Heparan Sulfate ELISA Kit

Antigen: Heparan Sulfate

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN367135

Additional Information

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

Alternative name: heparan sulfate (HS)

Sample Type: Serum, Plasma

Description: Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (PG) in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins. It is in this form that HS binds to a variety of protein ligands and regulates a wide variety of biological activities, including developmental processes, angiogenesis, blood coagulation and tumour metastasis. HS has been shown to serve as cellular receptor for a number of viruses including the respiratory syncytial virus.

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

Sensitivity: The minimum detectable dose of human HS is typically less than 5.3 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 HS is typically less than 5.3 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. An antibody specific to HS has been pre-coated onto a microplate. Standards or samples are added to the appropriate microtiter plate wells with HRP-conjugated HS and incubated. A competitive inhibition reaction is launched between HS (Standards or samples) and HRP-conjugated HS with the pre-coated antibody specific for HS. The more amount 3 of HS in samples, the less antibody bound by HRP-conjugated HS. Then the substrate solutions are added to the wells, respectively. And the color develops in opposite to the amount of HS in the sample. The color development is stopped and the intensity of the color is measured.

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. 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. Add 50μl of Standard or Sample per well. Standard need test in duplicate. 2 Add 50μl of HRP-conjugate to each well (not to Blank well), Mix well and then incubate for 1 hour at 37°C. 3 Fill each well with Wash Buffer (about 200 μl), 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 50μl 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 50μl 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. 9 6 Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.
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, 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 HS 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. 10.
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.If samples generate values higher than the highest standard, dilute the samples with the appropriate Diluent and repeat the assay.Any 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:
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. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. 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 (5), HRP-conjugate (1x6ml), Wash Buffer (20×concentrate) (1x15ml), Substrate A (1x6ml), Substrate B (1x6ml), Stop Solution (1x6ml)

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. 5 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