Ketone Body Assay Kit, BioAssay(TM)

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.\n\nSimple, direct and automation-ready procedures for measuring acetoacetic acid (AcAc) and 3-hydroxybutyric acid (BOH) are very desirable. 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,\n\nApplications:\nDirect assays of ketone body in serum, plasma, urine and other biological samples.\n\nKey Features:\nSensitive and accurate. Uses 10ul sample. Linear detection range of 0.12 to 8mM 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.\n\nKit Contents (200 tests in 96-well plates):\nAcAc Buffer: 20ml BOH Buffer: 20ml\nAcAc Reagent: 1ml BOH Reagent: 1ml\nAcAc Standard: 200ul 80mM BOH Standard: 200ul 80mM\nHBDH Enzyme: 120ul\nStorage conditions. Store all reagents at -20 degrees C. Shelf life: 6 months\nafter receipt.\nPrecautions: reagents are for research use only. Normal precautions\nfor laboratory reagents should be exercised while using the reagents.\nPlease refer to Material Safety Data Sheet for detailed information.\n\nProcedures:\nSamples: serum and plasma samples should be non-hemolyzed and assayed immediately. If not assayed, samples can be stored at -80 degrees C for up to 30 days.\nReagent preparation: bring all reagents to room temperature prior to assay. During experiment, keep the HBDH enzyme on ice or in refrigerator (2-8 degrees C).\nAcAc Assay\n1. Standards. Prepare 8mM standard by mixing 5ul AcAc standard with 45ul distilled H2O. Transfer 5ul H2O, 5ul 8mM AcAc standard in separate wells of a clear, flat-bottom, 96-well plate.\nSamples. Transfer 5ul sample into two wells, one Sample well and one sample Blank well.\n2. Reaction. Prepare Working Reagent for H2O, Standard and Sample wells, by mixing 195ul AcAc Buffer, 8ul AcAc Reagent and 0.5ul HBDH Enzyme for each well. The Blank Reagent is prepared by mixing, for each blank well, 195ul AcAc Buffer and 8ul AcAc Reagent (i.e., no enzyme).\nAdd 195ul Working Reagent to the H2O, Standard and Sample wells. Add 195ul Blank Reagent to Sample Blank wells. Gently tap plate to mix.\n3. Incubate 5 min at room temperature. Read OD340nm. Calculate the\nacetoacetic acid (AcAc) concentration from the OD values of the\nH2O, 8mM Standard, Sample and Sample Blank wells,\n[AcAc] =\nODBLANK

Prices direct from United States Biological

Quick response times

Exclusive Biosave savings/discounts

SPECIFICATIONS

Catalog Number

K0551-50

Size

1Kit

References

1. Nuwayhid, N.F., Johnson, G.F. and Feld, R.D. (1988). Kinetic measurement of the combined concentrations of acetoacetate and b-3-hydroxybutyrate in serum. Clin. Chem. 34/9, 1790-1793.\n2. Hansen, J.L. and Freier, E.F. (1978). Direct assays of lactate, pyruvate, b-3-hydroxybutyrate, and acetoacetate with a centrifugal analyzer. Clin. Chem. 24/3. 475-479.\n3. Siegel, L., Robin, N.I. and McDonald, L.J. (1977). New approach to determination of total ketone bodies in serum. Clin. Chem. 23/1, 46-49.