Triglyceride ELISA Kit

Antigen: Triglyceride

Reaktivität: Ratte (Rattus)

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN367570

Produktbeschreibung

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

Weitere Bezeichnung: Triglyceride (TG)

Proben: Serum, Plasma

Beschreibung: Triglycerides, as major components of very low density lipoprotein (VLDL) and chylomicrons, play an important role in metabolism as energy sources and transporters of dietary fat. Triglycerides are formed from a single molecule of glycerol, combined with three fatty acids on each of the OH groups, and make up most of fats digested by humans. Ester bonds form between each fatty acid and the glycerol molecule. This is where the enzyme pancreatic lipase acts, hydrolysing the bond and "releasing" the fatty acid. In triglyceride form, lipids cannot be absorbed by the duodenum. Fatty acids, monoglycerides (one glycerol, one fatty acid) and some diglycerides are absorbed by the duodenum, once the triglycerides have been broken down.

Spezifität: This assay recognizes rat triglyceride. No significant cross-reactivity or interference was observed.

Sensitivität: The minimum detectable dose of rat triglyceride is typically less than 1 μg/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 rat triglyceride is typically less than 1 μg/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.

Anwendungen

Prinzip: This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with goat-anti-rabbit antibody. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated triglyceride and antibody preparation specific for triglyceride, and incubated. 3 Then substrate solutions are added to each well. 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 triglyceride 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 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 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 without any solution. Add 5 0μl of Standard or Sample per well. 2. Ad d 50μl of HRP-Conjugate and 50μl of Antibody to each well. Not to Blank well! 7 3. Cover with the adhesive strip. Incubate for 2 hours at 37° C. 4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash: Fill each well with Wash Buffer (200μl) and let it stand for 2 minutes, then remove the liquid by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. Complete removal of liquid at each step is essential to good performance. 5. Add 50 μl of Substrate A and 50μl of 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. 6. 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. 7. Determine the optical density of each well within 30 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, control, and 8 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 triglyceride 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.Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. 9This 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. 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 (5x0.5ml), HRP-Conjugate (1x6ml), Antibody (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. 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar