Deoxypyridinoline Crosslinks ELISA Kit

Antigen: Deoxypyridinoline Crosslinks

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN367205

Additional Information

Alternative name: Deoxypyridinoline crosslinks (DPD)

Sample Type: Serum, Plasma

Specificity: This assay recognizes human DPD. No significant cross-reactivity or interference was observed.

Sensitivity: The minimum detectable dose of human DPD is typically less than 80 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 human DPD is typically less than 80 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: This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antibody specific to DPD. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated DPD and incubated. Then substrate solution is added to each well. And the color develops in opposite to the amount of DPD in the sample. 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 DPD in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Reagent Preparation:
1. Bring all reagents to room temperature before use. 2. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the 5 crystals have completely dissolved. Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
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. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well. 1. Set a Blank well without any solution. 2. Add 50μl of Standard or Sample to per well. 3. Add 50µl of HRP-conjugate to each well (not to the Blank!). 7 4. Cover with the adhesive strip. Incubate for 60 minutes at 37°C. 5. Aspirate each well and wash, repeating the process three or four times. Wash by filling each well with Wash Buffer (200 μl ) 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 50µl of Substrate A and 50µl Substrate B to each well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 7. Add 5 0μl of Stop Solution to each well when the first four wells containing the highest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 8. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. 8.
Calculation of Results:
Using the professional soft ",Curve Exert 1.3", to make a standard curve is recommended, which can be downloaded from our web. 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 DPD 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. 9Any variation in 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.
Sample collection and storage:
Urine Collect urine using a sterile collection tube. Remove any particulates by centrifugation and assay immediately or aliquot and store samples at -20°C. Avoid repeated 6 freeze-thaw cycles. Centrifuge again before assaying to remove any additional precipitates that may appear after storage.If the urine samples have a level high than 8000ng/ml after test, it is recommend to dilute the urine samples with physiological saline(1:50) and test again. The recommended dilution factor is for reference only. The optimal dilution factor should be determined by users according to their particular experiments.When calculate the high DPD level via standard curve, the OD value must high than 0.5, and the calculated DPD concentration no more than 80000ng/ml.

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

Application Notes: Detection Wavelength: 450 nm

Components: Reagent (Quantity): Assay plate (1), Standard 5x1ml HRP-conjugate (1x6ml), Wash Buffer (20×concentrate) (1x15ml), Substrate A (1x7ml), Substrate B (1x7ml), Stop Solution (1x10ml)

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. An incubator which can provide stable incubation conditions up to 37°C ± 0.5°C.

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, provided it is stored as prescribed above. Refer to the package label for the expiration date. 2. Opened test plate should be stored at 2-8 C in the aluminum foil bag with desiccants to minimize exposure to damp air. The 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