Hyaluronate Binding Protein (HABP) ELISA Kit

Antigen: Hyaluronate Binding Protein (HABP)

Reaktivität: Human

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN367025

Produktbeschreibung

Produktmerkmale: This immunoassay kit allows for the in vitro quantitative determination of human HABP concentrations in serum, plasma and other biological fluids. 

Beschreibung: Hyaluronic Acid (HA), also called hyaluronate or hyaluronan, is a mucopolysaccharide widely distributed throughout the body. HA is produced mainly by fibroblasts and other specialized connective tissue cells.HABP is hyaluronic acid binding protein. Detection of 34 kDa HABP in the serum-free supernatant culture medium of fibroblasts was further evident by immunoblot analysis, thus confirming the secretory nature of HABP and its occurrence in the extracellular matrix.HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. HABP efficiently prevented the basic fibroblast growth factor/epidermal growth factor (bFGF/EGF)-dependent proliferation of human umbilical vein endothelial cells.It also demonstrates that ITI-related proteins might be considered as HA-binding proteins.HABP staining revealed a stronger reactivity in the follicular keratinocytes surrounding the mucin-accumulated areas compared to the interfollicular keratinocytes. It was reported that human fibroblast 34-kDa hyaluronic acid-binding protein (HABP) is identical with P32, the protein co-purified with splicing factor SF-2. 3

Spezifität: This assay recognizes recombinant and natural human HABP . No significant cross-reactivity or interference was observed.

Anwendungen

Prinzip: The microtiter plate provided in this kit has been pre-coated with an antibody specific to HABP . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for HABP and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain HABP , biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. 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 HABP in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protokoll: Preparation of Reagents: Bring all reagents to room temperature before use. 1. 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. 6 2. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 1000 ng/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 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). 3. Biotin-antibody Dilute to the working concentration using Biotin-antibody Diluent(1:100), respectively. 4. HRP-avidin Dilute to the working concentration using HRP-avidin Diluent(1:100), respectively. 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 x 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 x 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: 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 HABP 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: Centrifuge vials before opening to collect contents. 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. 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.

Applikationshinweise: When mixing or reconstituting protein solutions, always avoid foaming. 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. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution. 12

Bestandteile: Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20 ml Biotin-antibody Diluent 1 x 10 ml HRP-avidin Diluent 1 x 10 ml Biotin-antibody 1 x 120μl HRP-avidin 1 x 120μl Wash Buffer 1 x 20 ml (25×concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 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 to minimize exposure to damp air. 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.

Lagerung: 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag to minimize exposure to damp air. 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar