This Klotho (KL) BioAssay™ ELISA Kit is a sandwich ELISA for in vitro quantitative measurement of KL in mouse serum, plasma and other biological fluids.
Specificity:Recognizes mouse KL. No significant crossreactivity or interference between mouse KL. and analogues was observed.
Test Principle:The microtiter plate provided in this kit has been pre-coated with an antibody specific to mouse KL. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to KL. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Free components are washed away. The substrate solution is added, only those wells that contain KL, biotin-conjugated antibody and enzyme-conjugated Avidin will appear blue in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450nm ± 2nm. The OD value is proportional to the concentration of mouse KL. The concentration of KL in the sample is then determined by comparing the O.D. of the sample to the standard curve.
Precaution:The Stop Solution (383515K) included for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Storage and Stability:Store *383515A, *383515B, *383515D and *383515E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months after receipt. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.
Assay Procedure Summary:1. Prepare all reagents, samples and standards
2. Add 100ul standard or sample to each well. Incubate 90 minutes at 37°C
3. Aspirate and add 100ul prepared Biotin Working Solution. Incubate 1 hour at 37°C
4. Aspirate and wash 3 times
5. Add 100ul preparedHRP Detection Working Solution. Incubate 30 minutes at 37°C
6. Aspirate and wash 5 times
7. Add 90ul Substrate. Incubate 15 minutes at 37°C
8. Add 50ul Stop Solution. Read at 450nm immediately.
