Allomyrina dichotoma Lectin

EnzyChromTM AF Cholesterol Assay Kit (E2CH-100)

Quantitative Colorimetric/Fluorimetric Determination of Cholesterol

DESCRIPTION

CHOLESTEROL is a sterol and lipid present in the cell membranes, and is transported in the bloodstream of all animals. It is used to form cell membranes and hormones, and plays important roles in cell signaling processes. Elevated levels (hypercholesterolemia) have been associated with cardiovascular diseases such as atherosclerosis; whereas, low levels (hypocholesterolemia) may be linked to depression, cancer and cerebral hemorrhage. Simple, direct and automation-ready procedures for measuring cholesterol are very desirable. BioAssay Systems' EnzyChromTM Cholesterol Assay uses 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 λem/ex = 585/530nm is directly proportional to total cholesterol concentration in the sample.

APPLICATIONS

Direct Assays: cholesterol in serum, plasma, and other biological samples.

Pharmacology: effects of drugs on cholesterol metabolism.

KEY FEATURES

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

KIT CONTENTS (100 tests in 96-well plates)

Assay Buffer: 20 mL Enzyme Mix: 120 uL

Dye Reagent: 120 µL Standard: 1 mL 300 mg/dL cholesterol

Storage conditions. Store reagents at -20°C. Shelf life of at least 6 months (see expiry dates on labels).

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.

COLORMETRIC PROCEDURE

Important: bring all reagents to room temperature prior to assay. Serum and plasma samples should be clear and free of turbidity or precipitates. If present, precipitates should be removed by filtration or centrifugation. If not assayed immediately, samples can be stored at 20 to -80°C for at least one year.

1. Standard Curve. Prepare a 10-fold diluted 100 mg/dL standard (STD) by mixing 15 L 300 mg/dL Standard and 435 µL Assay Buffer. Further dilute standard (STD) in Assay Buffer as shown below. Transfer 50 µL diluted standards into wells of a clear 96-well plate.

Samples: dilute samples 10-fold (e.g. 10 µL sample with 90 µL Assay Buffer). Transfer 50 µL diluted sample in separate wells.

2. 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.

3. Incubate 30 min at room temperature. Read OD at 570 nm.

Note: if the Sample OD is higher than the Standard OD at 100 mg/dL, dilute sample in assay buffer and repeat the assay. Multiply result by the dilution factor.

CALCULATION

Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The cholesterol concentration of Sample is calculated as

Note: since both the standards and samples were diluted 10-fold, no dilution factor is required.

FLUORIMETRIC PROCEDURE

For fluorimetric assays, the linear detection range is 0.2 to 10 mg/dL cholesterol. Dilute the Standards prepared in Colorimetric Procedure 1:10 in Assay Buffer. Transfer 50 µL standards and 50 µL samples into separate wells of a black 96-well plate. Add 50 µL Working Reagent (see Colorimetric Procedure). Tap plate to mix. Incubate 30 min at room temperature and read fluorescence at λex = 530nm and λem = 585nm. If assays in 384-well plate are desired, use 5µL Standards / samples and 45 µL Working Reagent. The cholesterol concentration of Sample is calculated as

MATERIALS REQUIRED, BUT NOT PROVIDED

Pipetting (multi-channel) devices, 96-well plate and plate reader.

LITERATURE

[1]. Kayamori, Y. et al (1999) Endpoint Colorimetric Method for Assaying Total Cholesterol in Serum with Cholesterol Dehydrogenase. Clin. Chem. 45: 2158-2163.

[2]. Sundvall J, Leiviska J, Alfthan G, Vartiainen E. (2007). Serum cholesterol during 27 years: assessment of systematic error and affecting factors and their role in interpreting population trends. Clin Chim Acta. 378:93-98.

[3]. Demacker PN, Hijmans AG, van Sommeren-Zondag DF, Jansen AP. (1982). Stability of frozen liquid control sera for assay of cholesterol in high-density lipoprotein. Clin Chem. 28:155-157.

EnzyChromTM Cholesterol Assay Kit (ECCH-100)

Quantitative Colorimetric Determination of Cholesterol at 340 nm

DESCRIPTION

CHOLESTEROL is a sterol and lipid present in the cell membranes, and is transported in the bloodstream of all animals. It is used to form cell membranes and hormones, and plays important roles in cell signaling processes. Elevated levels (hypercholesterolemia) have been associated with cardiovascular diseases such as atherosclerosis; whereas, low levels (hypocholesterolemia) may be linked to depression, cancer and cerebral hemorrhage. Simple, direct and automation-ready procedures for measuring cholesterol are very desirable. BioAssay Systems' EnzyChromTM Cholesterol Assay is based on cholesterol esterase hydrolysis of cholesterol esters to form free cholesterol and cholesterol dehydrogenase catalyzed conversion of cholesterol to cholest-4-ene- 3-one, in which 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.

APPLICATIONS

Direct Assays: cholesterol in serum, plasma, and other biological samples.

Pharmacology: effects of drugs on cholesterol metabolism.

KEY FEATURES

Sensitive and accurate. 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.

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

KIT CONTENTS (100 tests in 96-well plates)

Assay Buffer: 20 mL Enzyme Mix: 120 uL

NAD Solution: 2 x 1 mL Standard: 1 mL 300mg/dL cholesterol

Storage conditions. Store reagents at -20°C. Shelf life of at least 6 months (see expiry dates on labels).

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

Important: bring all reagents to room temperature prior to assay. Serum and plasma samples should be clear and free of turbidity or precipitates. If present, precipitates should be removed by filtration or centrifugation in a table centrifuge. If not assayed immediately, samples can be stored at -20 to -80°C for at least one year.

1. Standard Curve. Prepare a 10-fold diluted standard (STD) by mixing 40 μL 300 mg/dL Standard and 360 μL Assay Buffer. Further dilute standard (STD) in Assay Buffer as shown below.

Transfer 50 μL diluted standards into wells of the 96-well plate.

Samples: dilute samples 10-fold (e.g. 10 μL sample with 90 μL Assay Buffer). Transfer 50 μL diluted sample in separate wells.

2. Prepare enough NAD solution in Assay Buffer as follows: for each reaction well, mix 40 μL Assay Buffer with 18 μL the provided NAD Solution. Add 50 μL of diluted NAD to standards and sample wells. Tap plate to mix well. Let stand 5 min at room temperature. Read background optical density at 340nm (ODo).

3. Prepare enough enzyme mix as follows: for each reaction well, mix 10 μL Assay Buffer with 1 μL provided Enzyme Mix. Add 10 μL diluted enzyme mix per well. Tap plate to mix thoroughly. Note: the enzyme mix may appear to be turbid, but will be clear after mixing into the reaction mixture.

4. Incubate 30 min at room temperature. Read OD30 at 340nm.

5. Calculation. Subtract OD0 from OD30 for the standard and sample wells. Use the DOD values to determine sample cholesterol concentration from the standard curve. Note: since both the

standards and samples were diluted 10-fold, no dilution factor is required.

Note: if the sample OD value is higher than OD for the 300 mg/dL standard, dilute sample in water and repeat the assay. Multiply the results by the dilution factor.

MATERIALS REQUIRED, BUT NOT PROVIDED

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

EXAMPLES

Samples were run in duplicate according to the standard procedure. The cholesterol concentrations (mg/dL) were 105 ± 3 for a human serum, 155 ± 11 for human plasma, 157 ± 2 for a bovine serum, 68 ± 2 for a rat serum, 129 ± 3 for a mouse serum, 123 ± 2 for a goat serum sample.

PUBLICATIONS

[1]. Lee, S.M. et al (2008). GCG-Rich Tea Catechins are Effective in Lowering Cholesterol and Triglyceride Concentrations in Hyperlipidemic Rats. Lipids 43: 419-429.

[2]. Khan, M.A. et al (2009). Statins impair CD1d-mediated antigen presentation through the inhibition of prenylation. J Immunol 182(8): 4744-4750.

[3]. Mellado, M. et al (2008). Rough agave flowers as a potential feed resource for growing goats. Rangeland Ecol Manage 61: 640- 646.

EnzyChromTM AF HDL and LDL/VLDL Assay Kit (E2HL-100)

Quantitative Colorimetric/Fluorimetric Determination of HDL and LDL/VLDL

DESCRIPTION

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. BioAssay Systems' 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 λem/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 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.

KIT CONTENTS (100 assays in 96-well plates)

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

Storage conditions. Store PBS and Precipitation Reagent at room temperature and the rest reagents at -20°C. Shelf life of at least 6 months (see expiry dates on labels).

COLORMETRIC 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 20 µL serum into a 1.5-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”.

2. 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 55µL 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.

3. Calculation. Cholesterol concentrations in the Total, HDL and (LDL/VLDL) fractions are calculated as follows,

FLUORIMETRIC PROCEDURE

Dilute the Samples and Standard prepared in Colorimetric Procedure 1:10 in Assay Buffer. Transfer 50 µL diluted standards and 50 µL diluted samples into separate wells of a black 96-well plate. Add 50 µL Working Reagent (see Colorimetric Procedure). Tap plate to mix. Incubate 30 min at room temperature and read fluorescence at λex = 530nm and λem = 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

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.

LITERATURE

[1]. Viikari J. (1976). Precipitation of plasma lipoproteins by PEG 6000 and its evaluation with electrophoresis and ultracentrifugation. Scand J Clin Lab Invest 36:265-268.

[2]. Demacker, PMN et al. (1980). A study of the use of polyethylene glycol in estimating cholesterol in high density lipoprotein. Clin Chem 26:1775-1779.

[3]. Widhaim, K. and Pakosta, R. (1991). Precipitation with Polyethylene Glycol and Density-Gradient Ultracentrifugation Compared for Determining High-Density Lipoprotein Subclasses HDL2 and HDL3. Clin. Chem 37/2, 238-240.

EnzyChromTM HDL and LDL/VLDL Assay Kit (EHDL-100)

Quantitative Colorimetric Determination of HDL and LDL/VLDL Cholesterol

DESCRIPTION

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. BioAssay Systems' 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.

APPLICATIONS

Direct Assays: HDL and LDL/VLDL cholesterol in serum samples from any species.

Pharmacology: evaluation of drugs on cholesterol metabolism.

KEY FEATURES

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.

KIT CONTENTS (100 assays in 96-well plates)

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

Storage conditions. Store PBS and Precipitation Reagent at room temperature and the rest reagents at -20°C. Shelf life of at least 6 months (see expiry dates on labels).

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

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 20 μL serum into a 1.5-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”.

2. 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 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 multichannel pipettor is recommended.

Incubate 30 min at room temperature. Read OD values at 340nm.

3. Calculation. Cholesterol concentrations in the Total, HDL and (LDL/VLDL) fractions are calculated as follows,

MATERIALS REQUIRED, BUT NOT PROVIDED

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

EXAMPLES

Serum samples were run in duplicate according to the standard procedure.

LITERATURE

[1]. Viikari J. (1976). Precipitation of plasma lipoproteins by PEG 6000 and its evaluation with electrophoresis and ultracentrifugation. Scand J Clin Lab Invest 36:265-268.

[2]. Demacker PMN, Humans AGM, Vos-Janssen HE, van’t Laar A, Jansen AP. (1980). A study of the use of polyethylene glycol in estimating cholesterol in high density lipoprotein. Clin Chem 26:1775- 1779.