This Acetylcholine (ACH) BioAssay™ ELISA Kit is a competitive ELISA for the in vitro quantitative determination of ACH concentrations in serum, plasma and other biological fluids.
Specificity:Recognizes ACH. No significant cross-reactivity or interference between ACH and analogues was observed.
Test Principle:The micro ELISA plate provided in this kit has been pre-coated with ACH. During the reaction, ACH in the sample or standard competes with a fixed amount of ACH on the solid phase supporter for sites on the Biotinylated Detection Ab specific to ACH. 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 ACH 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.
