Mouse Endocrine 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 is a multiplex assay kit manufactured by Linco Research to be used for the simultaneous quantitative determination of the following five mouse endocrine hormones in any combination, Insulin, Amylin, Leptin, Glucagon, and Glucagon-Like Peptide-1 (GLP-1) (Active).
This kit is for research purposes only.
A. Antibody-Immobilized Beads
The following beads are supplied as requested:
1 bottle containing #05-Insulin
1 bottle containing #15-Amylin
1 bottle containing #16-Leptin
1 bottle containing #34-Glucagon
1 bottle containing #53-GLP-1 (Active)
Mix beads and dilute with HENDO-65K Bead Diluent as described in Section VIII. D.
Quantity: 0.2 ml antibody-immobilized beads/bottle
B. Bead Diluent
Quantity: 1 vial
C. Rat/Mouse Endocrine Standard Cocktail
1 vial containing endocrine standard cocktail, lyophilized
Quantity: 1 vial
D. Rat/Mouse Endocrine Controls
Control I – 1 vial containing mixed endocrine cocktail, lyophilized
Control II – 1 vial containing mixed endocrine cocktail, lyophilized
Quantity: 1 vial/Control
E. Serum Matrix, lyophilized
Serum containing 0.08% Sodium Azide
Reconstitute with 1.0 ml of deionized water before use.
Quantity: 1 ml/vial
F. Mouse Endocrine Detection Antibodies
1 bottle containing a cocktail of biotinylated detection antibodies in Assay Buffer
Quantity: 5.5 ml/bottle
G. Streptavidin-Phycoerythrin
1 bottle containing Streptavidin-Phycoerythrin prepared in Assay Buffer
Quantity: 5.5 ml/bottle
H. Assay Buffer
50 mM PBS with, 0.08% Sodium Azide, 0.05% Tween 20, Protease Inhibitor and 1% BSA, pH 6.8
Quantity: 30 ml/bottle
I. 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
J. Mixing Bottle
Quantity: 1 Bottle
K. Microtiter Filter Plate
Quantity: 1- 96 Well Filtration Plate
L. Plate Sealers
Quantity: 2 Plate Sealers
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 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, Bead Diluent, 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. 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 10 m l to 1000 m l
2. Multichannel Pipettes capable of delivering 10 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, Model #4625, or equivalent)
10. Vacuum Filtration Unit (Millipore Vacuum Manifold Catalogue #MAVMO960R, or equivalent)
11. Luminex Instrument
VI. SPECIMEN COLLECTION AND STORAGE
A. A 10 m l per well of serum or plasma can be used. Tissue culture or other media may also be used.
B. Preparation of Tissue Culture Supernatant:
Centrifuge the sample to remove debris and assay immediately or aliquot and store samples at £ -20şC. Avoid multiple (>2) freeze/thaw cycles. Tissue Culture Supernatant may require a dilution with the appropriate medium prior to assay.
C. Preparation of Serum Samples:
Note: For GLP-1 and Amylin measurements, plasma samples are preferred.
After collecting blood samples, immediately add the appropriate amount of a protease inhibitor according to the manufacturer’s instruction (see note). Invert tube several times to mix. Allow the blood to clot for at least 30 minutes before centrifugation for 10 minutes at 1000 xg. Remove serum and assay immediately or aliquot and store samples at £ -20şC. Avoid multiple (>2) freeze/thaw cycles. Use Serum Matrix Diluent as the diluent for samples requiring dilution prior to assay.
Note: A DPPIV inhibitor is required when testing samples for GLP-1 (Active) (if using Linco Catalogue #DPP4, add 10 ul DPP4 per milliliter of blood). A general protease inhibitor cocktail is required when testing for Amylin. If both GLP-1 and Amylin are being tested, then the DPPIV inhibitor and the protease inhibitor cocktail may be added to the same blood sample.
D. Preparation of Plasma Samples:
Plasma collection using EDTA as an anticoagulant is recommended. After collecting blood samples, immediately add the appropriate amount of a protease inhibitor according to manufacturer’s instruction (see note). Invert tube several times to mix. Centrifuge for 10 minutes at 1000 xg within 30 minutes of blood collection. Remove plasma and assay immediately or aliquot and store samples at £ -20şC. Avoid multiple (>2) freeze/thaw cycles. Use Serum Matrix as the diluent for samples requiring dilution prior to assay. It is recommended to centrifuge samples again at 3000 Xg for five minutes prior to assay set up.
Note: A DPPIV inhibitor is required when testing samples for GLP-1 (Active) (if using Linco Catalogue #DPP4, add 10 µl DPP4 per milliliter of blood). A general protease inhibitor cocktail is required when testing for Amylin. If both GLP-1 and Amylin are being tested, then the DPPIV inhibitor and the protease inhibitor cocktail may be added to the same blood sample.
E. Avoid using samples with gross hemolysis or lipemia.
F. Care must be taken when using heparin as an anticoagulant, since an excess will provide falsely high values. Use no more than 10 IU heparin per ml of blood collected.
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.
A. The Antibody-Immobilized Beads are light sensitive, so the assay plate containing beads must be covered with aluminum foil during all incubation steps.
B. The bottom of the Microtiter Filter Plate should not be in contact with any absorbent material during assay set-up or incubation times. Set the plate on a plate cover or any other plate holder to raise the plate from the surface.
C. After the wash steps, dry the bottom of the Microtiter Filter Plate by using paper towels or absorbent pads to prevent any leakage due to capillary action.
D. 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).
E. The standards prepared by serial dilution must be used within 1 hour of preparation. Discard any unused standards except the 10,000 pM stock standard which may be stored at £ -20° C for 1 month and at £ -80° C for greater than one month.
F. Mix only required amounts of beads prior to assay setup. Discard any unused premixed beads.
G. 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.
H. If the plate cannot be read on the same day as the assay was finished, do the final bead suspension in 100 ml Sheath Fluid, 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.
I. 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.
J. 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.
K. 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 Endocrine Standard Cocktail
1). Before use, reconstitute the Endocrine Standard Cocktail with 250 ml Deionized Water to give a 10,000 pM concentration of standard. Invert the vial several times to mix. Vortex for 10 seconds. Add 220 µl Assay Buffer into a microfuge tube and take 180 µl standard (10,000 pM) to prepare the 4500 pM concentration of standard. This will be used as the 4500 pM standard. Immediately transfer the standard to appropriately labeled polypropylene microfuge tube. The unused portions may be stored at £ -20° for up to one month.
2). Preparation of Working Standards
Label six polypropylene microfuge tubes 1500, 500, 166.7, 55.6, 18.5, and 6.2 pM. Add 200 ml of Assay Buffer to each of the six tubes. Prepare serial dilutions by adding 100 ml of the 4500 pM reconstituted standard to the 1500 pM tube, mix well and transfer 100 ml of the 1500 pM standard to the 500 pM tube, mix well and transfer 100 ml of the 500 pM standard to the 166.7 pM tube, mix well and transfer 100 ml of the 166.7 pM standard to 55.6 pM tube, mix well and transfer 100 ml of the 55.6 pM standard to 18.5 pM tube, mix well and transfer 100 ml of the 18.5 pMpg/ml standard to the 6.2 pM tube and mix well. The 0 pM standard (Background) will be Assay Buffer.
|
Standard Concentration |
Volume of Assay Buffer to Add |
Volume of Standard to Add |
|
4500 |
220 ml |
180 ml of 10,000 pM |
|
1500 |
200 ml |
100 ml of 4500 pM |
|
500 |
200 ml |
100 ml of 1500 pM |
|
166.7 |
200 ml |
100 ml of 500 pM |
|
55.6 |
200 ml |
100 ml of 166.7 pM |
|
18.5 |
200 ml |
100 ml of 55.6 pM |
|
6.2 |
200 ml |
100 ml of 18.5 pM |
B. Preparation of Controls
Before use, reconstitute Endocrine Control I and Endocrine Control II with 250 ml Deionized Water. Invert the vial several times to mix. Vortex well. 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, vortex for 1 minute. Add 0.15 ml from each antibody bead bottle to the Mixing Bottle and bring final volume to 3 ml with HENDO-65K Bead Diluent. Vortex well. Unused portions may be stored at 2-8° C for up to one month.
Example 1: when using 5 Endocrine antibody-immobilized beads, add 0.15 ml from each of the five bead sets to the mixing bottle and 2.25 ml Bead Diluent.
Example 2: when using 3 Endocrine antibody-immobilized beads, add 0.15 ml from each of the three bead sets to the mixing bottle. Add 2.55 ml Bead Diluent.
E. Preparation of Serum Matrix
Add 1.0 mL of deionized water to the vial containing the lyophilized Serum Matrix, gently swirl the bottle and then let sit for 5 to 10 minutes.
Prior to beginning this assay, it is imperative to read this protocol completely and to thoroughly understand the Technical Guidelines outlined in Section VII.
1. Diagram placement of Standards, 0 (Background) 6.2, 18.5, 55.6, 166.7, 500, 1500 and 4500 pM, 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 PLATES) and dry the bottom of the plate by using paper towels.
4. Add 10 µL of Assay Buffer to the 0 Standard (Background).
5. Add 10 µL of Assay Buffer to the Sample wells.
6. Add 10 µL of each Standard or Control into the appropriate wells.
7. Add 10 µL of appropriate matrix diluent to the Background, Standards, and Control wells. When assaying serum or plasma, use HENDO-65K Serum Matrix. When assaying Tissue Culture or other supernatant, use similiar medium as the diluent.
8. Add 10 µL of Sample into the appropriate wells.
9. Vortex Bead Bottle and add 25 µL of Mixed Beads to each well. (Note: during addition of Mixed Beads, shake bead mix intermittently to avoid settling)
10. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker overnight (16-18 hours) at 2-8oC. After overnight incubation, it is important to allow the plate and reagents to warm to room temperature (20-25° C) before continuing with the assay.
11. Gently remove fluid by vacuum.
12. Wash plate 3 times with 200 µL/well of Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Dry the bottom of the plate by using paper towels.
13. Pipet 50 µL of Detection Antibody Cocktail into each well. (Note: allow the Detection Antibody to warm to room temperature, 20-25° C, prior to addition.)
14. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 60 minutes at room temperature. DO NOT VACUUM AFTER INCUBATION.
15. Add 50 m L Streptavidin-Phycoerythrin (SAPE) to each well containing the 50 m L of Detection Antibody Cocktail. (Note: allow the SAPE to warm to room temperature prior to addition.)
16. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 30 minutes at room temperature.
17. Gently remove all contents by vacuum.
18. 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 tissue.
19. Add 100 µL of Sheath Fluid to all wells. Cover with aluminum foil and resuspend the beads on a plate shaker for 5 minutes.
20. Read plate on Luminex Instrument.
21. Save and evaluate the median data using a 5-parameter or spline fit data reduction.
Select the following equipment settings:
| Events: | 50, per bead | |
| Sample Size: | 50 ml | |
| Bead Set: | 05 for Insulin | |
| 15 for Amylin | ||
| 16 for Leptin | ||
| 34 for Glucagon | ||
| 53 for GLP-1 (Active) | ||
| **Gate (for IS System): | 8,000 to 15,000 | |
| **Gate (for 1.7 System): | 8,000 to 15,000 | |
**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.
Standard Unit Comparison
|
HormoneEndocrine |
Molecular |
pM |
ng/ml |
|
rInsulin |
5800 |
100 |
0.58 |
|
rAmylin |
3921 |
100 |
0.39 |
|
rLeptin |
16162 |
100 |
1.6 |
|
Glucagon |
3483 |
100 |
0.35 |
|
GLP-1 (Active) |
3297 |
100 |
0.33 |
B. Sensitivity
Sensitivity in serum matrix for Insulin is 55.6 pM and for all other analytes 6.2 pM.
C. Precision
Intra-assay: 3.8 – 10.6%
Inter-assay: 4.8 – 20.7%
D. Accuracy
|
Recovery in Serum Matrix |
Recovery in Serum Samples |
|
|
Insulin |
100.3% |
105.8% |
|
Amylin |
101.0% |
80.2% |
|
Leptin |
100.5% |
96.5% |
|
Glucagon |
99.7% |
105.9% |
|
GLP-1 (Active) |
101.1% |
115.2% |
E. Cross-Reactivity
Each antibody pair used in this panel is highly specific and does not show any significant cross-reactivity between any other analytes within this panel.
| REAGENTS | Catalogue # |
| Rat/Mouse Endocrine Standard Cocktail | LE-8085 |
| Rat/Mouse Endocrine Control I and II | LE-6085 |
| Mouse Endocrine Detection Antibodies | LME-1075 |
| Streptavidin-Phycoerythrin | L-SAPE |
| Assay Buffer | LE-ABGLP |
| Set of two 96-Well Filter Plates with sealers | L-PLATE |
| 10X Wash Buffer | L-WB |
| Bead Diluent | LE-BD |
| 05-Insulin Beads | RME-INS |
| 15-Amylin Beads | RME-AMLN |
| 16-Leptin Beads | RME-LPTN |
| 34-Glucagon Beads | RME-GLU |
| 53-GLP-1 (Active) Beads | HRE-GLP1 |
| Serum Matrix | LMC-SD |