Description
Beta Hydroxybutyrate (Ketone Body) Assay Kit (BA0124) (BA0124)
This Beta Hydroxybutyrate (Ketone Body) Assay Kit (SKU: BA0124) provides a simple, direct and automation-ready method for the quantitative determination of ketone bodies in serum, plasma, urine and other biological samples. Ketone bodies, namely acetoacetic acid (AcAc) and 3-hydroxybutyric acid (BOH), are produced in the liver mainly from the oxidation of fatty acids and are normally present at low concentrations in blood and urine. Increased ketone concentrations may lead to metabolic acidosis, which has been associated with diabetes, childhood hypoglycaemia, growth hormone deficiency, alcohol or salicylate intoxication and inborn errors of metabolism. The assay is based on 3-hydroxybutyrate dehydrogenase catalysed reactions in which the change in NADH absorbance, measured at 340 nm, is directly related to the AcAc and BOH concentrations. The procedure requires only 10 µL of sample and no pretreatment.
| Product Name: | Beta Hydroxybutyrate (Ketone Body) Assay Kit (BA0124) |
| SKU: | BA0124 |
| Detection Method: | Colorimetric (340 nm, NADH) |
| Detection Range: | 0.12 to 8 mM (0.6-40 nmoles/well) for each ketone body in 96-well plate assay |
| Sample Type: | Serum, plasma, urine and other biological samples |
| Species Reactivity: | All |
| Assay Time: | AcAc assay 5 minutes; BOH assay 15 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all reagents at -20°C. |
| Shelf Life: | 6 months after receipt, 3 weeks after reconstitution. |
| Shipping: | Gel Pack |
The assay is based on 3-hydroxybutyrate dehydrogenase catalysed interconversion of acetoacetic acid and 3-hydroxybutyric acid. The change in NADH absorbance at 340 nm is directly related to the concentrations of acetoacetic acid (AcAc) and 3-hydroxybutyric acid (BOH), and the total ketone body concentration is the sum of the two.
- Sensitive and accurate, using 10 µL of sample with a linear detection range of 0.12 to 8 mM for each ketone body in a 96-well plate assay.
- Convenient: no sample pretreatment is needed and the procedure involves adding a single working reagent and reading the optical density at room temperature.
- High-throughput and readily automated for many samples per day.
- Direct assay of ketone bodies in serum, plasma, urine and other biological 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 | Samples: serum and plasma should be non-haemolysed and assayed immediately; if not assayed, samples may be stored at -80°C for up to 30 days. |
| 2 | Reagent preparation: bring all reagents to room temperature, reconstitute the AcAc Reagent tube with 1000 µL distilled water (final 10 mM) and keep the HBDH Enzyme on ice or refrigerated during the experiment; unused reconstituted AcAc Reagent is stable for three weeks at -20°C. |
| 3 | AcAc assay: prepare an 8 mM standard by mixing 5 µL AcAc Standard with 45 µL distilled water, then transfer 5 µL water and 5 µL of the 8 mM AcAc standard into separate wells of a clear flat-bottom 96-well plate; transfer 5 µL of each sample into a sample well and a sample blank well. |
| 4 | For the AcAc reaction, prepare Working Reagent for the water, standard and sample wells by mixing, per well, 195 µL AcAc Buffer, 8 µL reconstituted AcAc Reagent and 0.5 µL HBDH Enzyme; prepare Blank Reagent (per blank well) with 195 µL AcAc Buffer and 8 µL AcAc Reagent (no enzyme). |
| 5 | Add 195 µL Working Reagent to the water, standard and sample wells and 195 µL Blank Reagent to the sample blank wells, gently tap to mix, incubate 5 minutes at room temperature and read OD at 340 nm. |
| 6 | BOH assay: prepare an 8 mM standard by mixing 5 µL BOH Standard with 45 µL distilled water and set up water, standard and sample wells as for the AcAc assay. |
| 7 | For the BOH reaction, prepare Working Reagent by mixing, per well, 195 µL BOH Buffer, 8 µL BOH Reagent and 0.5 µL HBDH Enzyme, and Blank Reagent with 195 µL BOH Buffer and 8 µL BOH Reagent (no enzyme). |
| 8 | Add 195 µL Working Reagent to the water, standard and sample wells and 195 µL Blank Reagent to the sample blank wells, gently tap to mix, incubate 15 minutes at room temperature and read OD at 340 nm. |
[AcAc] = (ODBLANK - ODSAMPLE) / (ODH2O - ODSTANDARD) x 8 (mM). [BOH] = (ODSAMPLE - ODBLANK) / (ODSTANDARD - ODH2O) x 8 (mM). Total ketone body [TKB] = [AcAc] + [BOH]. If a calculated value exceeds 8 mM, dilute the sample in water, repeat and multiply the result by the dilution factor.
| Component | Quantity | Storage |
| AcAc Buffer | 20 mL | -20°C |
| BOH Buffer | 20 mL | -20°C |
| AcAc Reagent | Dried | -20°C |
| BOH Reagent | 1 mL | -20°C |
| AcAc Standard | 200 µL | -20°C |
| BOH Standard | 200 µL | -20°C |
| HBDH Enzyme | 120 µL | -20°C |
