HUMAN Amylin (Total)
This kit is for non-radioactive quantification of Total Human Amylin in plasma. The capture antibody recognizes an epitope near the midpoint of the peptide. One kit is sufficient to measure 39 unknown samples in duplicate. This kit is for research purposes only.
The Total Human Amylin ELISA is a monoclonal antibody-based sandwich immunoassay for determining total Amylin levels in human plasma. The capture antibody recognizes reduced Human Amylin and Human Amylin Acid (deamidated amylin) but not the 1-20 fragment of amylin. The detection antibody binds to reduced or unreduced Human Amylin but not amylin acid and is complexed with Streptavidin-Alkaline Phosphatase. The substrate, 4-Methylumbelliferyl Phosphate (MUP), is applied to the completed sandwich and the fluorescent signal, monitored at 355 nm/460 nm, is proportional to the amount of amylin present in the sample.
Each kit is sufficient to run one 96-well microtiter plate and contains the following reagents:
A. Human Amylin (Total) ELISA Plate
Coated with Mouse anti-Human Amylin Antibody
Quantity: 1 plate
Preparation: Ready to use
B. Adhesive Plate Sealer
Quantity: 1 Sheet
Preparation: Ready to use
C. 10X TBS Wash Buffer Concentrate
10X concentrate of 50 mM Tris Buffered Saline with Tween 20 and Sodium Azide
Quantity: 50 ml
Preparation: Dilute 1:10 with deionized water
D. Human Amylin Standards
Human Amylin in Assay Buffer: 0, 1, 2, 5, 10, 40 and 100 pM
Quantity: Lyophilized, 250 µl /vial rehydrated
Preparation: Reconstitute with 250 µl deionized water
E. Human Amylin Quality Controls 1 and 2
Human Amylin in Assay Buffer.
Quantity: Lyophilized, 250 µl /vial rehydrated
Preparation: Reconstitute with 250 µl deionized water
F. Assay Buffer
0.05M PBS, pH 7.4, containing Proprietary Protease Inhibitors, with Tween 20,
0.08% Sodium Azide and 1% BSA
Quantity: 6 ml
Preparation: Ready to use
G. Human Amylin Detection Conjugate
Anti-Human Amylin-Alkaline Phosphatase Conjugate
Quantity: 11 ml
Preparation: Ready to use
H. Substrate (Light sensitive, avoid unnecessary exposure to light)
4-Methylumbelliferyl Phosphate
Quantity: 10 mg
Preparation: Hydrate in 1 ml deionized water just before use. Use at 1:200 dilution in substrate diluent (e.g. 105 µl hydrated substrate in 21 ml substrate diluent).
I. Substrate Diluent (Light sensitive, avoid unnecessary exposure to light)
Quantity: 21 ml
Preparation: Ready to use; warm to room temperature before use
J. Stop Solution
Quantity: 6 ml
Preparation: Bring to room temperature before use. Mix thoroughly to ensure no precipitate remains.
Upon receipt, all components of the kit should be stored at 2-8°C. 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 a large volume of water to prevent azide build up.
VI. MATERIALS REQUIRED BUT NOT PROVIDED
1. Pipet with Tips, 10µl-200µl
2. Multi-Channel Pipette, 50µl-300µl
3. Buffer and Reagent Reservoirs
4. Vortex Mixer
5. Absorbent Paper or Cloth
6. Refrigerator
7. Deionized Water
8. Orbital Microtiter Plate Shaker
9. Fluorescence Plate Reader
VII. SAMPLE COLLECTION AND STORAGE
1. For plasma collection, collect whole blood in ice-cooled Vacutainer® EDTA-plasma tubes. Centrifuge immediately at 1000 xg for 10 minutes in refrigerated centrifuge or place tubes on ice and centrifuge within one hour.
2. Specimens should be stored at less than or equal to -70°C. Aliquot samples before freezing if necessary.
3. Avoid using samples with gross hemolysis or lipemia.
ASSAY PROCEDURE
The assay should be run in duplicate using 50 µl of Assay Buffer and 50 µl of Standard, Control, or Sample in each well.
1. Dilute the concentrated Wash Buffer 10 fold by mixing the entire contents of the 10X Wash Buffer with 450 ml deionized water.
2. Reconstitute Standards and QC1 and QC2 (lyophilized) vials with 250 µl of deionized water. Mix gently and let sit for at least 5 minutes with occasional gentle mixing to assure complete reconstitution.
3. Remove the microtiter assay plate from the foil pouch and fill each well with 300 µl of diluted Wash Buffer. Incubate at room temperature for 10 minutes, no shaking.
4. Decant Wash Buffer from the plate and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times. Do not let wells dry before proceeding to the next step.
5. Add 50 µl Assay Buffer to each well.
6. Add in duplicates; 50 µl Standards, Samples and Controls. Refer to Section IX for suggested well orientations. Seal plate and incubate at room temperature on the shaker for one hour. (NOTE: Start incubation time as plate is loaded on the shaker, not from the time you start loading the plate with samples.) Decant and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.
7. Wash the plate 3 times with 300 µl per well Wash Buffer. Decant and tap after each wash to remove residual buffer.
8. Add 100 µl Detection Conjugate to each well. Cover the plate with sealer and incubate on the shaker at room temperature for 2 hours.
9. Near the completion of this incubation step, hydrate the Substrate (ESS-MUP) by adding 1 ml deionized water to 10 mg, mix well, and let stand 15 minutes (with occasional mixing) to assure complete dissolution. Remove 105 µl from the reconstituted substrate and add it to the 21 ml vial of Substrate Diluent (EDD-MUP), mix well. Referred to as Substrate Solution from here on.
10. Decant Detection Conjugate and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.
11. Wash the plate 3 times with 300 µl per well Wash Buffer. Decant and tap after each wash to remove residual buffer.
12. Add 100 µl Substrate Solution to each well. Incubate 15 minutes at room temperature in the dark, no shaking.
13. Read plate on a fluorescent plate reader with an excitation/emission wavelength of 355nm/460nm. Note the RFU of the top standard point; when the reading is 2000 RFU or greater, add 50 µl Stop Solution (ET-AP), gently mix, and read on the Fluorescence Plate reader after 5 minutes.
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 Total Human Amylin in the sample.
Note: When sample volumes assayed differ from 50 µl, an appropriate mathematical adjustment must be made to accommodate for the dilution factor (e.g., if 25 µl of sample is used, then calculated data must be multiplied by 2).
Sensitivity
The lowest level of Total Human Amylin that can be detected by this assay is 1 pM (50 µl plasma sample size).
Performance
The following parameters of assay performance are expressed as Mean ± Standard Deviation.
| ED 80 = 82 ± 2 pM |
| ED 50 = 54 ± 5 pM |
| ED 20 = 26 ± 4 pM |
Crossreactivity
| Human Glucagon | <1% |
| Human GLP-1 | <1% |
| Human Insulin | <1% |
| Human Pancreatic Polypeptide | <1% |
| Human Adrenomedullin | 1% |
| Human Calcitonin | <1% |
| Calcitonin Gene Related Peptide | <1% |
Note: This kit is not suitable for the determination of
Total Amylin levels in rat or feline plasma.
Precision
Within and Between Assay Variation