ACTIVE GHRELIN
This Active Ghrelin ELISA kit is used for the non-radioactive quantification of Active Ghrelin in EDTA plasma. This kit specifically measures the active form of Ghrelin based on the principle of 2 site Sandwich enzyme-linked immunosorbent assay (ELISA). It can detect not only octanoylated human Ghrelin (1-28) but also octanoylated rat/mouse Ghrelin (1-28). One kit is sufficient to measure 38 unknown samples in duplicate. This kit is for research purpose only.
II. SUMMARY AND EXPLANATION OF TEST
Ghrelin, a novel growth hormone releasing peptide is an acylated peptide that stimulates the release of growth hormone from pituitary. It was isolated from rat stomach and the structure was determined as a small peptide consisting of 28-amino acids by Dr. Kenji Kangawa (National Cardiovascular Center in Japan). The Ser3 residue of Ghrelin is modified by n-octanoic acid, a modification necessary for hormonal activity. Ghrelin has been implicated in regulation of hunger and long-term weight gain/loss.
Ghrelin is the endogenous ligand for the growth hormone secretagogue (GHS) receptor and has potent growth hormone-releasing activity. This peptide may constitute a new regulatory mechanism for GH-release. It is conceivable that Ghrelin may also have other functions in some tissues in addition to pituitary, because the GHS receptor is expressed in heart, lung, pancreas, intestine, and adipose tissue.
The Active Ghrelin ELISA kit recognizes the octanoyl-modified portion of Ghrelin and is manufactured using the highly specific antibody pairs generated by Dr. Kangawa and by following his protocol. (patent pending: PCT WO 01/07475 A1)
The Active Ghrelin ELISA is an enzymatically amplified "two-step" sandwich-type immunoassay. In the assay, standards, controls and unknown treated plasma samples are incubated in microtitration wells which have been coated with anti-acyl ghrelin monoclonal antibody. After incubation and washing, the wells are treated with another anti-ghrelin detection antibody labeled with the enzyme horseradish peroxidase (HRP). After a second incubation and washing step, the wells are incubated with the substrate tetramethylbenzidine (TMB). An acidic stopping solution is then added and the degree of enzymatic turnover of the substrate is determined absorbance measurement at 450 nm.
The absorbance measured is directly proportional to the concentration of active Ghrelin present. A set of active Ghrelin standards is used to plot a standard curve of absorbance versus active Ghrelin concentration from which the active Ghrelin concentrations in the unknowns can be calculated.
Each kit is sufficient to run one 96-well plate and contains the following reagents:
A. Antibody Coated Plate
Coated with mouse monoclonal antibody to N-terminal of active Ghrelin.
Quantity: 1 plate
Preparation: Ready to Use
B. Adhesive Plate Sealer
Quantity: 3 sheets
Preparation: Ready to Use
C. Washing Buffer Concentrate
Buffer solution containing sodium chloride, sodium phosphate,
2-methyl-4-isothiazoline-3-one preservative (<0.2%)
Quantity: 40 mL
Preparation: Dilute 1:20 with distilled or deionized water
D. Standard (lyophilized)
Lyophilized standard containing Active Ghrelin in sodium phosphate buffer containing a non-mercury preservative.
Preparation: Contents Lyophilized. Reconstitute with 1 mL distilled or deionized water. The actual concentration of Ghrelin present in the vial will be lot-dependent. Please refer to the analysis sheet for exact Ghrelin concentration present in a specific lot.
E. Quality Controls 1 and 2 (lyophilized)
One vial each, lyophilized, containing Active Ghrelin at two different levels.
Preparation: Contents Lyophilized. Reconstitute with 1 mL distilled or deionized water.
Note: Quality Controls 1 and 2 were manufactured by Linco Research
F. Assay Buffer
Buffer solution containing sodium phosphate buffer, protein and a non-mercury preservative.
Quantity: 22 mL
Preparation: Ready to Use
G. HRP Conjugated Antibody
Diluent stabilizer solution containing HRP labeled mouse monoclonal antibody to C-terminal of active Ghrelin in a protein buffer containing a non-mercury preservative.
Quantity: 250 µL
Preparation: Dilute 1:100 with HRP dilution buffer.
H. HRP Dilution Buffer
Phosphate buffer containing protein and a non-mercury preservative.
Quantity: 22 mL
Preparation: Ready to Use
I. Substrate Solution (Light sensitive, avoid unnecessary exposure to light)
Preparation containing 3,3’,5,5’-Tetramethylbenzidine (TMB) as a substrate.
Quantity: 22 mL
Preparation: Ready to Use.
J. Stop Reagent (Caution: Corrosive Solution)
0.5 M sulfuric acid solution
Quantity: 6 mL
Preparation: Ready to Use
Prior to use, all components in the kit can be stored at 2-8oC. Refer to expiration dates on all reagents prior to use. Do not mix reagents from different kits unless they have the same lot numbers.
A. Sulfuric Acid
Sulfuric Acid is corrosive and can cause eye and skin burns. It is harmful if swallowed and can cause respiratory and digestive tract burns. Avoid contact with skin and eyes. Do not swallow or ingest.
VII. MATERIALS REQUIRED BUT NOT PROVIDED
1. Pipettes and Pipette Tips: 50m l - 200 m L
2. Multi-Channel Pipettes and Pipette Tips: 50 ~ 300 µL
3. Buffer and Reagent Reservoirs
4. Vortex Mixer
5. Deionized Water
6. Microtiter Plate Reader capable of reading absorbency at 450 nm
7. Orbital Microtiter Plate Shaker
8. Absorbent Paper or Cloth
VIII. SAMPLE PREPARATION AND STORAGE
The active form of the Ghrelin molecule is very unstable due to the presence of the labile ester bond present on the amino acid serine-3 that links the peptide to the octanoyl group.
Samples should be processed as quickly as possible and kept on ice to prevent/minimize the breakdown of active Ghrelin.
1. To prepare plasma samples, whole blood is directly drawn into a centrifuge tube that contains 500U of Aprotinin and 1.25mg of EDTA-2Na per 1 mL of whole blood.
2. Rock the tubes gently and then immediately centrifuge the blood sample at 1,500 x g for 15 minutes at 4 oC.
3. Immediately add 100 µL of 1 mol/L HCl per mL of collected plasma.
4. Transfer and store plasma samples in separate tubes.
5. Use freshly prepared plasma or aliquot and store samples at £ –40oC for later use. Avoid freeze/thaw cycles.
IX. STANDARD AND QUALITY CONTROLS PREPARATION
A. Active Ghrelin Standard Preparation
1. Use care in opening the lyophilized Standard vial. Using an Eppendorf pipette, reconstitute the Active Ghrelin Standard with 1 mL distilled or deionized water to give a concentration prescribed in the analysis sheet. Invert and mix gently, let sit for 5 minutes then mix well.
2. Label six tubes 1, 2, 3, 4, 5, and 6. Add 0.5 mL Assay Buffer to each of the six tubes. Prepare serial dilutions by adding 0.5 mL of the reconstituted standard to tube 1, mix well and transfer 0.5 mL of tube 1 to tube 2, mix well and transfer 0.5 mL of tube 2 to tube 3, mix well and transfer 0.5 mL of tube 3 to tube 4, mix well and transfer 0.5 mL of tube 4 to tube 5, mix well and transfer 0.5 mL of tube 5 to tube 6 and mix well.
Note: Do not use a Repeater pipette. Change tip for every dilution. Wet tip with Standard before dispensing. Unused portions of the reconstituted last standard should be aliquotted and stored at £ -20°C. Avoid multiple freeze/thaw cycles.
| Volume of Deionized Water to Add |
Volume of Standard to Add |
Standard Concentration fmol/mL | |
| 1 mL | 0 | X(Refer to analysis sheet for exact concentration) |
|
Tube # |
Volume of Assay Buffer to Add | Volume of Standard to Add | Standard Concentration fmol/mL |
| 1 | 0.5 mL | 0.5 mL of reconstituted standard | X/2 |
| 2 | 0.5 mL | 0.5 mL of tube 1 | X/4 |
| 3 | 0.5 mL | 0.5 mL of tube 2 | X/8 |
| 4 | 0.5 mL | 0.5 mL of tube 3 | X/16 |
| 5 | 0.5 mL | 0.5 mL of tube 4 | X/32 |
| 6 | 0.5 mL | 0.5 mL of tube 5 | X/64 |
Active Ghrelin Quality Control 1 and 2 Preparation
1. Use care in opening the lyophilized Quality Control vials. Using an Eppendorf pipette, reconstitute each of the Active Ghrelin Quality Control 1 and Quality Control 2 with 1 mL distilled or deionized water. Invert and mix gently, let sit for 5 minutes then mix well.
Note: For exact concentration of Quality Control 1 and 2, refer to Analysis Sheet. Unused portions of the reconstituted Quality Controls should be stored at £ -20°C. Avoid multiple freeze/thaw cycles.
Pre-warm all reagents to room temperature prior to setting up the assay.
1. Dilute the 20X concentrated Wash Buffer 20 fold by mixing the entire content of buffer with 760 mL deionized or distilled water.
2. Dilute only the required volume of HRP conjugated antibody with appropriate volume of HRP dilution buffer such that the conjugate is diluted 1:100 (example: 10 µL added to 990 µL). Unused portion of the HRP conjugated antibody should be stored at 4°C in the original concentrated form.
3. Remove the required number of strips of the microtiter plate from the foil pouch. Store unused strips in the pouch at 4°C
4. Add 150 µL Assay Buffer to all wells.
5. Add in duplicate 50 µL Assay Buffer to blank wells.
6. Add in duplicate 50 m L Active Ghrelin Standards in the order of ascending concentration to the appropriate wells. Add in duplicate 50 m L QC1 and 50 m L QC2 to the appropriate wells. Add sequentially 50 µL of the unknown samples in duplicate to the remaining wells.
7. Cover the plate with plate sealer and incubate at room temperature for 2 hours.
8. Remove plate sealer and decant solutions from the plate. Tap to remove residual solutions in the wells. Wash wells 3 times with diluted Wash buffer, 300 µL per well per wash, with 1 minute soak time with the wash buffer between each wash. Decant and tap firmly after each wash to remove residual buffer.
9. Add 200 µL diluted HRP conjugated antibody to all wells.
10. Cover the plate with plate sealer and incubate at room temperature for 1 hour.
11. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells. Wash wells 4 times with diluted Wash buffer, 300 µL per well per wash, with 1 minute soak time with the wash buffer between each wash. Decant and tap firmly after each wash to remove residual buffer.
12. Add 200 µL Substrate Solution to each well. Cover plate with sealer and incubate in the dark at room temperature for 30 minutes.
13. Remove sealer and add 50 µL Stop Reagent [CAUTION: CORROSIVE SOLUTION] and shake plate by hand to ensure complete mixing of solution in all wells. The blue color should turn to yellow after acidification. Read absorbance at 450 nm in a plate reader within a few minutes and ensure that there are no air bubbles in any well. Record the difference of absorbance units. .