Description
Diamine Oxidase Assay Kit (Fluorometric) (BA0172) (BA0172)
The Diamine Oxidase Assay Kit (Fluorometric) (SKU: BA0172) provides a rapid, non-radioactive method for the quantitative determination of diamine oxidase (DAO) activity in serum or plasma. Diamine oxidase, also known as histaminase, is an enzyme involved in the metabolism, oxidation and inactivation of histamine in animals, with the highest levels observed in the digestive tract and placenta. An imbalance between histamine intake and the capacity for its degradation can lead to histamine intolerance, so measuring DAO activity in serum can be useful in research into this condition. This assay is based on the oxidation of putrescine to pyrroline plus ammonia and hydrogen peroxide; the generated hydrogen peroxide is then used by horseradish peroxidase to oxidise a dye, making it fluorescent. The increase in fluorescence at 530/585 nm is directly proportional to the enzyme activity. It is intended for research use only.
| Product Name: | Diamine Oxidase Assay Kit (Fluorometric) (BA0172) |
| SKU: | BA0172 |
| Detection Method: | Fluorimetric (lambda ex/em = 530/585 nm) |
| Detection Range: | 0.5 to 6 U/L for a 30 min reaction at 25C |
| Sample Type: | Serum and plasma |
| Species Reactivity: | All |
| Assay Time: | 30 min |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Room Temperature |
A fast, sensitive, non-radioactive fluorimetric assay for the determination of diamine oxidase activity in serum or plasma. DAO oxidises putrescine to produce hydrogen peroxide, which is used by horseradish peroxidase to oxidise a dye to a fluorescent product measured at 530/585 nm. The linear detection range is 0.5-6 U/L for a 30-minute reaction at 25C, and the room-temperature assay requires no 37C heater.
- Fast and sensitive; uses only 10 uL sample with a linear range of 0.5 to 6 U/L (30 min at 25C)
- Convenient single working-reagent procedure with readings at 0 and 30 minutes
- Room-temperature assay requiring no 37C heater
- Readily automated as a high-throughput 96-well plate assay
- Direct measurement of DAO activity in serum or plasma samples
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Internal Standard: prepare 500 uL of 8.82 mM H2O2 by mixing 5 uL of the H2O2 Standard (882 mM) with 495 uL dH2O, then mix 20 uL of the 8.82 mM H2O2 with 960 uL dH2O to make a 180 uM internal standard (use within 1 hour). |
| 2 | Prepare Working Reagent (WR) for the sample wells by mixing, per well: 85 uL Assay Buffer, 1 uL HRP Enzyme, 1 uL Substrate and 1 uL Dye Reagent. Prepare Blank Working Reagent (BWR) for sample blank and internal standard wells by mixing, per well: 85 uL Assay Buffer, 1 uL HRP Enzyme and 1 uL Dye Reagent (no Substrate). |
| 3 | Transfer 10 uL of each sample into three separate wells of a black flat-bottom 96-well plate: Sample (FS), Sample Blank (FSB) and Internal Standard (FIS). Add 10 uL dH2O to the Sample and Sample Blank wells and 10 uL of the 180 uM H2O2 to the Internal Standard wells. |
| 4 | Transfer 80 uL WR to each Sample well and 80 uL BWR to each Sample Blank and Internal Standard well. |
| 5 | Read fluorescence at 530/585 nm at time 0 and again at 30 min. |
Subtract the time-0 fluorescence from the time-30 fluorescence for the Sample and Sample Blank wells to obtain delta FS and delta FSB. DAO Activity = [(delta FS - delta FSB) / (FIS30 - FSB30)] x (180 uM / t) x n (U/L), where FIS30 and FSB30 are the 30-minute readings for the Internal Standard and Sample Blank, t is the reaction time (30 min) and n is the dilution factor. One unit (U) of DAO catalyses the conversion of 1 umole of putrescine to pyrroline plus NH3 and H2O2 per minute at pH 7.5.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| HRP Enzyme | 120 uL | -20C |
| Substrate | 120 uL | -20C |
| H2O2 Standard | 100 uL | -20C |
| Dye Reagent | 120 uL | -20C |
