This Desacyl-Ghrelin ELISA kit is used for the non-radioactive quantification of Desacyl-Ghrelin in human, mouse or rat plasma. This kit specifically measures only the Desacyl-Ghrelin based on the principle of 2 site Sandwich enzyme-linked immunosorbent assay (ELISA). One kit is sufficient to measure 38 unknown samples in duplicate. This kit is for research purpose only.


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 Desacyl-Ghrelin ELISA kit recognizes the non-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 Desacyl-Ghrelin ELISA is an enzymatically amplified "two-step" sandwich-type immunoassay. In the assay, standards, controls and unknown plasma samples are incubated in microtitration wells which have been coated with anti-desacyl 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 by absorbance measurement at 450 nm.

The absorbance measured is directly proportional to the concentration of desacyl ghrelin present. A set of desacyl ghrelin standards is used to plot a standard curve of absorbance versus desacyl ghrelin concentration from which the desacyl 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 Desacyl-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 Desacyl-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 Desacyl-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, Inc.

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 desacyl-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.


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


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. Transfer and store plasma samples in separate tubes.

4. Use freshly prepared plasma or aliquot and store samples at £ –40oC for later use. Avoid freeze/thaw cycles.

Note: Acidified plasma prepared for Active Ghrelin ELISA measurement can also be used for this assay.


A. Desacyl-Ghrelin Standard Preparation

1. Use care in opening the lyophilized Standard vial. Using an Eppendorf pipette, reconstitute the Desacyl-Ghrelin Standard with 1 mL distilled or deionized water to give a concentration prescribed in 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.