Carbohydrate-Deficient Transferrin (CDT) ELISA Kit

Antigen: Carbohydrate-Deficient Transferrin (CDT)

Reaktivität: Ratte (Rattus)

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN367558

Produktbeschreibung

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

Proben: Serum, Plasma

Beschreibung: Transferrin is a plasma protein that carries iron through the bloodstream to the bone marrow, where red blood cells are manufactured, as well as to the liver and spleen. Structurally, transferrin is a polypeptide with two N-linked polysaccharide chains. These polysaccharide chains are branched with sialic acid residues. Sialic acid is a monosaccharide carbohydrate. Various forms of transferrin exist, with differing levels of sialylation. The most common form is tetrasialotransferrin, with four sialic acid chains. In persons who consume significant quantities of alcohol, the proportion of transferrin with zero, one, or two sialic acid chains is increased. These are referred to as carbohydrate-deficient transferrins(CDT). These carbohydrate-deficient transferrins can be measured in the bloodstream, and are an important marker for alcohol abuse.

Spezifität: This assay recognizes recombinant and natural rat CDT. 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 CDT. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody 3 preparation specific for CDT 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 CDT, 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 CDT 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 up 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. 2. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 40 nmol/ml. Allow the standard to sit for a minimum of 15 minutes 6 with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (40 nmol/ml). The Sample Diluent serves as the zero standard (0 nmol/ml). Prepare fresh for each assay. Use within 4 hours and discard after use. 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 7 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. 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 CDT 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 10 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.

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

Applikationshinweise: Detection Wavelength: 450 nm

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 STORAGE 5 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.

Lagerung: 5 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar