Cholesterol Assay Kits

In biochemical tests, total cholesterol, HDL-cholesterol, and LDL-cholesterol in the blood are the main objects of measurement. Abnormalities in cholesterol synthesis in the liver, lipid metabolism, and nutritional status are screened by measuring cholesterol. It is also a useful indicator for assessing the risk of atherosclerosis.

Fujifilm Wako’s LabAssay™ Cholesterol is a kit designed to determine total cholesterol using the cholesterol oxidase-DAOS method. It is intended for use with a microplate, facilitating quick and convenient measurements of total cholesterol in samples. For specific measurements of HDL-cholesterol or LDL-cholesterol, an effective method involves pretreatment with blocking polymers to protect the targeted cholesterol and eliminate the other types. Fujifilm Wako’s LabAssay™ HDL-Cholesterol and LabAssay™ LDL-Cholesterol kits include a pretreatment solution containing a blocking polymer. In addition, our kits do not require fractionation of lipoproteins by centrifugation.

Principle of the total cholesterol assay (cholesterol oxidase-DAOS method)

Cholesterol in the blood mainly exists in the forms of esterified cholesterol (cholesterol esters) and free cholesterol. When measuring total cholesterol, it is necessary to break down the cholesterol esters into free cholesterol using cholesterol esterase²⁾.

When a chromogenic reagent is added to the sample, the cholesterol esters in the sample are broken down into free cholesterol and fatty acids due to the action of cholesterol esterase. The produced cholesterol, along with the preexisting free cholesterol, is oxidized by the action of cholesterol oxidase and produces hydrogen peroxide in the process. The resulting hydrogen peroxide induces quantitative oxidative condensation of N-ethyl-N(-2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium) (DAOS) and 4-aminoantipyrine through the action of peroxidase (POD), producing a blue dye. By measuring the absorbance of this blue color, the total cholesterol concentration in the sample can be determined.

Principle of the HDL-cholesterol assay

To specifically measure HDL cholesterol, a two-step process is required. This process involves the elimination of non-HDL-cholesterol, followed by a colorimetric reaction to detect HDL-cholesterol.

1) The first reaction（elimination of non-HDL cholesterol）

The block polymer containing hydrophilic and hydrophobic moiety in Pretreatment selectively binds to HDL in the specimens, and protects it from enzyme (CHE, CO) reactions. CHE and CO react with non-HDL lipoprotein [chylomicron (CM), very low density lipoprotein (VLDL), low density lipoprotein (LDL)]. Hydrogen peroxide produced by the enzyme reactions with non-HDL cholesterol is decomposed to water by catalase in Pretreatment reagent.

2) The second reaction（color reaction of HDL-cholesterol)

When Reacting Solution is added, the cholesterol and its derivatives in HDL produce hydrogen peroxide by CHE and CO. Hydrogen peroxide produced by the enzyme reactions with HDL-cholesterol yields a blue color pigment upon oxidative condensation with N-(3-sulfopropyl)-3-methoxy-5-methylaniline (HMMPS) and 4-aminoantipyrine in the presence of peroxidase (POD). The amount of HDL-cholesterol contained in the sample is determined by measuring the absorbance of the blue color.

Principle of the LDL-cholesterol assay

As well as HDL-cholesterol, a two-step process is required.

1) The first reaction（elimination of non-LDL cholesterol）

The block polymer containing hydrophilic and hydrophobic moiety in Pretreatment selectively binds to LDL in the specimens, and protects it from enzyme (CHE, CO) reactions. CHE and CO react with non-LDL lipoprotein [chylomicron (CM), very low density lipoprotein (VLDL), high density lipoprotein (HDL)]. Hydrogen peroxide produced by the enzyme reactions with non-LDL cholesterol is decomposed to water by catalase in Pretreatment reagent.

2) The second reaction（color reaction of LDL-cholesterol)

When Reacting Solution is added, the cholesterol and its derivatives in LDL produce hydrogen peroxide by CHE and CO. Hydrogen peroxide produced by the enzyme reactions with LDL-cholesterol yields a blue color pigment upon oxidative condensation with N-(3-sulfopropyl)-3-methoxy-5-methylaniline (HMMPS) and 4-aminoantipyrine in the presence of peroxidase (POD). The amount of LDL-cholesterol contained in the sample is determined by measuring the absorbance of the blue color.

LabAssay™ Cholesterol comes with all the necessary reagents and samples for cholesterol measurement, and can be readily used as a training kit. Students can acquire measurement skills and deepen their knowledge of the assay principle and related subjects through the measurement of control samples.

Why it is ideal for educational training

• Measurement can be completed in a short time (about 10 minutes).
• Samples can be prepared just by dissolving and mixing.
• The kit capacity is 500 assays, making it possible to conduct measurements with a large number of participants.

What can be learned with this kit

Kit Operation Procedures (for Educational Training)

Sample Preparation (for Calibration Curve Creation)

Mix the standard solution and purified water according to the table below to prepare samples.

Sample No.	For sample preparation		For measurement
	Standard solution	Purified Water	Amount of use	Concentration
1	5 μL	15 μL	4 μL	50 mg/dL
2	10 μL	10 μL	4 μL	100 mg/dL
3	Undiluted	-	4 μL	200 mg/dL
4	Undiluted	-	8 μL	392 mg/dL*

*Concentration is corrected because the amount of liquid increases during measurement

Measurement

1. Add each sample to the microplate according to the table 2 (2 wells for each).
2. Add 200 μL of the chromogenic reagent to the wells containing the samples.
3. Mix well using a shaker and allow it to stand at 37℃ for 5 minutes.
4. Using a microplate reader, measure the absorbance with the blank as a control (primary wavelength 600 nm / reference wavelength 700 nm).

Table 2. Amount of sample and chromogenic reagent to be used

Name	Sample	Chromogenic reagent
Unknown sample X	4 μL	200 μL each
Unknown sample Y	4 μL
Sample 1	4 μL
Sample 2	4 μL
Sample 3	4 μL
Sample 4	8 μL
Blank	4 μL

Data Analysis

Example of Data Recording Sheet

Record the measurement results (absorbance) on the sheet below.
Correct the data using the average value of the blank (in the case of n=2).

Example of data analysis

1. Based on the absorbance of the blank and samples 1-4, create a calibration curve as shown in figure below.
2. Calculate the cholesterol concentration of unknown samples X and Y based on the regression equation of the calibration curve.

1. Yamada, K.: Journal of Lipid Nutrition, 14(1), 27(2005).
   Cholesterol metabolism in the human body (Japanese)
2. Japan Atherosclerosis Society: https://www.j-athero.org/jp/general/5_colqa/ (Apr. 25th, 2023) (Japanese)
3. Allain, C. C. et al.: Clin. Chem., 20(4), 470(1974).
   Enzymatic determination of total serum cholesterol.