HUMAN CARDIOVASCULAR DISEASE (CVD) PANEL 3 LINCOplex

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 [email protected]

 

I. INTENDED USE

This multiplex assay kit manufactured by LINCO Research, Inc. is to be used for the simultaneous quantification of the following analytes in any combinations: IL-1ß, IL-6, IL-8, IL-10, IFNg , MCP-1, TNFα, NT-proBNP, and VEGF. This kit can be used for the analysis of the above analytes in tissue extract, cell/tissue culture samples, and human serum or plasma samples.

This kit is for research purposes only.

II. REAGENTS SUPPLIED

A. Human CVD Panel 3 Beads (20X concentrated): Dilution is required before the assay. The following Antibody-Immobilized Beads are available for your customized kits:

#01-IL-1ß

#12-IL-6

#20-IL-8

#23-IL-10

#35-IFNg

#40-TNFα

#46-MCP-1

#71-VEGF

#75-NT-proBNP

Quantity: 200 m l per tube

 

B. Human CVD Panel 3 Standard

1 vial containing mixed analytes in a cocktail, lyophilized

Quantity: 1 vial

C. Human CVD Panel 3 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 3 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. Serum Matrix (optional for serum/plasma samples)

1 bottle containing Serum Diluent for Standard and Controls

Quantity: 1.0 ml, lyophilized/bottle

H. LINCOplex Assay Buffer

PBS with 0.08% Sodium Azide and 1% BSA, pH 7.4

Quantity: 30 ml/bottle

 

I. 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

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 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.

IV. REAGENT PRECAUTIONS

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 per well of tissue extract or cell/tissue culture supernatant samples, serum, or 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:

For serum, allow the blood to clot for at least 30 minutes. For plasma, use appropriate anti-coagulant. After centrifugation for 10 min at 1000 X g, remove serum or plasma and assay immediately or aliquot and store samples at a temperature £ -20ºC. Avoid multiple (>2) freeze/thaw cycles.

If dilutions are required, serum or plasma samples should be diluted using the LINCOplex Serum Matrix as the sample diluent.

D. All samples must be stored in polypropylene tubes. DO NOT STORE SAMPLES IN GLASS.

VII. TECHNICAL GUIDELINES

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 1 to 10 minutes. Long 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 supernatants or tissue extraction 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 Cocktails

1). Reconstitute the Human CVD Panel 3 Standard with 250 ml deionized water to give a 50 ng/ml concentration for NT-proBNP and a 10 ng/mL concentration for all other analytes. 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. 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° C for up to one month.

2). Preparation of Working Standards

Label six polypropylene microfuge tubes Standard 1, Standard 2, Standard 3, Standard 4, Standard 5, and Standard 6. Add 200 ml of Assay Buffer to each of the six tubes. Prepare serial dilutions by adding 50 ml of the Original reconstituted Standard to the Standard 6 tube, mix well and transfer 50 ml of the Standard 6 tube 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
to Add

Original

250 ml

0

Standard Dilution

Volume of Assay Buffer to Add

Volume of Standard to Add

Standard 6

200 µl

50 µl of Original

Standard 5

200 ml

50 µl of Standard 6

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

IL-1ß
(pg/ml)

IL-6
(pg/ml)

IL-8
(pg/ml)

IL-10(pg/ml)

IFNg (pg/ml)

MCP-1(pg/ml)

TNFα(pg/ml)

NT-proBNP(pg/ml)

VEGF(pg/ml)

Original

10,000

10,000

10,000

10,000

10,000

10,000

10,000

50,000

10,000

1:5

2,000

2,000

2,000

2,000

2,000

2,000

2,000

10,000

2,000

1:25

400

400

400

400

400

400

400

2,000

400

1:125

80

80

80

80

80

80

80

400

80

1:625

16

16

16

16

16

16

16

80

16

1:3,125

3.2

3.2

3.2

3.2

3.2

3.2

3.2

16

3.2

1:15,625

0.64

0.64

0.64

0.64

0.64

0.64

0.64

3.2

0.64

B. Preparation of Controls

Control 1:

Reconstitute Human CVD Panel 3 Control 1 with 250 µl water. Invert the vial several times to mix. Allow the vial to set for 5-10 minutes, and then transfer the reconstituted Control 1 to an appropriately-labeled polypropylene microfuge tube. Unused portions may be stored at £ -20° C for up to one month.

Control 2:

Reconstitute Human CVD Panel 3 Control 2 with 250 µl water. Invert the vial several times to mix. Allow the vial to set for 5-10 minutes, and then transfer the reconstituted Control 2 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 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, 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 9-plex assay, add 150 µl of each of the 9 beads to the Mixing Bottle, add 1.65 ml Bead Diluent.

E. Preparation of Serum Matrix

This step is required for serum or plasma samples only.

Add 1.0 mL deionized water to the bottle containing lyophilized Serum Matrix. Mix well. Allow at least 10 minutes for complete reconstitution. Left-over reconstituted Serum Matrix should be stored at £ -20° C for up to one month.

IX. IMMUNOASSAY PROCEDURE

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 the end of the protocol.

2. Block the filter plate by pipetting 200 µL of Assay Buffer into each well of the microtiter plate. Seal plate with plate sealer and agitate 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.

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 Sample into the appropriate wells.

8. Add 25 µL of an appropriate matrix solution to the Background, Standards, and Control wells. If assaying serum or plasma samples, use the Serum Matrix provided in the kit as the matrix solution and sample diluent if needed. If assaying tissue/cell extract or tissue/cell culture medium samples, use identical extraction buffer or control medium respectively as the matrix solution and sample diluent, if needed.

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 for 1 hour at room temperature (i.e. 22-25° C). [Alternatively, for better assay sensitivities, this incubation step may be done at 4° C with agitation on a shaker overnight (16-18 h)].

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 30 minutes. (Alternatively, for better assay sensitivities, the plate may be incubated room temperature with agitation on a plate shaker 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 re-suspend 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.