Description

Ketone bodies (acetoacetic acid and 3-hydroxybutyric acid) are produced in the liver mainly from oxidation of fatty acids, and are normally present at low concentrations in urine and blood. Increased ketone concentrations in the blood may lead to metabolic acidosis, which has been associated with diabetes, childhood hypo-glycaemia, growth hormone deficiency, alcohol or salicylate intoxication and inborn errors of metabolism.

Simple, direct and automation-ready procedures for measuring acetoacetic acid (AcAc) and 3-hydroxybutyric acid (BOH) are very desirable. BioAssay Systems EnzyChrom^TM ketone body assay is based on 3-hydroxybutyrate dehydrogenase catalyzed reactions, in which the change in NADH absorbance, measured at 340nm, is directly related to the AcAc and BOH concentrations,

Applications

Direct assays of ketone body in serum, plasma, urine and other biological samples.

Key features

Sensitive and accurate. Uses 10 mL sample. Linear detection range of 0.12 to 8 mM for each ketone body in 96-well plate assay.

Convenient. The procedure involves adding a single working reagent, and reading the optical density at room temperature.

High-throughput. Can be automated as a high-throughput 96-well plate assay for many samples per day.

Kit Contents (200 tests in 96-well plates)

Storage conditions. Store all reagents at -20°C. Shelf life: 6 months after receipt.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

ProcedureS

Samples: serum and plasma samples should be non-hemolyzed and assayed immediately. If not assayed, samples can be stored at -80°C for up to 30 days.

Reagent preparation: bring all reagents to room temperature prior to assay. During experiment, keep the HBDH enzyme on ice or in refrigerator (2-8°C).

AcAc Assay

1. Standards. Prepare 8 mM standard by mixing 5 mL AcAc standard with 45 mL distilled H₂O. Transfer 5 mL H₂O, 5 mL 8 mM AcAc standard in separate wells of a clear, flat-bottom, 96-well plate.

    Samples. Transfer 5 mL sample into two wells, one Sample well and one sample Blank  well.

2. Reaction. Prepare Working Reagent for H₂O, Standard and Sample wells, by mixing 195 mL AcAc Buffer, 8 mL AcAc Reagent and 0.5 mL HBDH Enzyme for each well. The Blank Reagent is prepared by mixing, for each blank well, 195 mL AcAc Buffer and 8 TUNEology 波长可调检测卡盒-->