Human Melatonin ELISA Kit (HUFI02640)
- SKU:
- HUFI02640
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Sensitivity:
- 4.688pg/ml
- Range:
- 7.813-500pg/ml
- ELISA Type:
- Competitive
- Synonyms:
- Melatonin
- Reactivity:
- Human
- Research Area:
- Metabolism
Description
Human Melatonin ELISA Kit
Melatonin is a fascinating hormone that plays a crucial role in regulating the sleep-wake cycle, also known as the circadian rhythm. It is naturally produced by the pineal gland, a small pea-sized gland located deep within the brain. Understanding and accurately measuring melatonin levels can provide valuable insights into sleep disorders, chronobiological research, and overall health assessment. The Assay Genie Human Melatonin ELISA Kit is a highly sensitive assay for the quantitative measurement of MT in serum, plasma, cell and tissue lysates.
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 Melatonin ELISA Kit |
| Product Code: | HUFI02640 |
| Size: | 96 Assays |
| Alias: | Melatonin |
| Detection Method: | Competitive ELISA, Coated with Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human MT concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 4.688pg/ml |
| Range: | 7.813-500pg/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 MT and the recovery rates were calculated by comparing the measured value to the expected amount of Human MT in samples.
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| Linearity | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human MT 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
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. |
Melatonin Background
Melatonin
Melatonin, also known as 5-methoxy-N-acetyltryptamine, was first discovered and isolated from bovine pineal in 1958 by Aaron Lerner. Primarily produced by the pineal gland, melatonin serves as the main hormone secreted by this gland. Interestingly, melatonin can also be found in various other tissues and organs outside the pineal gland, such as the retina, bone marrow cells, platelets, skin, lymphocytes, Harderian gland, cerebellum, and notably, the gastrointestinal tract in vertebrate species. In newborns and infant mammals, melatonin in the gastrointestinal tract is derived from the mother, as it easily passes through the placenta and is subsequently secreted into the mother's milk. There have even been suggestions of melatonin's involvement in meconium production. The synthesis and release of melatonin are stimulated by darkness and suppressed by light, demonstrating its dependence on light exposure for proper regulation.
Role of Melatonin
Melatonin hormone plays a crucial role in regulating the sleep-wake cycle, as well as other circadian and seasonal rhythms. It acts as both an immunostimulator and a cytoprotective agent. In the body, melatonin is primarily metabolized by hepatic P450 monooxygenases, undergoing 6-hydroxylation, and then excreted as 6-sulfatoxymelatonin. This hormone finds applications in sedation, hypnosis, immune stimulation, anti-tumor activities, and as an antioxidant.
Melatonin ELISA Kit FAQs
What is the Melatonin ELISA Kit used for?
The Melatonin ELISA kit is designed for the quantitative measurement of melatonin levels in human samples. Melatonin is a hormone primarily synthesized and secreted by the pineal gland in the brain. It plays a crucial role in regulating the sleep-wake cycle (circadian rhythm) and is involved in various physiological processes such as sleep regulation, antioxidant activity, and immune function. The Melatonin ELISA kit enables researchers and healthcare professionals to accurately measure melatonin levels, facilitating investigations into the role of melatonin in sleep disorders, circadian rhythm disturbances, and other melatonin-related conditions.
What are the advantages of using MT ELISA Kit?
The MT ELISA Kit offers several advantages for accurate and precise MT measurements. It provides high sensitivity and specificity, ensuring reliable results. The kit is user-friendly, offering a streamlined protocol for easy handling and efficient analysis. Additionally, it provides a wide dynamic range, allowing for the detection of both low and high MT levels, and it is suitable for both research and clinical applications.
What sample types are compatible with MT ELISA kit?
The MT 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 MT levels in different biological matrices.
What are the storage requirements for the M ELISA Kit?
The MT 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.
Q: Question?
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 MT ELISA Kit.
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