Human Ghrelin ELISA Kit (HUFI00296)- High Sensitivity

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Human Ghrelin ELISA Kit
Product Code:	HUFI00296
Size:	96 Assays
Alias:	GHRL, Ghrelin, Obestatin
Detection Method:	Competitive ELISA, Coated with Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human GHRL concentrations in serum plasma and other biological fluids
Sensitivity:	1.125pg/ml
Range:	1.875-120pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Human GHRL and the recovery rates were calculated by comparing the measured value to the expected amount of Human GHRL in samples.


Matrix	Recovery Range (%)	Average (%)
serum(n=5)	89-99	94
EDTA plasma(n=5)	90-104	96
UFH Plasma(n=5)	86-102	96

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human GHRL and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.


Sample	1:2	1:4	1:8
serum(n=5)	85-96%	88-97%	87-101%
EDTA plasma(n=5)	87-101%	83-100%	82-98%
UFH Plasma(n=5)	83-100%	86-98%	85-96%

CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate (SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Protein Information

Heading 1	Heading 2
Uniprot	Q9UBU3
Uniprot Protein Function	ghrelin: a hormone that binds to the growth hormone secretagogue receptor type 1 (GHSR). Secreted by the stomach
NCBI GenInfo Identifier	17865471
NCBI Gene ID	51738
Molecular Weight	9,972 Da

Protocol

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!
2.	Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).
3.	Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.
4.	HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.
5.	Wash: Repeat the aspiration/wash process for five times.
6.	TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.
7.	Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.
8.	OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testing parameters.

Sample Type

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Ghrelin Background

Ghrelin

Ghrelin is a peptide hormone primarily produced in the stomach and plays a crucial role in regulating appetite and energy balance. It is known as the 'hunger hormone' due to its ability to stimulate appetite and increase food intake. Ghrelin levels in the body are influenced by various factors, including fasting, meal patterns, and stress. Through its interactions with the hypothalamus and other regions of the brain, Ghrelin helps modulate the release of other hormones involved in hunger and satiety. Measuring Ghrelin levels can provide valuable insights into understanding appetite regulation, metabolic disorders, and the potential impact on overall health.

Ghrelin Peptide

Ghrelin peptide, a bioactive molecule derived from the preproghrelin gene, is a versatile regulator of multiple physiological processes beyond appetite control. Apart from its role in stimulating hunger, Ghrelin peptide has been implicated in the regulation of growth hormone secretion, glucose metabolism, and cardiovascular functions. It has also shown potential involvement in modulating inflammation, immune responses, and cognitive functions. With its diverse physiological effects, Ghrelin peptide has emerged as a fascinating target for therapeutic interventions aimed at addressing conditions such as obesity, diabetes, cardiovascular diseases, and neurodegenerative disorders.

Ghrelin Pathway

The Ghrelin pathway represents a complex network of interactions and signaling cascades involved in the regulation of appetite, energy balance, and various physiological processes. Ghrelin binds to its receptor, the growth hormone secretagogue receptor (GHSR), predominantly located in the hypothalamus, but also found in other tissues throughout the body. Activation of GHSR by Ghrelin triggers a series of intracellular signaling events, including activation of G-proteins and subsequent activation of downstream pathways such as the protein kinase A (PKA) pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphoinositide 3-kinase (PI3K)/Akt pathway. These signaling pathways mediate the effects of Ghrelin on appetite regulation, growth hormone release, glucose metabolism, and other physiological functions.

Ghrelin and Obesity

Ghrelin has emerged as a key player in the complex relationship between appetite regulation and obesity. Studies have shown that obese individuals often exhibit altered Ghrelin levels and dysregulated Ghrelin signaling. While Ghrelin is known to stimulate appetite and promote food intake, its role in obesity is multifaceted. It has been proposed that individuals with obesity may develop a resistance to the appetite-suppressing effects of Ghrelin, leading to increased food consumption and weight gain. Moreover, obesity-associated changes in body composition and metabolic abnormalities can impact Ghrelin production and secretion. Understanding the intricate interplay between Ghrelin, obesity, and related metabolic disorders is crucial for developing targeted interventions aimed at addressing appetite dysregulation and managing obesity. By measuring Ghrelin levels and investigating its signaling pathways, researchers can gain valuable insights into the underlying mechanisms of obesity and potentially identify novel therapeutic strategies to combat this global health issue

Ghrelin ELISA Kit FAQs

What is the Ghrelin ELISA kit used for?

The Ghrelin ELISA kit is a valuable tool used for the quantitative measurement of Ghrelin levels in human samples. It enables researchers and healthcare professionals to study and understand the role of Ghrelin in appetite regulation, energy balance, metabolic disorders, and various physiological processes. By accurately measuring Ghrelin levels, the kit assists in unraveling the complexities of Ghrelin's functions and its potential implications in health and disease.

What are the advantages of using the Ghrelin ELISA Kit?

The Ghrelin ELISA kit offers several advantages for researchers and healthcare professionals. Firstly, it provides a highly sensitive and specific method for quantifying Ghrelin levels in human samples, ensuring accurate and reliable results. Additionally, it offers a wide dynamic range, enabling the detection of both low and high Ghrelin concentrations. The Ghrelin ELISA kit thus facilitates precise and robust measurements, enhancing the quality and reliability of research findings.

What sample types are compatible with the Ghrelin ELISA kit?

The FSH ELISA Kit is compatible with various sample types, including serum, plasma, cell lysates, and tissue homogenates. It provides flexibility in sample selection, allowing researchers to analyze FSH levels in different biological matrices.

What are the storage requirements for the FSH ELISA Kit?

The Ghrelin ELISA Kit components should be stored according to the instructions provided in the kit manual. Generally, it is recommended to store the kit components at the recommended temperature to ensure their stability and optimal performance.

What should I do if my assay results are not optimal?

If you encounter any issues or have suboptimal assay results, we recommend contacting our dedicated support team for assistance. They will be available to provide troubleshooting guidance, answer your questions, and ensure you achieve the best possible results with the Fc epsilon RI ELISA Kit.

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Human Ghrelin ELISA Kit

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