HUMAN CARDIOVASCULAR DISEASE (CVD) PANEL 2 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 info@lincoresearch.com.
This multiplex assay kit manufactured by LINCO Research may be used for the simultaneous quantification of the following acute-phase proteins: C-Reactive Protein (CRP), Serum Amyloid A (SAA), and Serum Amyloid Protein P Component (SAP). 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 (fully validated). SAA showed cross-reactivity in non-human primate samples (not fully validated).
This kit is for research purposes only.
A. Human CVD Panel 2 Beads (20X concentrated): Dilution is required before the assay. The following Antibody-Immobilized Beads are available for your customized kits:
#48-anti CRP
#67-anti SAA
#71-anti SAP
Quantity: 200 µl per tube
B. Human CVD Panel 2 Standard
1 vial containing mixed analytes in a cocktail, lyophilized
Quantity: 1 vial
C. Human CVD Panel 2 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 2 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 per well of tissue extract or cell/tissue culture supernatant samples or diluted serum, plasma samples can be used. Samples may require appropriate dilutions before the assay. Dilution factor varies with sample types and treatment.
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 1,000 X g, remove serum or plasma and assay immediately or aliquot and store samples at a temperature £ -20ºC. Avoid multiple (>2) freeze/thaw cycles.
Serum and Plasma Sample Dilution: Concentrations of acute phase proteins can change dramatically depending upon specific treatments or pathological conditions. Customers may need to determine the optimal dilution factors for their samples depending on the expected biological range. In general, samples from normal subjects should be diluted 1:2,000 or more for quantification of CRP, SAA, and SAP. LINCOplex Assay Buffer provided in the kit should be used as the sample diluent.
Sample dilutions can be done in two steps:
For samples diluted at 1:2,000, Step 1: add 5 µl plasma to 495 µl Assay Buffer (1:100); Step 2: add 10 µl 1:100 diluted sample to 190 µl of Assay Buffer (1:2,000).
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. 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.
I. 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.
J. 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.
K. Ensure the needle probe is clean and unclogged. This may be achieved by sonication and/or Alcohol Flushes. Adjust probe height to the filter plate prior to reading an assay. For instrument maintenance, please refer to instrument manual for detailed instructions.
L. For cell culture supernatants or tissue extracts, use the culture or extraction medium as matrix in blank, standard points and controls.
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 all analytes (CRP, SAA, and SAP). 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 (250 ng/ml). The unused portions of this stock may be stored at £ -20° C for up to one month.
2). Preparation of Working Standards
Label five polypropylene microfuge tubes 50, 10, 2, 0.4, and 0.08 ng/ml. Add 200 ml of Assay Buffer to each of the five tubes. Prepare serial dilutions by adding 50 ml of the 250 ng/ml reconstituted standard to the 50 ng/ml tube, mix well and transfer 50 ml of the 50 ng/ml standard to the 10 ng/ml tube, mix well and transfer 50 ml of the 10 ng/ml standard to the 2 ng/ml tube, mix well and transfer 50 ml of the 2 ng/ml standard to 0.4 ng/ml tube, mix well and transfer 50 ml of the 0.4 ng/ml standard to the 0.08 ng/ml tube and mix well. The 0 standard (Background) will be the Assay Buffer.