Description
Sorbitol Dehydrogenase Assay Kit (BA0056) (BA0056)
The Sorbitol Dehydrogenase Assay Kit (SKU: BA0056) provides a fast, sensitive and automation-ready colorimetric method for determining sorbitol dehydrogenase (SDH) activity in biological samples. SDH is an enzyme that catalyses the interconversion of sorbitol and fructose, and elevated serum levels indicate liver damage, making it important in the diagnosis of liver disease, especially in combination with aminotransferases. SDH levels are also measured to evaluate diabetic complications such as proliferative diabetic retinopathy. The non-radioactive assay is based on the reduction of the tetrazolium salt MTT in an NADH-coupled enzymatic reaction to a reduced form of MTT, which absorbs maximally at 565nm. The increase in absorbance at 565nm is directly proportional to the enzyme activity. The homogeneous mix-incubate-measure format can be readily automated for high-throughput screening.
| Product Name: | Sorbitol Dehydrogenase Assay Kit (BA0056) |
| SKU: | BA0056 |
| Detection Method: | Colorimetric (kinetic) |
| Detection Range: | 0.1 to 125 U/L for a 12-minute reaction (20 µL sample) |
| Sample Type: | Plasma, serum, urine, tissue and culture media |
| Species Reactivity: | All |
| Assay Time: | 12-minute kinetic reaction (readings at 3 and 15 minutes) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | The kit is shipped at room temperature. Store all components at -20°C upon receipt. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Room Temperature |
This non-radioactive, colorimetric assay is based on the reduction of the tetrazolium salt MTT in an NADH-coupled enzymatic reaction to a reduced form of MTT, which absorbs maximally at 565nm. The increase in absorbance at 565nm is directly proportional to the enzyme activity.
- Fast and sensitive: linear detection range (20 µL sample) 0.1 to 125 U/L for a 12-minute reaction
- Convenient and high-throughput: homogeneous mix-incubate-measure assay, readily automated on HTS liquid handling systems for thousands of samples per day
- SDH activity determination in biological samples (e.g. plasma, serum, urine, tissue and culture media)
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 | Equilibrate reagents to the desired reaction temperature (37°C recommended) and briefly centrifuge tubes before use. |
| 2 | Transfer 100 µL dH2O (ODH2O) and 100 µL Calibrator (ODCAL) into wells of a clear flat-bottom 96-well plate. |
| 3 | Transfer 20 µL dH2O into one well (blank) and 20 µL of each sample into separate wells. |
| 4 | Prepare Working Reagent (per 96-well assay: 2 µL Substrate, 8 µL NAD/MTT Solution, 1 µL Diaphorase and 75 µL Assay Buffer) and add 80 µL to all sample and blank wells; tap plate briefly to mix. |
| 5 | Incubate at the desired temperature and read OD565nm at 3 minutes (OD3) and 15 minutes (OD15). |
Subtract OD3 from OD15 for each sample well to compute ∆ODS, and do the same for the blank to compute ∆ODB. SDH activity = [(∆ODS − ∆ODB) / t] × [(ODCAL − ODH2O) / (∆ODCAL time factor)] × n (U/L), using the calibrator to determine the light pathlength; t is the difference in reading times (12 minutes recommended), reaction and sample volumes are 100 µL and 20 µL, and n is the dilution factor. Unit definition: 1 Unit (U) catalyses the conversion of 1 µmole of D-sorbitol to fructose per minute at pH 8.2.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| Diaphorase | 120 µL | -20°C |
| Substrate | 250 µL | -20°C |
| Calibrator | 1.5 mL | -20°C |
| NAD/MTT Solution | 1 mL | -20°C |
