Histamine (HIS) ELISA Kit

Antigen: Histamine (HIS)

Reactivity: Rat (Rattus)

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN368162

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of rat HIS concentrations in cell culture supernates, serum, plasma and other biological fluids.

Sample Type: Serum, Plasma

Description: Histamine is a biogenic amine involved in local immune responses as well as regulating physiological function in the gut and acting as a neurotransmitter. Histamine triggers the inflammatory response. As part of an immune response to foreign pathogens, histamine is produced by basophils and by mast cells found in nearby connective tissues. Histamine increases the permeability of the capillaries to white blood cells and other proteins, in order to allow them to engage foreign invaders in the affected tissues. It is found in virtually all animal body cells.

Specificity: This assay recognizes recombinant and natural rat HIS. No significant cross-reactivity or interference was observed.

Sensitivity: The minimum detectable dose of rat HIS is typically less than 0.1 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of rat HIS is typically less than 0.1 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with an goat-anti-rabbit antibody. Standards or samples are then added to the appropriate microtiter plate wells with a HRP-conjugated HIS and antibody preparation specific for HIS and incubated. Then substrate solutions 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 3 spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of HIS in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. 8 1. Set a Blank well without any solution. Add 50 µ l of Standard or Sample per well. Standards need test in duplicate. 2. Add 50 µ l of HRP-conjugate to each well (not to Blank well), then add 50 µ l Antibody to each well. Mix well and then incubate for 1 hour at 37°C. 3. Fill each well with Wash Buffer (about 200 µ l), stay for 10 seconds and Spinning. Repeat the process for a total of three washes. 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. 4. Add 50 µ l of Substrate A and Substrate B to each well, mix well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 5. Add 50 µ l of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 6. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm. 9.
Calculation of Results:
Average the duplicate readings for each standard, Blank, and sample and subtract the optical density of Blank. 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 HIS 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 sources. 10 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. 11 Notes 12 13 14 15 16 17.
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 x g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repea ted 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. Note: Grossly hemolyzed samples are not suitable for use in this assay.

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

Application Notes: Detection Wavelength: 450 nm

Components: Reagent (Quantity): Assay plate (1), Standard (5x0.5ml), HRP-conjugate (1x6ml), Antibody (1x6ml), Wash Buffer (20×concentrate) (1x15ml), Substrate A (1x7ml), Substrate B (1x7ml), Stop Solution (1 x7ml)

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer.

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. 5 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.

Restrictions: For Research Use only