This 5-Hydroxyindoleacetic Acid (5-HIAA) BioAssay™ ELISA Kit is a competitive ELISA for the in vitro quantitative determination of 5-HIAA concentrations in serum, plasma and other biological fluids.
Specificity:Recognizes 5-HIAA. No significant cross-reactivity or interference between 5-HIAA and analogues was observed.
Test Principle:The micro ELISA plate provided in this kit has been pre-coated with 5-HIAA. During the reaction, 5-HIAA in the sample or standard competes with a fixed amount of 5-HIAA on the solid phase supporter for sites on the Biotinylated Detection Ab specific to 5-HIAA. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 2nm. The concentration of 5-HIAA in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Storage and Stability:Unused kit is stable for 6 months after receipt. Store Microtiter Strips, Standard, and Antibody-Biotin at 4°C if kit will be used within 30 days; otherwise store at -20°C until expiration date. Store the rest of the components at 4°C. Protect HRP Conjugate and Substrate Reagent from light. For maximum recovery, 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 prepared HRP 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.
