This package insert follows the Pharmaceuticals, Medical devices and Other Therapeutic Products Act of Japan.
In Vitro Diagnostics
Marketing Notification No. 13A2X00197218044**Revised: January 2017 (12th edition)*Revised: September 2016 (11th edition)

This package insert must be read carefully prior to use.
LDL-cholesterol assay kit(Classification No.: 30173000)

standard method of measuring LDL-cholesterol, thismethod is difficult to use for laboratory tests inroutine clinical practice as requires special equipmentand time. The direct method, therefore, is widely
employed in recent years.

Cholestest LDL

Assay Principle
1. Assay Principle
The principle of this method is that eachlipoprotein reacts differently with surfactants
depending on its physicochemical properties.
Therefore, two different surfactants are employed.
Surfactant 1 is added in the first reaction. It canchange the structure of lipoproteins other thanLDL, including chylomicron (CM), very VLDLand HDL. In the presence of this surfactant,lipoproteins other than LDL are eliminated by theaction of cholesterol oxidase and cholesterolesterase. Surfactant 2, which promotes theenzymatic reaction of all types of lipoproteins, isused in the second reaction. The enzymatic
reaction with LDL-cholesterol left from the firstreaction is initiated to produce color development.

General Precautions
1. This product is for in vitro diagnostic use, andmust not be used for any other purposes.
2. Clinicians should make a comprehensive clinicaldecision based on assay results in conjunction withclinical symptoms and other examination results.
3. This product should be used only as directed in thispackage insert. Reliability of results cannot beguaranteed if there are any deviations from theinstructions in this package insert.
4. If reagent accidentally comes in contact with eyes
and/or mouth, rinse immediately with ample water
as first aid, and consult the doctor if required.
5. Carefully read the operating instructions for eachtype of automated analyzers prior to using thisproduct. Parameters for each type of analyzers areavailable, and can be requested from SEKISUIMEDICAL CO., LTD. if required.
6. Perform a quality control test prior to assay toensure accuracy.

First reaction:
HDL, VLDL, CM

Description (Kit Components) ＊Component: Ingredients
Enzyme Solution: 4-Aminoantipyrine
Cholesterol oxidase
Cholesterol esterase
Peroxidase
Coloring Solution: N,N-bis(4-sulfobutyl)-m-toluidine
disodium (DSBmT)

Surfactant 1

Micellar cholesterol

Cholesterol esterase

H2O2

Cholesterol oxidase
H2O2 + 4-Aminoantipyrine
LDL

Surfactant 1

Peroxidase

ColorlessLDL

Second reaction:

Intended Use
Measurement of LDL-cholesterol in serum orplasma

LDL

Surfactant 2
Cholesterol esterase

Micellar cholesterol

H2O2
Cholesterol oxidase

Cholesterol that exists in LDL (Low Density
Lipoprotein) fraction is called LDL-cholesterol.
Elevation of LDL-cholesterol is known to be a risk
factor for arteriosclerotic diseases, especiallycoronary artery disease. Total cholesterol has
commonly been measured for diagnosis of
hyperlipidemia, which is considered to be a cause ofarteriosclerotic diseases. However, according to the“Research Report 1986 on Specified Disease PrimaryHyperlipidemia by Investigation and Research Groupof the Ministry of Health and Welfare”1), theLDL-cholesterol level is more closely correlated withischemic heart disease than the total cholesterol level.
The LDL-cholesterol concentration has beencalculated according to the Friedwald equation basedon measurement of 3 other variables (total cholesterol,HDL-cholesterol, and triglycerides). However, theaccuracy of this method is insufficient. Even thoughultracentrifugation has been also performed as theH2O2 + 4-Aminoantipyrine + DSBmTRed-purple color

Peroxidase

2. Features
1) The direct method does not requirecomplicated procedures
2) Results obtained by this method are closelycorrelated with those by ultracentrifugation.
3) Analytical results are not affected by chyle (upto TG 1500 mg/dL).
Procedural Precautions ＊1. Properties of Samples and Sampling Methods
1) Samples
Serum and plasma may be used.
2) Storage of samples4)
If the isolated serum or plasma sample cannot

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be tested on the same day, specimens should be
stored as follows:
2–10C: for tests within 1 week
≤ -20C: for tests after more than 1 week
Do not refreeze.
Bring samples to room temperature (15–30C)
before use.
2. Interfering substances
Assay results are not affected by free bilirubin (upto 20 mg/dL), conjugated bilirubin (up to 20
mg/dL), hemoglobin (up to 500 mg/dL), ascorbic
acid (up to 50 mg/dL), formazin turbidity (up to2500 FTU), or Intralipos (up to 5%).
3. Others
1) Always use Cholestest N Calibrator orQUALIGENT N Calibrator for calibration.
2) Precautions for assay range
If the concentration of sample exceeds assay
range, dilute the sample with saline and repeat
the measurement.

100 mg/dL of LDL-cholesterol.
Accuracy: 90–110% of the expected assay value
Within-run Reproducibility:
Coefficient of variation ≤ 5%
(Test methods used for 1. –3. are in-housemethods.)
4. Measurement Range4):(On Hitachi 7150
automated analyzer)
1–450 mg/dL
5. Correlation4)
1) Serum N=60 r=0.992 y=0.95x＋3.66
Control method: Ultracentrifugation
2) Plasma
N=76 r=0.998 y=1.01x –4.03
Control method: Comparison with the values
for plasma samples obtained simultaneouslywith the serum samples.
6. Standard Material
Certified Reference Material for Measurement ofHDL Cholesterol, LDL Cholesterol andTriglycerides in Human Serum (Reference2.
3.

Material Institute for Clinical ChemistryStandards)

Dosage/Administration (Assay Procedure) ＊1. Preparation of reagents
Reagent (1): Enzyme Solution is ready to use.
Reagent (2): Coloring Solution is ready to use.
2. Assay Procedure
This product is compatible with various types ofautomated analyzer. An example of the assay
procedure is indicated below.
Sample Reagent (1)+3 μL
300 μL
Reagent (2)100 μL

37C
5 min
37C
5 min

Precautions for Use or Handling ＊1. Precautions for Handling (to Ensure Safety)
1) All samples used in the test should be handled
as a material possibly infected with HIV, HBV,HCV, or other viruses. To prevent infection, use
disposable gloves and avoid mouth pipetting
during the test.
2) Cholestest N Calibrator and QUALIGENT N
Calibrator for LABOSPECT 008 containhuman-derived components that have beenshown to be negative for HBs antigens, HIVantibodies (AIDS virus antibodies), and HCV
antibodies. However, these reagents (as well asthe samples) should be considered potentially
infectious and handled with great care.
3) Proclin 300, which possesses skin-irritative
potential, is added as an antiseptic agent in theEnzyme Solution and Coloring Solution.
Therefore, if the reagent comes in contact withskin or clothes, rinse immediately with ample
water, and consult the doctor if skin irritation
develops.
2. Precautions for use
1) This product should be stored as directed,without freezing. Freezing can deteriorate thereagents, which can produce inaccurate results.
Therefore, avoid using the reagents which havebeen previously frozen.
2) Do not use expired reagents. Use of suchreagents cannot guarantee the reliability ofmeasurement values.
3) Do not replenish the reagents.
4) Do not perform the assay under direct sunlight
3. Precautions for Disposal
1) Before disposal, used samples and theircontainers must be immersed in sodium
hypochlorite solution at a concentration ofgreater than 0.1% for longer than 1 hour or
autoclaved at 121ºC for 20 minutes.
2）To prevent infections from spilled samples orsolutions containing samples, wipe the spilledarea thoroughly with disinfectants such as
sodium hypochlorite solution at a concentration

Measurement(Absorbance I※)
Measurement(Absorbance II※)
Calculation ofconcentration※

Absorbance I and II: The difference in absorbance
between 660 nm and 546 nm.
Calibration material: Cholestest N Calibrator(in-house indicated values) or QUALIGENT N
Calibrator for LABOSPECT 008 (Manufacture'sassigned value)
Reagent blank: Purified water or saline

Assessment of Assay Results ＊＊1. Reference standard range2)
65–163 mg/dL
2. Diagnostic criterion3)
Hyper-LDL cholesterolemia: ≥ 140 mg/dL
3. There may be reactions with non-target substancesin the samples or interfering reactions. Especially
if abnormal lipid metabolism is suspected,
including cases of biliary obstruction, accurate
measurements may not be obtained due to theinfluence of lipoprotein abnormalities. If assay
results appear to be unreliable, repeat themeasurement (if necessary, after dilution) or try
another analytical methods.
Performance
1. Sensitivity
1) Reagent blank: absorbance being equal to or
lower than 0.05
2) Sensitivity: The absorbance is 0.18–0.28 per2

of greater than 0.1%.
3) The reagents and treated samples should bediscarded as medical waste or industrial waste
according to the waste disposal regulations.
4) The reagents should be disposed of inaccordance with the Water Pollution Control act
or related regulations.
4. Other precautions
Do not use the containers for other purposes.
Storage and Shelf Life ＊1. Storage temperature: 2–10C
2. Shelf life: 2 years from the date of manufacture(The expiration date is printed on the outerpackage.)
Packaging
NamePackage
Cholestest (1) Enzyme Solution 2 × 6 0mL
LDL(2) Coloring Solution 2 × 20 mL
Reagents are available in other configurations. For
further details please contact SEKISUI MEDICAL
CO., LTD.
References ＊＊1) Tarui S.: Research Report 1986 on Specified
Disease Primary Hyperlipidemia by Investigation
and Research Group of the Ministry of Health andWelfare (group leader: Seiichi Tarui), 17–26,
1987.
2) Kanai M. (supervising editor): Kanai’s manual ofclinical laboratory medicine. 34th ed. 522,
Kanehara Shuppan, 2015.
3) Japan Atherosclerosis Society, ed. 2012 Japan
Atherosclerosis Society (JAS) Guidelines forPrevention of Atherosclerotic Cardiovascular
Diseases, 13.
4) In house data, SEKISUI MEDICAL CO., LTD.
Contact
SEKISUI MEDICAL CO., LTD.
international@sekisui.com
Manufacturer ＊＊SEKISUI MEDICAL CO., LTD.
1-3, Nihonbashi 2-chome, Chuo-ku, Tokyo, Japan

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