55 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 BG (Background), Calibrators at Original, 1:5, 1:25, 1:125, 1:625, 1:3125, 1:15625, 1:78125, 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. Prewet the filter plate by adding 200 µL of 1x Wash 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 the 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.
4. Add 65 µL of Assay Buffer to all wells (Background, Calibrators, Controls and Samples)
5. Add 10 µL Assay Buffer to BG (Background).
6. Add 10 µL of each Calibrator or Control into the appropriate wells.
7. Add 10 µL of appropriately diluted tissue culture medium or 10,000x diluted serum/plasma samples into the appropriate wells.
8. Vortex Bead Bottle containing mixed beads for approximately 1 min and add 25 mL of the Bead suspension to each well. (Note: during addition of Mixed Beads, shake bead mix intermittently to avoid settling).
9. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker at room temperature (20-25° C) for exactly 1 hour. Prolonged incubation time will result in higher background and decrease assay sensitivities.
10. Gently remove fluid by vacuum filtration. (NOTE: DO NOT INVERT PLATES).
11. Wash plate 3 times with 200 µL/well of Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Completely remove any excess Wash Buffer from the bottom of the plate by blotting on an absorbent pad or paper towels.
12. Add 50 µL of Detection Antibody Cocktail into each well. (Note: allow the Detection Antibody to warm to room temperature prior to addition)
13. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for exactly 30 minutes at room temperature (20-25° C). Prolonged incubation time will result in higher background and decrease assay sensitivities.
14. Gently remove all contents by vacuum.
15. 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 paper towel or tissue.
16. Add 50 m L Streptavidin-Phycoerythrin to each well.
17. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for exactly 30 minutes at room temperature (20-25° C). Prolonged incubation time will result in higher background and decrease assay sensitivities.
18. Gently remove all contents by vacuum.
19. 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 paper towel or tissue.
20. 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.
21. Run plate on Luminex Instrument.
22. Save the data and evaluate the median fluorescence units using appropriate curve-fitting software. A 5 or 4-parameter logistic method or cubic spline method is recommended.
Select the following equipment settings:
| Events: | 50, per bead |
| Sample Size: | 50 ml |
| Bead Set: | 08 for Apo AI |
| 04 for Apo AII | |
| 06 for Apo B | |
| 09 for Apo CII | |
| 11 for Apo CIII | |
| 14 for Apo E |
|
**Gate (for IS System): |
8061 - 13011 |
|
**Gate (for 1.7 System): |
8061 - 13011 |
**These specifications are for the Luminex100 or Luminex200 with software v. 1.7 or IS. 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. Sensitivity (Minimum Detectable Concentrations, MinDC)
|
Apolipoproteins |
MinDC (ng/mL) |
|
Apo AI |
0.50 |
|
Apo AII |
0.50 |
|
Apo B |
3.00 |
|
Apo CII |
0.25 |
|
Apo CIII |
0.10 |
|
Apo E |
0.10 |
B. Precision
|
Apolipoprotein |
Intra-Assay Precision (%CV) |
Interassay Precision (%CV) |
|
Apo AI |
5 |
11 |
|
Apo AII |
5 |
13 |
|
Apo B |
8 |
14 |
|
Apo CII |
5 |
15 |
|
Apo CIII |
5 |
13 |
|
Apo E |
5 |
22 |
Intra-assay precision is generated from the mean of the %CV’s from 8 reportable results across three different concentrations of calibrators in a single assay. Interassay precision is generated from the mean of the %CV’s from 8 reportable results across three different concentrations of calibrators across six different assays.
C. Accuracy
|
Apolipoprotein |
Average |
|
Apo AI |
93.0 |
|
Apo AII |
85.3 |
|
Apo B |
95.3 |
|
Apo CII |
91.0 |
|
Apo CIII |
100.0 |
|
Apo E |
92.2 |
Accuracy, defined as the measured apolipoprotein as a percentage of spiked calibrator, was determined by taking the average of different concentrations of apolipoprotein in the assay matrix across six different assays.
D. Dilution Linearity
|
Apolipoprotein |
%Recovery* (1:4000) |
%Recovery* (1:8000) |
%Recovery* (1:16000) |
|
Apo AI |
132.6 |
115.6 |
117.6 |
|
Apo AII |
99.7 |
113.7 |
140.2 |
|
Apo B |
105.9 |
109.8 |
----- |
|
Apo CII |
99.5 |
114.7 |
108.2 |
|
Apo CIII |
79.5 |
82.3 |
79.1 |
|
Apo E |
104.9 |
116.3 |
116.7 |
*The concentration at 1:2000 dilution was used as 100%.
E. Cross-Reactivity
To test cross-reactivity among the assays in the panel, individual apolipoprotein calibrator was prepared at a concentration 4 times greater than the highest concentration in the calibration curve and tested in the 6-plex assays using multiplexed beads immobilized with capture antibodies and using individual detection antibodies. There was no or negligible cross-reactivity within the panel.
| REAGENTS |
Cat # |
| Human Apolipoprotein Calibrator Cocktail |
LA-8062 |
| Human Apolipoprotein Quality Controls 1,2 |
LA-6062 |
| Human Apolipoprotein Detection Antibodies |
LA-1062 |
| Streptavidin-Phycoerythrin |
L-SAPE5 |
| Assay Buffer |
L-AB |
| 10X Wash Buffer |
L-WB |
| Bead Diluent |
LA-BD |
| Set of two 96 Well Filter Plates with sealers |
L-PLATE |
| 08-Human Apolipoprotein AI Beads |
APOA-1 |
| 04-Human Apolipoprotein AII Beads |
APOA-2 |
| 06-Human Apolipoprotein B Beads |
APOB |
| 09-Human Apolipoprotein CII Beads |
APOC-2 |
| 11-Human Apolipoprotein CIII Beads |
APOC-3 |
| 14-Human Apolipoprotein E Beads |
APOE |
WELL MAP
Assay Method for Apolipoprotein Lincoplex Kit