AF HDL and LDL/VLDL Assay Kit

Produktnummer ABIN1000283

Produktdetails

Target: AF HDL, LDL/VLDL

Applikation: Biochemical Assay (BCA)

Proben: Serum

Produktmerkmale: Sensitive and accurate. Linear detection range in 96-well plate: 1 to 100 mg/dL cholesterol for colorimetric assays and 0.2 to 10 mg/dL for fluorimetric assays.
Convenient. Room temperature assay. No 37°C heater is needed.

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

Benötigtes Material: Pipetting devices, 96-well plate and plate reader.

Anwendungsinformationen

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

Protokoll: Assay: Transfer 50 µL Assay Buffer (Blank), 50 µL Standard, 50 µL Total, 50µL HDL and 50 µL LDL/VLDL into wells of a clear flat-bottom 96-well plate. If desired, run assays in duplicate. For each reaction well, mix 55 µL Assay Buffer with 1 µL Enzyme Mix and 1 µL Dye Reagent. Add 50 µL of this Working Reagent to each standard and sample well. Tap plate to mix well. Incubate 30 min at room temperature. Read OD values at 570 nm. Note: if the Sample OD is higher than the Standard OD, dilute sample in assay buffer and repeat the assay. Multiply result by the dilution factor.

Aufbereitung der Reagenzien:

Important: bring all reagents except enzyme mix to room temperature prior to assay. Non-hemolyzed serum samples should be used.

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 5 µL 300 mg/dL cholesterol and mix with 145 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).

Nagajyothi, Weiss, Silver, Desruisseaux, Scherer, Herz, Tanowitz: "Trypanosoma cruzi utilizes the host low density lipoprotein receptor in invasion." in: PLoS neglected tropical diseases, Vol. 5, Issue 2, pp. e953, (2011) (PubMed).

Wan, Lim, Lionakis, Rivollier, McDermott, Kelsall, Farber, Murphy: "Genetic deletion of chemokine receptor Ccr6 decreases atherogenesis in ApoE-deficient mice." in: Circulation research, Vol. 109, Issue 4, pp. 374-81, (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).

Takakuwa, Kurokawa, Ooka, Sato, Nagai, Arito, Suematsu, Okamoto, Nagafuchi, Yamada, Ozaki, Kato: "AC13, a C-terminal fragment of apolipoprotein A-I, is a candidate biomarker for microscopic polyangiitis." in: Arthritis and rheumatism, Vol. 63, Issue 11, pp. 3613-24, (2011) (PubMed).

Tucci, Primassin, Ter Veld, Spiekerkoetter: "Medium-chain triglycerides impair lipid metabolism and induce hepatic steatosis in very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient mice." in: Molecular genetics and metabolism, Vol. 101, Issue 1, pp. 40-7, (2010) (PubMed).

Tam, Vemuri, Liu, Bátkai, Mukhopadhyay, Godlewski, Osei-Hyiaman, Ohnuma, Ambudkar, Pickel, Makriyannis, Kunos: "Peripheral CB1 cannabinoid receptor blockade improves cardiometabolic risk in mouse models of obesity." in: The Journal of clinical investigation, Vol. 120, Issue 8, pp. 2953-66, (2010) (PubMed).

Antigendetails

Target: AF HDL, LDL/VLDL

Hintergrund: Quantitative determination of HDL and LDL/VLDL cholesterol by colorimetric (570nm) or fluorimetric (530nm/590nm) methods.
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 a single Working Reagent that combines cholesterol ester hydrolysis, oxidation and color reaction in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at gamma em/ex = 585/530nm is directly proportional to total cholesterol concentration in the sample.