Glucagon-Like Peptide-1 (Active) ELISA KIT
This kit is for non-radioactive quantification of biologically active forms of Glucagon-Like Peptide-1 [i.e. GLP-1
(7-36 amide) and GLP-1 (7-37)] in plasma and other biological media. It is highly specific for the immunologic measurement of active GLP-1 and will not detect other forms of GLP-1 (e.g., 1-36 amide, 1-37, 9-36 amide, or
9-37). The GLP-1 sequence is highly conserved between the species, with no sequence variation occurring at all in mammals. One kit is sufficient to measure 39 unknown samples in duplicate. This kit is for research purposes only.
This assay is based, sequentially, on: 1) capture of active GLP-1 from sample by a monoclonal antibody, immobilized in the wells of a microwell plate, that binds specifically to the N-terminal region of active GLP-1 molecule, 2) washing to remove unbound materials, 3) binding of an anti GLP-1-alkaline phosphatase detection conjugate to the immobilized GLP-1, 4) washing off unbound conjugate, and 5 ) quantification of bound detection conjugate by adding MUP (methyl umbelliferyl phosphate) which in the presence of alkaline phosphatase forms the fluorescent product umbelliferone. Since the amount of fluorescence generated is directly proportional to the concentration of active GLP-1 in the unknown sample, the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of active GLP-1.
Each kit is sufficient to run one 96-well microtiter plate and contains the following reagents:
A. GLP-1 (Active) ELISA Plate
Coated with anti-GLP-1 Monoclonal Antibody
Quantity: 1 plate
Preparation: Ready to use
Adhesive Plate Sealer
Quantity: 1 Sheet
Preparation: Ready to use
C. 10X Wash Buffer Concentrate
10X concentrate of 10 mM PBS Buffer containing Tween 20 and Sodium Azide.
Quantity: 50 ml
Preparation: Dilute 1:10 with deionized water
D. GLP-1 (7-36) amide ELISA Standards
GLP-1 (7-36 amide) in Assay Buffer: 2, 5, 10, 20, 50 and 100 pM
Quantity: 1 ml/vial
Preparation: Ready to use
E. ELISA GLP-1 (Active) Quality Controls 1 and 2
Various peptides including GLP-1 (7-36 amide) in QC Buffer.
Quantity: 1 ml/vial
Preparation: Ready to use
F. GLP-1 (Active) Assay Buffer
0.05M PBS, pH 6.8, containing proprietary protease inhibitors, with Tween 20, 0.08% Sodium Azide and 1% BSA.
Quantity: 25 ml
Preparation: Ready to use
G. GLP-1 (Active) Detection Conjugate
Anti GLP-1-Alkaline Phosphate Conjugate.
Quantity: 21 ml
Preparation: Ready to use
H. Substrate (Light sensitive, avoid unnecessary exposure to light)
MUP
Quantity: 10 mg
Preparation: Hydrate in 1 ml deionized water just before use. Use at 1:200 dilution in substrate diluent (e.g. 100 m l hydrated substrate in 20 ml substrate diluent). Dilute fresh each time just before use. Undiluted substrate may be used within one week after hydration if stored at £ - 20ºC. Do not reuse diluted substrate.
I. Substrate Diluent (Light sensitive, avoid unnecessary exposure to light)
Quantity: 21 ml
Preparation: Ready to use
J. Stop Solution
Quantity: 6 ml
Preparation: Bring to room temperature before use. Mix thoroughly to ensure no precipitate remains.
All components of the kit should be stored at £ -20° C. Refer to expiration dates on all reagents prior to use. Do not mix reagents from different kits unless they have the same lot numbers.
Diethanolamine
Substrate diluent contains diethanolamine. This compound can be harmful through ingestion, inhalation, and skin contact. May be irritating to eyes and skin. If skin/eye contact occurs flush thoroughly with water.
Sodium Azide
Sodium Azide has been added to reagents as a preservative at a concentration of 0.08%. Although it is at a minimum concentration, Sodium Azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with large volume of water to prevent azide build up.
VI. MATERIALS REQUIRED BUT NOT PROVIDED
1. Pipet with Tips, 10m l-200m l
2. Multi-channel Pipette, 50m l-300m l
3. Reagent Reservoirs
4. Vortex mixer
5. Refrigerator
6. Deionized Water
7. Fluorescence Plate Reader
8. DPP-IV Inhibitor (Linco Cat# DPP4 recommended)
VII. SAMPLE COLLECTION AND STORAGE
1. For plasma collection, collect blood in ice-cooled Vacutainerâ EDTA-plasma tubes. Immediately (< 30 seconds) after collection, add appropriate amount of DPP-IV inhibitor according to manufacturer’s directions. Invert tube to mix and store tubes in ice bath. (If using Linco Cat # DPP4, add 10 m l DPP-IV inhibitor per milliliter of blood.) Centrifuge immediately at 1000 xg for 10 minutes in refrigerated centrifuge or place tubes on ice and centrifuge within one hour.
2. Specimens can be stored at 4°C if they will be tested within 3 hours of collection. For longer storage, specimens should be stored at -70°C. Avoid multiple (>3) freeze/thaw cycles. Aliquot samples before freezing if necessary.
3. Avoid using samples with gross hemolysis or lipemia.
The assay should be run in duplicate in a 200m l total volume.
First Day
1. Add 300m l diluted Wash Buffer (for preparation refer to Section III C) in each well. Incubate at room temperature for 5 minutes. Decant and tap out excess buffer on absorbent towels.
2. Add 200m l Assay Buffer to NSB (non-specific binding) wells A1, A2. Refer to Section IX for suggested microtiter plate arrangement.
3. Add 100m l Assay Buffer to the remaining wells.
4. Add 100m l standards in ascending order to wells A3, A4, etc.
5. In the next set of wells, add 100m l EQC 1 (wells B3 and B4) and EQC 2 (wells B5 and B6).
6. Add 100m l samples in the remaining wells. Shake plate gently for proper mixing.
7. Cover the plate with plate sealer. Incubate overnight (20 to 24 hours) at 4oC.
Second Day
8. Decant liquid from plate and tap out excess fluid on absorbent towels.
9. Wash the plate 5 times with 300m l Wash Buffer per well with 5-minute incubation at room temperature in Wash Buffer with the fourth wash. Tap out excess buffer on absorbent towels after the fifth wash.
10. Immediately add 200m l Detection Conjugate in each well. Incubate 2 hours at room temperature. Decant.
11. Wash 3 times with 300m l diluted Wash Buffer. Tap out excess buffer on absorbent towels.
12. Add 200m l diluted Substrate (for preparation, refer to Section III H) in each well. Incubate at least 20 minutes at room temperature in the dark. Monitor to see if there is significant signal-to-noise ratio with the lowest point on standard curve (i.e. 2 pM), and the highest standard point (i.e. 100 pM) within the maximum relative fluorescence unit (RFU) read-out of plate reader. Incubate longer if necessary.
13. If sufficient fluorochrome has been generated, add 50m l Stop Solution to each well in the same order as the Substrate was added. Incubate 5 minutes at room temperature in the dark to arrest phosphatase activity.
14. Read plate on a fluorescence plate reader with an excitation/emission wavelength of 355 nm/460 nm.
IX. MICROTITER PLATE ARRANGEMENT
Standard GLP-1 ELISA
The RFU can be fitted directly to the concentration. If curve fitting software is available, the best fit can be obtained with a linear-linear spline fit.
Since this assay is a direct ELISA, the RFU is directly proportional to the concentration of GLP-1 in the sample.
Sensitivity
The lowest level of GLP-1 that can be detected by this assay is 2 pM (100 m l plasma sample size).
Performance
| ED 80 = 81 ± 4 pM |
| ED 50 = 55 ± 7 pM |
| ED 20 = 28 ± 1 pM |
Crossreactivity
| GLP-1 (7-36 amide) | 100% |
| GLP-1 (7-37) | 100% |
| GLP-1 (9-36 amide) | ND |
| GLP-2 | ND |
| Glucagon | ND |
| ND- Not Detectable |
Precision
Within and Between Assay Variation
|
Sample |
Mean |
Within |
Between |
|
1 |
4 | 8 | 13 |
|
2 |
8 | 7 | 12 |
|
3 |
12 | 6 | 7 |
|
4 |
28 | 7 | 7 |
|
5 |
76 | 9 | <1 |
Within and between assay variation was performed on five human plasma samples containing varying concentrations of GLP-1. Data shown are from four duplicate determinations for within and four duplicate determinations for between.
Recovery
Spike & Recovery of GLP-1 in Human Plasma
|
Sample # |
GLP-1 added pM |
Expected pM |
Observed pM |
% of Recovery |
|
0 |
5 |
5 |
100 |
|
|
10 |
15 |
13 |
87 |
|
|
20 |
25 |
21 |
84 |
|
|
50 |
55 |
43 |
78 |
|
|
0 |
13 |
13 |
100 |
|
|
10 |
23 |
20 |
87 |
|
|
20 |
33 |
29 |
88 |
|
|
50 |
63 |
50 |
80 |
|
|
0 |
12 |
12 |
100 |
|
|
10 |
22 |
20 |
91 |
|
|
20 |
32 |
28 |
88 |
|
|
50 |
62 |
53 |
85 |
|
|
0 |
37 |
37 |
100 |
|
|
10 |
47 |
44 |
94 |
|
|
20 |
57 |
55 |
96 |
|
|
50 |
87 |
74 |
85 |
Varying concentrations of GLP-1 were added to four human plasma samples and the GLP-1 content was determined by ELISA. Mean of the observed levels from four duplicate determinations are shown.
Percent recovery = observed ÷ expected x 100%.
Linearity
Effect of Plasma Dilution
|
Sample |
Volume |
Expected |
Observed |
% Of |
|
1 |
100 µl | 5 | 5 | 100 |
| 50 µl | 3 | 3 | 100 | |
| 25 µl | < 2 | < 2 | --- | |
|
2 |
100 µl | 13 | 13 | 100 |
| 50 µl | 7 | 7 | 100 | |
| 25 µl | 3 | 4 | 133 | |
|
3 |
100 µl | 11 | 11 | 100 |
| 50 µl | 6 | 7 | 116 | |
| 25 µl | 3 | 4 | 133 |
Aliquots of pooled human plasma containing varying concentrations of GLP-1 were analyzed in the volumes indicated. The mean GLP-1 levels and percent of expected from four duplicate determinations are shown.
The ranges for Quality Control 1 and 2 are provided on the card insert or can be located at the Linco Research website www.lincoresearch.com.
Low or No Signal with Standards
* Standards were left at room temperature. Standards should be stored at £ -20ºC.
* Insufficient time for reaction with substrate. Allow substrate to react longer.
* Kit reagents have expired.
* Inadequate plate washing after sample incubation.
* Too much washing after conjugate incubation can however reduce signal.
High Background
* Inadequate plate washing. After conjugate incubation, tap out plate on absorbent towels after decanting.
* Plate was not kept in dark after substrate addition.
* Cross contamination between neighboring wells.
* Substrate has been diluted too long or exposed to light before use, or diluent has been contaminated with old substrate. Check only substrate in a well.
Samples too High
* Dilute sample 1:10 with assay buffer to bring GLP-1 concentration within standard range.
Signal too High on Highest Standard
* Plate incubated too long with substrate. Discard substrate, wash plate once and add freshly prepared substrate. Check RFU in less time.
High Variance in RFU of Duplicates
* Cross contamination in wells
* Bubbles in substrate at time of reading
* Loss of reagent or faulty pipetting in duplicates
| Reagents | Cat. # |
| GLP-1 (Active) ELISA Plate | EP35 |
| 10X Wash Buffer Concentrate | EWB |
| GLP-1 (7-36) amide ELISA Standards | E8035-K |
| ELISA GLP-1 (Active) Quality Controls 1 & 2 | E6016-K |
| GLP-1 (Active) Assay Buffer | AB-GLPHK |
| GLP-1 (Active) Detection Conjugate | E1035 |
| Substrate | ESS-MUP |
| Substrate Diluent | EDD-MUP |
| Stop Solution | ET-AP |