EnzyLight™ HDL and LDL/VLDL Assay Kit

Produktnummer ABIN1000315

Produktdetails

Target: HDL/LDL/VLDL

Applikation: Biochemical Assay (BCA)

Proben: Serum

Spezifität: 5 mg/dL

Produktmerkmale: Sensitive and accurate. Requires only 20 µL serum sample. Detection limit of 5 mg/dL, linearity up to 300 mg/dL cholesterol in 96- well plate assay.
Convenient. Room temperature assay. No 37°C heater is needed.

Bestandteile: PBS: 1.5 mL. Precipitation Reagent: 1.5 mL. Assay Buffer: 20 mL. Enzyme Mix: 120 uL. NAD Solution: 2 mL. Standard: 1 mL 300mg/dL cholesterol.

Benötigtes Material: Pipetting (multi-channel) devices, clear bottom 96-well plate and plate reader.

Anwendungsinformationen

Applikationshinweise: Direct Assays: HDL and LDL/VLDL cholesterol in serum samples from any species.
Pharmacology: evaluation of drugs on cholesterol metabolism.

Protokoll: Important: bring all reagents except enzyme mix to room temperature prior to assay. Non-hemolyzed serum samples should be used. The following procedure is designed for duplicate determinations.
1. Sample Preparation.
Transfer 50 µL Assay Buffer (Blank), 50 µL Standard, 50 µL Total, 50µL HDL and 50 µL LDL/VLDL into wells of a clear bottom 96-well plate. If desired, run assays in duplicate. Prepare enough Working Reagent. For each reaction well, mix 50 µL Assay Buffer, 18 µL NAD Solution and 1 µL Enzyme Mix. Transfer 60 µL of the Working Reagent to each reaction well. Tap plate to mix well. Note: addition of Working Reagent to all wells should be rapid and mixing should be thorough. Use of a multi- channel pipettor is recommended. Incubate 30 min at room temperature. Read OD values at 340nm.

Aufbereitung der Proben:

Transfer 20 µL serum into a1.5-mL centrifuge tube, add 20 µL Precipitation Reagent. Vortex to mix and centrifuge 5 min at 9,500 x g (e.g. 9,500 rpm in an Eppendorf 5415C tabletop centrifuge). Carefully transfer 24 µL supernatant into a clean tube, add 96 µL Assay Buffer. Label this tube HDL. Carefully remove all remaining supernatant from the pellet. Transfer 40 µL PBS to the pellet and mix by repeated pipetting. Transfer 24 µL mixture into another clean tube, add 96 µL Assay Buffer. Label this tube LDL/VLDL. In a third tube, transfer 12 µL serum sample and mix well with 108 µL Assay Buffer. Label this tube Total. Cholesterol Standard: transfer 12 µL 300 mg/dL cholesterol and mix with 108 µL Assay Buffer. Label this tube Standard.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C

Referenzen

Bourdon, Halappanavar, Saber, Jacobsen, Williams, Wallin, Vogel, Yauk: "Hepatic and pulmonary toxicogenomic profiles in mice intratracheally instilled with carbon black nanoparticles reveal pulmonary inflammation, acute phase response, and alterations in lipid homeostasis." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 127, Issue 2, pp. 474-84, (2012) (PubMed).

Shanmugasundaram, Selvaraj: "Dietary lutein and fish oil interact to alter atherosclerotic lesions in a Japanese quail model of atherosclerosis." in: Journal of animal physiology and animal nutrition, Vol. 95, Issue 6, pp. 762-70, (2011) (PubMed).

Shon, Park, Nahrendorf, Schellingerhout, Kim, Kang, Jeong, Kim, Ryu, Kim, Kwon, Lee, Lee, Kim: "Exercise attenuates matrix metalloproteinase activity in preexisting atherosclerotic plaque." in: Atherosclerosis, Vol. 216, Issue 1, pp. 67-73, (2011) (PubMed).

Klusoňová, Pátková, Ergang, Mikšík, Zicha, Kuneš, Pácha: "Local metabolism of glucocorticoids in Prague hereditary hypertriglyceridemic rats--effect of hypertriglyceridemia and gender." in: Steroids, Vol. 76, Issue 12, pp. 1252-9, (2011) (PubMed).

Uddin, Duy, Cinar, Tesfaye, Tholen, Juengst, Looft, Schellander: "Detection of quantitative trait loci affecting serum cholesterol, LDL, HDL, and triglyceride in pigs." in: BMC genetics, Vol. 12, pp. 62, (2011) (PubMed).

Ponda, Barash, Feig, Fisher, Skolnik: "Moderate kidney disease inhibits atherosclerosis regression." in: Atherosclerosis, Vol. 210, Issue 1, pp. 57-62, (2010) (PubMed).

Oliver, Rosa, Milne, Pontello, Borntrager, Heydari, Galassetti: "Increased oxidative stress and altered substrate metabolism in obese children." in: International journal of pediatric obesity : IJPO : an official journal of the International Association for the Study of Obesity, Vol. 5, Issue 5, pp. 436-44, (2010) (PubMed).

Antigendetails

Target: HDL/LDL/VLDL

Hintergrund: Quantitative determination of HDL and LDL/VLDL cholesterol at 340nm.
Procedure: 60 min.

Cholesterol concentrations in High-Density Lipoprotein (HDL) and Low-Density (LDL)/Very-Low-Density (VLDL) Lipoproteins are strong predictors for coronary heart disease. Functional HDL offers protection by removing cholesterol from cells and atheroma. Higher concentrations of LDL and lower concentrations of functional HDL are strongly associated with cardiovascular disease due to higher risk of atherosclerosis. The balances between high- and low-density lipoproteins are solely genetically determined, but can be changed by medications, food choices and other factors. Simple, direct and automation-ready procedures for measuring HDL and LDL/VLDL concentrations are very desirable. This HDL and LDL/VLDL quantification kit is based on our improved PEG precipitation method in which HDL and LDL/VLDL are separated, and cholesterol concentrations are determined using cholesterol esterase/cholesterol dehydrogenase reagent. In this reaction, NAD is reduced to NADH. The optical density of the formed NADH at 340 nm is directly proportionate to the cholesterol concentration in the sample.