The Phenylalanine Hydroxylase (PAH) BioAssay™ ELISA Kit is a quantitative sandwich assay for the detection of PAH in human serum, plasma and other biological fluids.
Specificity:Recognizes natural and some recombinant human PAH. No significant crossreactivity or interference between human PAH and analogues was observed.
Test Principle:The microtiter plate provided in this kit has been pre-coated with an antibody specific to PAH. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to PAH. 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 PAH, biotin-conjugated antibody and enzyme-conjugated Avidin will apear 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 human PAH. The concentration of PAH in the sample is then determined by comparing the O.D. of the sample to the standard curve.
Precaution:The Stop Solution (383954K) 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 *383954A, *383954B, *383954D and *383954E 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.
