85-1588-67 Lipoprotein, High Density (HDL) and Lipoprotein, Low Density (LDL)/Lipoprotein, Very Low Density (VLDL) BioAssay™ Kit, Colorimetric/Fluorometric 96Tests L2600-57A
85-1588-67 Lipoprotein, High Density (HDL) and Lipoprotein, Low Density (LDL)/Lipoprotein, Very Low Density (VLDL) BioAssay™ Kit, Colorimetric/Fluorometric 96Tests L2600-57A
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. 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 lem/ex=585/530nm is directly proportional to total cholesterol concentration in the sample.
Applications:Direct Assays: HDL and LDL/VLDL cholesterol in serum samples
Pharmacology: evaluation of drugs on cholesterol metabolism.
Key Features:Sensitive and accurate. Linear detection range in 96-well plate: 1 to 100mg/dL cholesterol for colorimetric assays and 0.2 to 10 mg/dL for fluorometric assays
Convenient. Room temperature assay. No 37°C heater is needed.
Kit Contents: (100 assays in 96-well plates)PBS: 2 × 1.5ml Precipitation Reagent: 1.5mlAssay Buffer: 20ml Enzyme Mix: 120uLDye Reagent: 120ul Standard: 1ml 300mg/dL cholesterolStorage conditions. Store PBS and Precipitation Reagent at room temperature and other 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.
Colorimetric Procedures:Important: bring all reagents except enzyme mix to room temperature prior to assay. Non-hemolyzed serum samples should be used
1. Sample Preparation. Transfer 20ul serum into a 1.5-mL centrifuge tube, add 20ul 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 24ul supernatant into a clean tube, add 96ul Assay Buffer. Label this tube “HDL”
Carefully remove all remaining supernatant from the pellet. Transfer 40ul PBS to the pellet and mix by repeated pipetting. Transfer 24ul mixture into another clean tube, add 96ul Assay Buffer. Label this tube “LDL/VLDL”
In a third tube, transfer 12ul serum sample and mix well with 108ul Assay Buffer. Label this tube “Total”. Cholesterol Standard: transfer 5ul 300 mg/dL cholesterol and mix with 145ul Assay Buffer. Label this tube “Standard”
2. Assay. Transfer 50ul Assay Buffer (“Blank”), 50ul Standard, 50ul “Total”, 50ul “HDL” and 50ul “LDL/VLDL” into wells of a clear flat-bottom 96-well plate. If desired, run assays in duplicate
For each reaction well, mix 55ul Assay Buffer with 1ul Enzyme Mix and 1ul Dye Reagent. Add 50ul 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 inassay buffer and repeat the assay. Multiply result by the dilution factor
3. Calculation:. Cholesterol concentrations in the Total, HDL and (LDL/VLDL) fractions are calculated as follows,[Total] =ODTOTAL–ODBLANKODSTANDARD–ODBLANK× 100 (mg/dL)[HDL] =ODHDL–ODBLANKODSTANDARD–ODBLANK× 100 (mg/dL)[LDL/VLDL] =ODLDL/VLDL–ODBLANKODSTANDARD–ODBLANK× 100 (mg/dL)
Fluorometric Procedure:Dilute the Samples and Standard prepared in Colorimetric Procedure: 1:10 in Assay Buffer. Transfer 50ul diluted standards and 50ul diluted samples into separate wells of a black 96-well plate
Add 50ul Working Reagent (see Colorimetric Procedure:). Tap plate to mix. Incubate 30 min at room temperature and read fluorescence at lex=530nm and lem=585nm
Note: if the Sample F is higher than the Standard F, dilute sample in assay buffer and repeat the assay. Multiply result by the dilution factor. The cholesterol concentration of Sample is calculated as[Total] =FTOTAL–FBLANKFSTANDARD–FBLANK× 100 (mg/dL)[HDL] =FHDL–FBLANKFSTANDARD–FBLANK× 100 (mg/dL)[LDL/VLDL] =FLDL/VLDL–FBLANKFSTANDARD–FBLANK× 100 (mg/dL)
Materials Required, But Not Provided:Pipetting devices, 96-well plate and plate reader.
Examples:Serum samples were run in duplicate according to the standard procedure.
