Human ACTH ELISA Kit
- SKU:
- HUFI00819
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P01189
- Sensitivity:
- 9.375pg/ml
- Range:
- 15.625-1000pg/ml
- ELISA Type:
- Competitive
- Synonyms:
- ACTH, Adrenocorticotropic Hormone, Corticotropin, Adrenocorticotropin
- Reactivity:
- Human
- Research Area:
- Cell Biology
Description
Human ACTH ELISA Kit
Adrenocorticotropic hormone (ACTH) is a peptide hormone that is secreted by the anterior pituitary gland and stimulates the production and release of glucocorticoids from the adrenal cortex. The ACTH is released in response to a stressor, such as an exercise, hypoglycemia, or pain. ACTH plays a key role in the function of the sympathetic nervous system. It is used as a marker for stress and has been associated with the symptoms of chronic stress such as anxiety, depression, and insomnia. The most common cause of ACTH deficiency is surgical removal or destruction of part or all of one or both adrenal glands.
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 ACTH ELISA Kit |
| Product Code: | HUFI00819 |
| Size: | 96 Assays |
| Alias: | ACTH, Adrenocorticotropic Hormone, Corticotropin, Adrenocorticotropin |
| Detection Method: | Competitive ELISA, Coated with Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human ACTH concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 9.375pg/ml |
| Range: | 15.625-1000pg/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 ACTH and the recovery rates were calculated by comparing the measured value to the expected amount of Human ACTH in samples.
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ACTH and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
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| CV(%) | Intra-Assay: CV<8% |
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
| Uniprot: | |
| UniProt Protein Function: | POMC: ACTH stimulates the adrenal glands to release cortisol. Defects in POMC may be associated with susceptibility to obesity (OBESITY). It is a condition characterized by an increase of body weight beyond the limitation of skeletal and physical requirements, as the result of excessive accumulation of body fat. Defects in POMC are the cause of pro-opiomelanocortinin deficiency (POMCD). Affected individuals present early-onset obesity, adrenal insufficiency and red hair. Belongs to the POMC family. |
| UniProt Code: | |
| NCBI GenInfo Identifier: | |
| NCBI Gene ID: | |
| NCBI Accession: | |
| UniProt Related Accession: |
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. |
ACTH Background
ACTH stands for Adrenocorticotropic Hormone. It is a hormone produced by the anterior pituitary gland in the brain. ACTH plays a crucial role in regulating the release of cortisol from the adrenal glands, which are located on top of the kidneys.
Regulation of ACTH
The production and release of ACTH are controlled by a complex feedback system involving the hypothalamus and the adrenal glands. The hypothalamus produces a hormone called corticotropin-releasing hormone (CRH), which stimulates the pituitary gland to release ACTH into the bloodstream. ACTH then travels to the adrenal glands and stimulates the production and release of cortisol.
Functions of ACTH and Cortisol
Cortisol plays a vital role in various physiological processes, including metabolism, immune function, and the body's response to stress. It helps regulate blood sugar levels, maintain blood pressure, and modulate inflammation. ACTH is responsible for the stimulation of cortisol production, ensuring appropriate levels of cortisol in the body.
Disorders and Dysregulation of ACTH
Dysregulation of ACTH can lead to various disorders. Excessive production of ACTH can result in conditions like Cushing's disease, characterized by elevated cortisol levels and associated symptoms such as weight gain, high blood pressure, and muscle weakness. Insufficient production of ACTH can lead to conditions such as adrenal insufficiency or Addison's disease, characterized by low cortisol levels and symptoms such as fatigue, weight loss, and low blood pressure.
ACTH FAQs
What is the ACTH ELISA Kit?
The Human ACTH ELISA Kit accurately measures Adrenocorticotropic Hormone levels in human samples, providing insights into its role in regulating cortisol release.
What are the advantages of using the ACTH ELISA Kit?
Using the ACTH ELISA Kit offers precise and reliable measurement of ACTH levels. It provides a user-friendly workflow and facilitates insights into the HPA axis regulation and its implications in physiological processes.
Where can I find more information about the - ELISA Kit?
For more detailed information about the ACTH ELISA Kit, including technical specifications, performance characteristics, and ordering details, please refer to the product brochure or contact our customer support team. We are here to assist you with any inquiries you may have.
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