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  • 產(chǎn)品名稱：Orexin(OX) ELISA Kit

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  • 產(chǎn)品廠商：國內(nèi)供應(yīng)3
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Orexin(OX) ELISA Kit
詳情介紹：
Purpose This immunoassay kit allows for the specific measurement of Human Orexin A concentrations in cell culture supernates, serum and plasma.
Sample Type Cell Culture Supernatant, Serum, Plasma
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This assay recognizes recombinant and natural Human Orexin A.
Characteristics Homo sapiens,Human,Orexin,Hypocretin,Hcrt,HCRT,OX,PPORX,PPOX
Components Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1x120μl), Detection Reagent B (1x120μl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)
Alternative Name HCRT (OX ELISA Kit Abstract)
Background Orexins, also called hypocretins, are the common names given to a pair of highly excitatory neuropeptide hormones that were simultaneously discovered by two groups of researchers in rat brains.The two related peptides (orexin A and B, or hypocretin-1 and -2), with approximately 50% sequence identity, are produced by cleavage of a single precursor protein. Studies suggest that orexin A/hypocretin-1 may be of greater biological importance than orexin B/hypocretin-2. Although these peptides are produced by a very small population of cells in the lateral and posterior hypothalamus, they send projections throughout the brain. Orexin A peptide is 100% conserved between human, mouse, rat, canine, with BSA bovinew and porcine.The orexins/hypocretins are strongly conserved peptides, found in all major classes of vertebrates. The peptides are thought to have arisen early in vertebrate evolution.The orexin/hypocretin system was initially suggested to be primarily involved in the stimulation of food intake, based on the finding that central administration of orexin A/hypocretin-1 increases food intake. The discovery that orexin/hypocretin dysregulation causes the sleep disorder narcolepsy subsequently indicated a major role for this system in sleep regulation. Leptin is a hormone produced by fat cells and acts as a long-term internal measure of energy state. Ghrelin is a short term factor secreted by the stomach just before an expected meal, and strongly promotes food intake.Orexin-producing cells have recently been shown to be inhibited by glucose but are activated by ghrelin. Hypocretin-producing cells have recently been shown to be inhibited by leptin (by leptin receptors) but are activated by ghrelin and hypoglycemia. Orexin/hypocretin (as of 2007) is therefore a very important link between metabolism and sleep regulation. Such a relationship has been long suspected based on the observation that long-term sleep deprivation in rodents dramatically increases food intake and energy metabolism, i.e. catabolism, with lethal consequences on a long term basis.
Pathways Synaptic Membrane, Feeding Behaviour
Sample Volume 100 μL
Plate Pre-coated
Protocol This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Orexin A has been pre-coated onto a microplate. Standards and samples are 2 pipetted into the wells and any Orexin A present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for Orexin A is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Orexin A bound in the initial step. The color development is stopped and the intensity of the color is measured.
Reagent Preparation

3 Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 1000 pg/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (1000 pg/ml). The Sample Diluent serves as the zero standard (0 pg/ml). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.
Sample Collection Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.
Assay Procedure

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:
1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available. 4
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
Calculation of Results

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Orexin A concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Restrictions For Research Use only
Handling Advice 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Storage 4 °C/-20 °C
Storage Comment The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.