This package insert follows the Pharmaceuticals, Medical devices and Other Therapeutic Products Act of Japan.
In Vitro Diagnostics
Marketing Approval No. 21900AMZ00070000*Revised: January 2017 (2nd edition)
Prepared: April 2008 (1st edition)

This package insert must be read carefully prior to use.
MDA-LDL (oxidized LDL) kit(Classification No.: 83018000)(A diagnostic aid for predicting the risk ofcomplications of coronary artery disease in patientswith diabetes who have a history of coronary arterydisease.)

Oxidized LDL ELISA“Daiichi”

Assay Principle
1. Assay Principle
Serum is processed with Sample Dilution
Solution containing a surfactant to alter the
structure of MDA−LDL. MDA-LDL in a sample
binds to anti-MDA-LDL mouse monoclonal
antibody (ML25) immobilized on the plate.
Subsequently, β-galactosidase-labeled anti-apo B
mouse monoclonal antibody (AB16) is added toform immune complexes consisting of theimmobilized
antibody,
MDA-LDL,andenzyme-labelled
antibody.
ThenO-nitrophenyl-β-D-galactopyranoside is added fordevelopment of color and the absorbance of thesample is measured to determine the MDA−LDL
level.
2. Features
1) Thisproduct
measuresmalondialdehyde-modified LDL (MDA-LDL),
which is a representative form of oxidized
LDL.
2) ML25 is an antibody that recognizes
MDA-modified
proteins,
while
AB16
recognizes MDA-LDL and LDL combinedwith apoB. An enzyme-linked immunosorbent
assay (ELISA) combining both antibodies isused to specifically measure MDA-LDL.

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. For the effects of an administered drug on themeasured value, carefully read the Precautions for
Use in the package insert of the drug, especiallythe section about the effects on laboratory test
results.
4. This product should be used only as directed in thispackage insert. Reliability of results cannot be
guaranteed if there are any deviations from the
instructions in this package insert.
5. If the reagent accidentally comes in contact witheyes and/or mouth, rinse immediately with ample
water as first aid, and consult the doctor if
required.
6. The calibrator, one of the components of thisproduct, contains human-derived ingredients thathave been confirmed to be negative for HBs
antigens, HIV antibodies, and HCV antibodies.
The calibrator should be considered potentially
infectious and handled with great care in the samemanner as the samples.
7. Perform a quality control test prior to assay toensure accuracy.

Procedural Precautions *1. Properties of Samples and Sampling Methods
1) Samples(1) Always use serum. Do not use plasma.
(2) Serum should be isolated after the blood iscompletely clotted. Serum should be
isolated within 6 hours after bloodcollection.
2) Storage of samples(1)Fresh samples
Measure the sample within 8 hours after
blood collection if it is stored at 15–25°C
and within 3 days if it is stored at 2–8°C.
(2) Stored samples
In order to store a sample, either of thefollowing procedures should be performed
within 8 hours after blood collection.
(1) Store the sample after adding Sample
Preservation Solution. Dilute thesample with Sample Preservation
Solution at a 3:1 ratio (e.g., 300 μL of
the sample and 100 μL of Sample
Preservation Solution), and then freeze
the diluted sample for storage. Measure
the sample within 5 months if it isstored at –80°C and within 1 month if

Description (Kit Components)
Component
Antibody-Coated Plate

Enzyme-Labelled
Antibody Solution

Wash Buffer Concentrate
Substrate Solution
Stop solution
Sample Dilution Solution
Sample Preservation
Solution
Calibrator

IngredientWells coated with
anti-MDA-LDL mousemonoclonal antibody
β-galactosidase-labelled
anti-apolipoprotein B(apo B) mousemonoclonal antibody
Phosphate Buffer
Solutiono-Nitrophenyl-β-D-galactopyranoside
Sodium carbonate
solution
HEPES Buffer Solution
Saccharose
Human serum

Intended Use
Measurement of MDA−LDL (oxidized LDL) in
serum

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it is stored at –20°C.
(2) Freeze the undiluted sample at –20°C or
below
for
storage.
Avoid
thawing/refreezing during storage.
When the sample is stored at –20°C, thefollowing procedures should be
performed within 1 week. After
thawing, dilute the sample with Sample
Preservation Solution at a 3:1 ratio (e.g.,
300 μL of the sample and 100 μL of
Sample Preservation Solution) before
measurement. When the sample isrefrozenforstorage,perform
measurement within 5 months if it isstored at –80°C and within 1 month if itis stored at –20°C.
(3) Diluted samples should be brought 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), or
formazin turbidity (up to 3000 FTU).
3. Others
1) Prepare a new calibration curve for every
assay.
2) When measuring multiple samples, be sure to
maintain the specified constant reaction timefor each well.
3) Completely remove the Wash Buffer Solution
after the washing step.
4) Do not perform the assay under direct
sunlight.
5) Place the sample, the diluted calibrator
solution, and each reagent in the center of eachwell while avoiding contact with the walls.
6) If the MDA-LDL concentration in the sample
exceeds the measurement range, dilute thesample with a Sample Dilution Solution andperform re-measurement.

water to the calibrator to make the calibrator
solution. Dilute the solution with the dilution
solution according to the following example
and let it stand for 1 hour at room temperature(15–30°C) before use.
Example:(Indicated value for calibrator a U/L)
Concentration (U/L)
4a 2a
Dilution factor (times)
1 10 500 1000
Calibrator solution (μL)
q.s. 100 20 500
Sample Dilution Solution (μL)
0 900 980 500
Concentration (U/L)
Concentration (U/L)
10-fold calibrator solution (μL)
Sample Dilution Solution (μL)

a
1/2a
1/4a 1/8a2000 4000 8000 16000
500
500 500 500
500 500 500 500

8a
4a
250-fold 500-fold
40
500
960
500

※

As a reagent blank, use unmodified Sample Dilution
Solution together with diluted calibrator solution
prepared according to the above example.
※When the indicated value for the calibrator is lessthan 83 U/L, prepare a calibration curve startingfrom a 250-fold dilution that includes the upper
limit of measurement (330 U/L) in the calibration
range.
3. Assay Procedure
Preparation of reagents
1) Fresh samples
To make a sample, take serum and dilute itwith Sample Dilution Solution to a final
dilution ratio of 1:2000 according to thefollowing steps. Let it stand for 1 hour at room
temperature (15–30°C) before use.
(1) Dilute the sample 50-fold by adding 980µL of Sample Dilution Solution to 20 µLof the sample.
(2) Dilute the 50-fold diluted sample preparedin Step (1) by a further 40-fold by adding
780 µL of Sample Dilution Solution to 20µL of the 50-fold diluted sample.
(3) Let it stand for 1 hour at room temperature(15–30°C) and use as the sample.

Dosage/Administration (Assay Procedure) ＊1. Instruments and reagents required formeasurement
1) Variable micropipettes (20, 200, and 1000 μL)
2) Test tube
3) Measuring cylinder (1000 mL)
4) Plate seal or plate cover5) Microplate reader (dominant wavelength of
415 nm and complementary wavelength of
600 nm, or single wavelength of 415 nm )6) Purified water
2. Preparation of reagents
1) Antibody-Coated Plate: Ready to use Unused
wells are stable at 2–10°C for 2 weeks whenstored in an air-tight container.
2) Enzyme-Labelled Antibody Solution: Ready touse3) Wash Buffer: Add 400 mL of purified water to
100 mL of Wash Buffer Concentrate beforeuse.
4) Substrate Solution: Ready to use5) Stop Solution: Ready to use6) Sample Dilution Solution: Ready to use
7) Sample Preservation Solution: Ready to use8) Calibrator Solution: Add 0.5 mL of purified

Sample dilution
Solution
Sample(50-fold
dilution)(40-fold
dilution)(Final dilution
ratio 1:2000)

980 μL780 μL

20 μL

20 μL

Let it stand for
1 hour at room
temperature(15–30°C) and
use as the
sample.

2) Stored samples
Before use, stored samples to which Sample
Preservation Solution was added should bediluted 1500-fold with Sample Dilution
Solution to achieve a final dilution ratio of
1:2000 according to the following steps. Let it
stand for 1 hour at room temperature (15–30°C) before use.
(1) Dilute the stored sample 50-fold by adding
980 µL of Sample Dilution Solution to 20µL of the stored sample.
(2) Dilute the 50-fold diluted stored sample
prepared in Step (1) by a further 30-fold byadding 580 µL of Sample Dilution
Solution to 20 µL of the 50-fold diluted

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stored sample.
(3) Let it stand for 1 hour at room temperature(15–30°C) and use as the sample.
(30-fold
dilution)(Final dilution
ratio 1:2000)

980 μL580 μL

20 μL

20 μL

Let it stand for 1
hour at room
temperature (15–30°C) and use as
the sample.

Absorbance (415/600 nm)

Sample Dilution
Solution
Sample(50-fold
dilution)

Example of a calibration curve

MDA-LDL concentration

5. Procedural precautions
1) All reagents should be brought to room
temperature (15–30°C) before use. If insoluble
matter has formed in a refrigerated Sample
Dilution Solution, ensure complete dissolution
before use.
(2) Add exactly 0.5 mL of purified water to 1 vialof the calibrator, fit the inner stopper, mix
gently, and let stand for 10 minutes at room
temperature (15–30°C). After confirming thatthe contents of the vial have been completely
dissolved, gently mix the solution by inversion
before use. At this time, avoid vigorous
mixing.
3) Dilute the sample with the Sample Dilution
Solution through gentle mixing by inversion.
Do not mix vigorously with a vortex mixer.
4) When the antibody-coated plate is let stand formeasurement, cover it with a plate seal or plate
cover to avoid evaporation of the Reactant
Solution.

Measurement(1) All reagents should be brought to room
temperature (15–30°C) before use.
(2) Dispense 300 µL of the Wash Buffer into eachwell of the antibody-coated plate and then discard
the buffer. Repeat this step three times to wash thewells. Remove the Wash Buffer completely, andthen follow the steps below.
(3) Dispense 100 µL of the sample or diluted
calibrator solution into each well and then let theplate stand for 2 hours at room temperature (15–30°C). Make sure that the difference in reaction
time among the wells does not exceed 10 minutes.
Duplicate assays are required for diluted
calibrator solution.
(4) Remove the solution from the wells, and dispense
300 μL of Wash Buffer into each well and wash
three times as in Step (2).
(5) Remove the Wash Buffer, dispense 100 µL of
Enzyme-Labeled Antibody Solution into eachwell, and then let the plate stand for 1 hour atroom temperature (15–30°C).
(6) Remove the solution from the wells, and dispense
300 μL of Wash Buffer into each well and wash
three times as in Step (2).
(7) Remove the Wash Buffer, dispense 100 μL of
Substrate Solution into each well, and then let theplate stand for 2 hours at room temperature (15–30°C).
(8) Dispense 100 μL of Stop Solution to terminate thereaction and maintain a constant response time
among the wells.
(9) Read the absorbance at a dominant wavelength of
415 nm and a complementary wavelength of 600
nm, or only at the dominant wavelength of 415
nm, using a microplate reader with a reagent
blank as the control. Sample Dilution Solution isused as the reagent blank.
4. Method of calculating the MDA-LDL
concentration
1) Create a graph with the concentration of
MDA−LDL on the X-axis and the absorbanceon the Y-axis. Then plot the parameters for the
diluted calibrator solutions and fit a smooth
curve to the data points to obtain thecalibration curve.
2) Read the concentration corresponding to themeasured absorbance of the sample on thecalibration curve to obtain the concentration of
MDA-LDL.

Assessment of Assay Results ＊1. Reference standard range10)
Men aged < 45 years or women aged <55 years:
64 ± 18 U/L (mean ± SD) (N = 134)
Men aged ≥ 45 years or women aged ≥ 55 years:
83 ± 22 U/L (mean ± SD) (N = 122)
2. Precautions for Assessment
There may be reactions or interfering reactions
with non-target substances. If assay results appear
to be unreliable, repeat the measurement (ifnecessary, after dilution) or try another analyticalmethods.
Clinical Significance10) ＊Oxidized LDL is a general term for LDL that has
undergone oxidative degradation. Lipid peroxidation
products modify apo B, the main protein in LDL,
resulting in oxidized LDL. Malondialdehyde (MDA)
has been identified as a representative lipid
peroxidation product, and LDL in which apo B has
been
modifiedbyMDA
iscalledmalondialdehyde-modified
LDL(MDA−LDL).
MDA-LDL is considered to be a typical form ofoxidized LDL.
In patients with diabetes mellitus undergoing
percutaneous coronary intervention (PCI), therelationship between the pre-procedural MDA-LDL
level and restenosis after PCI was investigated.
MDA-LDL levels were higher in patients withrestenosis than in those without restenosis andpatients with an MDA-LDL level ≥ 110 U/L had ahigher risk of restenosis than those with an

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MDA-LDL level < 110 U/L (relative risk = 5.3).
In patients with diabetes and a history of coronary
artery disease (CAD), the relationship between the
MDA-LDL level and cardiac events was investigatedduring a 4-year follow-up period, revealing asignificantly higher frequency of cardiac events inpatients with an MDA-LDL level ≥ 110 U/L than inthose with an MDA-LDL level < 110 U/L.
These findings demonstrated that MDA-LDL is a
useful prognostic marker for cardiac events inpatients with diabetes and a history of CAD.

greater 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 concentrationof 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.

Performance10) ＊1. Sensitivity
1) When the test is performed using Sample
Dilution Solution, the absorbance is  0.15.
2) When the test is performed using a sample
with a known concentration, the absorbance is
0.4–1.8 per 200 U/L of MDA-LDL.
2. Accuracy: 80–120 % of the expected assay value
3. Within-run Reproducibility:
Coefficient of variation ≤ 15 %(Test methods used for 1.–3. are in-housemethods.)
4. Measurement Range10):
10–330 U /L
5. Standard Material
MDA−LDL (in-house standard material)

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 outer
package.)
Packaging
For 96 assays＊

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) Proclin 300, which possesses skin-irritative
potential, is added as an antiseptic agent in the
Enzyme-Labelled Anti-body Solution, Wash
Buffer Concentrate, Substrate Solution, Sample
Dilution Solution, Sample Preservation Solution,
and Calibrator. Therefore, if the reagent comesin contact with skin or clothes, rinse
immediately with ample water, and consult thedoctor 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 mix materials from different kit lot
numbers.
5) Do not exchange caps between reagents.
6) Do not allow the cap of the reagent bottle tocome into contact with other reagents or
samples.
7) 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 of

OxidizedLDL
ELISA“Daiichi”

Name
Antibody-Coated
Plate
Enzyme-Labelled
Antibody Solution
Wash Buffer
Concentrate
Substrate Solution
Stop solution
Sample Dilution
Solution
Sample Preservation
Solution
Calibrator

Package
1 × 96 wells
1 × 11 mL
For 2 × 500
mL
1 × 11 mL
1 × 11 mL
2 × 100 mL
1 × 50 mL
For 1 × 0.5
mL

References ＊1) Kotani K. et al：Biochim. Biophys. Acta 1215,
121（1994）
2) Kotani K. et al.: Jpn J Clin Pathol, 45, 47, 1997.
3) Kotani K. et al.: J Anal Bio-Sci, 20, 111, 1997.
4) Kondo A. et al.: Clin Chem, 47, 893, 2001.
5) Kanno T. et. al.: The lipid, 9, 32, 1998.
6) Tanaga K. et al.: Arterioscler Thromb Vasc Biol, 2,662, 2002.
7) Kotani K.: Oxidized stress marker, 243, Japan
Scientific Societies Press, 2005.
8) Shigematsu S. et al：Circ J. 71, 1697（2007）9) Kotani K, et al：Clin. Chim. Acta 450, 145（2015）10) 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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