Carboxyterminal Propeptide of Type 1 Procollagen (P1CP) ELISA Kit

Antigen: Carboxyterminal Propeptide of Type 1 Procollagen (P1CP)

Reaktivität: Schwein

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN368425

Produktbeschreibung

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

Proben: Serum, Plasma

Beschreibung: Type I collagen is found in several different types of connective tissues throughout the rat body, but most of it is present in bones, where it forms more than 95% of the organic matrix. Many diseases are associated with an increase in the amount of collagen synthesized i.e. an increase in the rate of bone matrix formation and turnover. In particular it would be desirable to be able to assess this rate since osteoporosis and other metabolic bone diseases are a common health problem and such assessment would provide guidance in formulating a therapy for these disorders. Type I collagen is synthesized as a procollagen containing propspride extensions at both ends of the molecule. Cultured skin fibroblasts always produce both type I and III procollagens. However, it is technically difficult to completely separate the two types from each other. Salt fractionation, the most commonly used separation method, does not result in complete separation. The concentration of type III procollagen is often monitored using an assay for the aminoterminal propeptide of type III procollagen. However, this is not sufficiently sensitive 3 since it does not recognize molecules that contain the carboxyterminal propeptide but have lost the aminoterminal one (such molecules are known as type III pC-collagen). Both PICP and PIIICP contain mannose-rich oligosaccharide side chains and they thus bind to concanavalin A lectin.

Spezifität: This assay recognizes recombinant and natural porcine PICP. 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 PICP. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for PICP 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 PICP, 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 4 concentration of PICP in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protokoll: 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. 9 1. Add 100µl of Standard, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37°C. 2. Remove the liquid of each well, don’t wash. 3. Add 100µl of Biotin-antibody working solution to each well. Incubate for 1 hour at 37°C. Biotin-antibody working solution may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform. 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 100µl of HRP-avidin working solution to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C. 6. Repeat the aspiration and wash five times as step 4. 7. Add 90µl of TMB Substrate to each well. Incubate for 10-30 minutes at 37°C. Keeping the plate away from drafts and 10 other temperature fluctuations in the dark. 8. 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. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.

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

Applikationshinweise: Detection Wavelength: 450 nm

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. 6 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