Human Ghrelin Acylated ELISA Kit

Sample

Cell culture supernatant

Species Reactivity

Human

Detection Method

sELISA

Intended Use

1. This assay has been developed to screen GOAT activity by monitoring Acylated Ghrelin production in cell culture media. Cells over-expressing GOAT are cultivated in large excess of Unacylated Ghrelin, GOAT transforming Unacylated Ghrelin into Acylated Ghrelin. The kit detects Acylated Ghrelin with 103 to 105 more Unacylated Ghrelin in the sample.
2. Alternatively, it can be used for measuring Acylated Ghrelin in human biological samples. For this application, please contact technical support at CD.

Contents of Kit

1. Conjugate Solution (Tracer for 384 well plate), Green, lyophilized (2)
2. Human Acylated Ghrelin Standard, Blue with red septum, lyophilized (1)
3. Quality Control, Green with red septum, lyophilized (1)
4. Dilution Buffer (EIA buffer), Blue, lyophilized (1)
5. Wash Solution Conc. (400x), Silver, liquid (1)
6. Substrate Solution (Ellman' s reagent), Black with red septum, lyophilized (2)
7. Tween 20, Transparent, liquid (1)
Each kit contains sufficient reagents for 384 wells. This allows for the construction of one standard curve in duplicate and the assay of 176 samples in duplicate or screening of 352 samples in simplicate.

Storage

Store the kit at -20°C. Under these conditions, the kit is stable until expiration date (see label on the box).

Detection Range

2-250 pg/ml

Detection Limit

Limit of detection in the sample before dilution <8 pg/mL

Sensitivity

Sensitivity: <5 pg/mL

General Description

Acetylcholinesterase (AChE) the enzymatic label for ELISA, has the fastest turnover rate of any enzymatic label. This specific AChE is extracted from the electric organ of the electric eel, Electrophorus electricus, and it' s capable of massive catalytic turnover during the generation of the electrochemical discharges.
AChE assays are revealed with Substrate Solution (Ellman' s reagent) , which contains acetylthiocholine as a substrate. The final product of the enzymatic reaction (5-thio-2-nitrobenzoic acid), is bright yellow and can be read at 405-414 nm. AChE offers several advantages compared to enzymes conventionally used in EIAs:
a. Kinetic superiority and high sensitivity: AChE shows true fi rst-order kinetics with a turnover of 64,000 sec-1. That is nearly 3 times faster than Horse Radish Peroxidase (HRP) or alkaline phosphate. AChE allows a greater sensitivity than other labeling enzymes.
b. Low background: non-enzymatic hydrolysis of acetylthiocholine in buffer is essentially absent. So, AChE allows a very low background and an increased signal/noise ratio compared to other substrate of enzymes which is inherently unstable.
c. Wide dynamic range: AChE is a stable enzyme and its activity remains constant for many hours as, unlike other enzymes, its substrate is not suicidal. This permits simultaneous assays of high diluted and very concentrated samples.
d. Versatility: AChE is a completely stable enzyme, unlike peroxidase which is suicidal. Thus, if a plate is accidentally dropped after dispatch of the AChE substrate solution (Ellman' s reagent) or if it needs to be revealed again, one only needs to wash the plate, add fresh Substrate Solution (Ellman' s reagent) and proceed with a new development. Otherwise, the plate can be stored at 4°C with Wash Buffer in wals while waiting for technical advice from the Bioreagent Department.
Ghrelin Ghrelin discovered in 1999, is fast becoming an endokrinology target of the millennium. Ghrelin, identifi ed in rat stomach as an endogenous ligand for the GH secretagogue receptor, is mainly produced in stomach, but has been demonstrated in many other organs. In addition to GH-releasing properties and its orexant action, Ghrelin could act as an hormone having effects on gastric motility (similarity with the peptide hormone motilin), acidic secretion, cardiovascular action, antiproliferative effects, pancreatic and glucose metabolism function, sleep.
Ghrelin gene raises to mRNA prepro-ghrelin of 117 amino acids. This precursor is processed into Ghrelin, 28 amino acids (human). Before being secreted, this peptide is octanoylated at Ser 3 by GOAT (Ghrelin Octanoyl Acyl Transferase). This step is Essentials for biological activity making GOAT a perfect target for drugs in feeding behaviour. Interestingly, the potential therapeutic importance of this hormone is not restricted to regulation of food intake but also in cachexia (related to cancer treatment, anorexia nervosa or ischemia) gastrin motility and may be involved in osteoporosis, somatopause, infertility and ovulation induction, neurological disorders (Alcoholism, Post Traumatic Stress disorders...) and cardiovascular diseases.

Standard Curve