C-Peptide ELISA Kit

Antigen: C-Peptide

Reaktivität: Maus

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN366344

Produktbeschreibung

Produktmerkmale: This immunoassay allows for the in vitro quantitative determination of mouse C-Peptide concentrations in serum, plasma and other biological fluids.

Proben: Serum, Plasma

Beschreibung: C Peptide is part of the molecule of Proinsulin, that consists of three parts: C Peptide and two long strands of amino acids (called the alpha and beta chains) that later become linked together to form the insulin molecule. From every molecule of proinsulin, one molecule of insulin plus one molecule of C Peptide are produced. C peptide is released into the blood stream in equal amounts to insulin. A test of C peptide levels will show how much insulin the body is making. Insulin decreases blood glucose concentration. It increases cell permeability to monosaccharides, amino acids and fatty acids. It accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in liver.

Spezifität: This assay recognizes recombinant and natural mouse C-Peptide. No significant cross-reactivity or interference was observed.

Sensitivität: The minimum detectable dose of mouse C-Peptide is typically less than 1ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of mouse C-Peptide is typically less than 1ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest concentration that could be differentiated from zero.

Anwendungen

Prinzip: The microtiter plate provided in this kit has been pre-coated with an antibody specific to C-Peptide. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated monoclonal antibody preparation specific for C-Peptide and incubated. Then substrate solution A and B are added to each well. Only those wells that contain C-Peptide, HRP-conjugated antibody will exhibit a 3 change in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of C-Peptide in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protokoll: Reagent Preparation:
1. Bring all reagents and plate to room temperature for at least 30 minutes before use. Unused wells need store at 2-8°C and avoid sunlight. 2. Standard Reconstitute the every Standard with 0.5 ml of distilled water. 3. Wash Buffer If crystals have formed in the concentrate, warm to room temperature and mix gently until the 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. 7 1. Set a Blank well without any solution. Add 50 µ l of Standard or Sample per well. Standard need test in duplicate. 2. Add 50 µ l of HRP-conjugate to each well (not to Blank well). Mix well and then incubate for 2 hour at 37°C. 3. Complete remove the liquid. Then 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 50 µ l of Substrate B to each well, mix well. Incubate for 15 minutes at 37°C. Ke eping 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.
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. 8 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 C-Peptide 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. If samples generate values higher than the highest standard, dilute the samples with the appropriate Diluent and repeat the assay. 9 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 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 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.

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

Applikationshinweise: Detection Wavelength: 450 nm

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

Benötigtes Material: 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.

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. 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. 5 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.

Forschungsgebiet: Diabetes

Beschränkungen: Nur für Forschungszwecke einsetzbar