Oxytocin (OXT) ELISA Kit

Antigen: Oxytocin (OXT)

Synonyms: OT, Oxy

Reactivity: Mouse (Murine)

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN365185

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of mouse oxytocin concentrations in serum, plasma and other biological fluids.

Alternative name: Oxytocin (OT)

Sample Type: Serum, Plasma

Description: Oxytocin is a mammalian hormone that also acts as a neurotransmitter in the brain. It is best known for its roles in female reproduction: it is released in large amounts after distension of the cervix and vagina during labor, and after stimulation of the nipples, facilitating birth and breastfeeding, respectively. Recent studies have begun to investigate oxytocin's role in various behaviors, including social recognition, bonding, anxiety, trust, and maternal behaviors. Oxytocin is a peptide of nine amino acids (a nonapeptide). The sequence is cysteine - tyrosine - isoleucine - glutamine - asparagine - cysteine - proline - leucine - glycine (CYIQNCPLG). The cysteine residues form a sulfur bridge. Oxytocin has a molecular mass of 1007 daltons. Oxytocin is made in magnocellular neurosecretory cells of the supraoptic and paraventricular nuclei of the hypothalamus and is stored in Herring bodies at the axon terminals in the posterior pituitary. It is then released into the blood from the posterior lobe of the pituitary gland. Oxytocin is also made by some neurons in the paraventricular nucleus that project to other parts of the brain and to the spinal cord. Depending on the species, oxytocin-expressing cells are located in other areas, including the amygdala and bed nucleus of the stria terminalis. 3

Specificity: 4 This assay recognizes recombinant and natural mouse oxytocin. No significant cross-reactivity or interference was observed.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with a goat-anti-rabbit antibody. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated oxytocin and anti-oxytocin antibody and incubated. Then substrate solution A and B are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of oxytocin in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Preparation of Reagents: 1. Bring all reagents and plate to room temperature for at least 30 minutes before use. Unused wells need store at 2-8°C and avoid sunlight. 2. Wash Buffer If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer. 6 Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. Sample Collection and Storage: Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles. 7 Assay Procedure: 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 oxytocin 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. Limitations of the Procedure: The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or 9 sources. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator 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. This assay is designed to eliminate interference by soluble receptors, 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.

Assay Procedure: Assay Time: 1-3h
Sample Volume: 50-100ul

Application Notes: Detection Wavelength: 450 nm

Components: Reagent Quantity Assay plate 1 Standard(S 1 -S 5 ) 5 Antibody 1 x 6 ml HRP-conjugate 1 x 6 ml Wash Buffer 1 x 15 ml (20×concentrate) Substrate A 1 x 7 ml Substrate B 1 x 7 ml Stop Solution 1 x 7 ml 5 STORAGE 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Storage: 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. Expiration date: six months from the date of manufacture.

Restrictions: For Research Use only