This package insert follows the Pharmaceuticals, Medical devices and Other Therapeutic Products Act of Japan.
In Vitro Diagnostics
Marketing Notification No. 13A2X00197225002**Revised: January 2017 (5th edition)*Revised: August 2015 (4th version)

This package insert must be read carefully prior to use.
Methotrexate assay kit for blood tests(Classification No.: 30418001)

ability to actively take up methotrexate, a high dose isadministered so that excess methotrexate is passively
incorporated. After a certain time, normal cells thatcan actively take up leucovorin are salvaged byadministering leucovorin to reverse the toxicity ofmethotrexate. When methotrexate/leucovorin salvage
therapy is performed, the risk of serious adverse
events is high when the methotrexate concentration is
not lower than 10, 1, and 0.1 μmol/L at 24, 48, and 72
hours after starting methotrexate treatment,respectively. Therefore, it is important to frequently
measure the blood concentration of methotrexate fora certain period after administration.1)

Nanopia eTDM Methotrexate
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. Please read carefully the "PRECAUTIONS"
section, particularly under "Effects on Laboratory
Tests" of the package insert of each drug, forinformation on the influence to the assay result that
medications administered to the patient have.
Please also read carefully the "2) Cross-reactivity,"
under "2. Interfering Substances," in the"Procedural Precautions" section, as well as "2.
Precautions for Assessment" in the "Assessment ofAssay Results" section, of this package insert.
4. 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.
5. If the reagent accidentally comes in contact witheyes and/or mouth, rinse immediately with ample
water as first aid, and consult the doctor ifrequired.
6. 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.
7. Perform a quality control test prior to assay toensure accuracy.

Assay Principle
1. Assay Principle
This product is a homogenous enzyme
immunoassay kit that employs a competitive
reaction between methotrexate in a sample andglucose-6-phosphate
dehydrogenase(G6PDH)-labeled methotrexate. When a certain
amount of anti-methotrexate antibody is added tothesample,areaction
occursandanti-methotrexate
antibodyisconsumed
depending on the amount of methotrexate in thesample. If G6PDH-labeled methotrexate is added,
the residual anti-methotrexate antibody that has
not bound to methotrexate in the sample binds toG6PDH-labeled
methotrexate,
while
G6PDH-labeled methotrexate that does not bindto the anti-methotrexate antibody reduces NAD toNADH.
Since the amount of reduced NADH that isproduced from NAD depends on the methotrexate
concentration in the sample, the methotrexate
concentration can be obtained by measuring the %change of absorbance. Because NAD only acts on
the microorganism-derived enzyme used in thisproduct, endogenous G6PDH in the sample has
no influence on the assay results.

Description (Kit Components)
Component: Ingredients
MTX Antibody Solution 1:
Anti-methotrexate
rabbitpolyclonal antibody
Glucose-6-phosphate
Nicotinamide
adeninedinucleotide (oxidized form)
MTX Enzyme Solution 2:
Glucose-6-phosphate
dehydrogenase-labeled
methotrexate

Sample (methotrexate) + Anti-methotrexate
antibody
Antigen-antibody reaction
Unreacted
anti-methotrexate
antibody+G6PDH-labeled methotrexate
Antigen-antibody reaction
Glucose-6-phosphate + NAD
Unbound G6PDH-labeled methotrexate

Intended Use
Measurement of methotrexate in serum or plasma

6-phosphogluconate + NADH
2. Features
1) Liquid reagents, ready-to-use.
2) It can be measured using the automatedanalyzers commonly used.

Methotrexate suppresses cell growth by inhibiting
dihydrofolate reductase (DHFR), which reduces folicacid to produce the active form of folic acid required
for nucleic acid synthesis, and inhibiting thethymidylic acid and purine synthesis enzyme systems.
Methotrexate is used for methotrexate/leucovorin
salvage therapy. Because certain cancer cells lack theProcedural Precautions ＊＊1. Properties of Samples and Sampling Methods
1) Samples

1Serum and plasma (heparin plasma and EDTAplasma) may be used.
2) Storage of samples(1) After separation of serum (plasma),samples may be stored for up to 7 days ina refrigerator. If samples cannot bemeasured within 7 days of serum(plasma) separation, store them at −30Cand measure them within 35 days. Storedsamples should be brought to room
temperature (15–30C) before use.
(2) Samples may be frozen and thawed up totwice.
3) Sampling should be performed after removing
insoluble matter from the sample. Very cloudy
specimens should be centrifuged before assay.
2. Interfering substances
1) Assay results are not affected by free bilirubin(up to 19.5 mg/dL), conjugated bilirubin (up to20.9 mg/dL), hemoglobin (up to 491 mg/dL),
formazin turbidity (up to 1410 FTU), or
rheumatoid factors (up to 550 IU/mL).
2) Cross-reactivity
The following table summarizes thecross-reactivity with the methotrexate
metabolites and other drugs.
(1) Methotrexate metabolites
Metabolite7-Hydroxymethotrexate
2,4-Diamino-N10-methylpteroic
acid (DAMPA)

Concentration
tested(μmol/L)

Methotrexate
concentration(μmol/L)

Crossreactivity※(%)

50.05< 0.02500.50< 0.070.05

0.00< 100.0

0.50

0.00

< 80.7cross-reactivity was calculated by thefollowing equation.
Cross-reactivity (%) = ({[Concentration ofmethotrexate in the sample after adding themetabolite or drug] – [concentration ofmethotrexate in the sample without the
metabolite or concomitant drug]} /[Concentration of the metabolite or drug in thesample]) × 100
3. Others
1) Always use Methotrexate Calibrator forcalibration.
2) Precautions for assay range
If the methotrexate concentration in thesample exceeds the measurement range, dilutethe sample with a Methotrexate Sample
Dilution
Solution,andperform
re-measurement.
Dosage/Administration (Assay Procedure) ＊＊1. Preparation of reagents
Reagent (1): MTX Antibody Solution 1 is readyto use.
Reagent (2): MTX Enzyme Solution 2 is readyto use.
Check that there are no bubbles with thereagents before using this product.
2. Assay Procedure
This product is compatible with various types ofautomated analyzer. An example of the assay
procedure is indicated below.
37CSample + Reagent (1)8.5 μL150 μL Approx.
5 min
37C

(2) Other drugs
If each drug is at the concentration shown in
the table below when the methotrexate
concentration is 0.00, 0.05, or 0.50 μmol/L,
cross-reactivity is as follows.
ConcentraCrosstion tested reactivity
Drug name※(μmol/L)(%)
Adriamycin
1000< 0.01
Cyclophosphamide
1500
0.00
Cytosine
1000
0.00
Dihydrofolic acid
1000
0.01
DL-6-Methyl-5,6,7,81000< 0.01
tetrahydropterin
Folic acid1000
< 0.01Folinic acid (leucovorin)
1000
0.00
5-Fluorouracil
3000
0.00
6-Mercaptopurine
1000
0.00
5-Methyltetrahydrofolate
1000
0.00
Prednisolone
1000
0.00
Pyrimethamine
1000
0.00
Sulfamethoxazole
1600
0.00
Tetrahydrofolic acid1000
< 0.01
Vinblastine
1000
0.01
Vincristine
1000
0.01
Triamterene
25> 2.80
Trimethoprim
100< 0.54※
Based on the Clinical and Laboratory
Standards
Institute(CLSI)
EP7-A2,

Measurement
Reagent (2)Approx.
※
75 μL 50–180 (Absorbance )secCalculation ofconcentration※Absorbance : Absorbance at 340 nm
Calibration material: Methotrexate Calibrator(Manufacture's assigned value)

Assessment of Assay Results
1. Reference standard range
The 24-, 48-, and 72-hour values for blood
methotrexate concentration limits for toxicity are
specified as 10, 1, and 0.1 μmol/L, respectively.
2 . Precautions for Assessment
1) There may be reactions or interfering reactions
with non-target substances. If assay resultsappear to be unreliable, repeat the measurement(if necessary, after dilution) or try anotheranalytical methods.
2) This product shows cross-reactivity withDAMPA (a metabolite of methotrexate),accurate assay values cannot be obtained withsamples collected from patients on gulcarpidase(carboxypeptidase G2) therapy.

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accordance with the Water Pollution Control act
or related regulations.
4. Other precautions
Do not use the containers for other purposes.

Performance
1. Sensitivity
The difference between the change of absorbanceper minute with the standard solution (0.00µmol/L) and that with another standard solution(1.20 µmol/L) is ≥ 50.0 mAbs./min.
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 Range2): (On Hitachi 7180
automated analyzer)
0.04–1.20μmol /L
5. Correlation2)
1) Serum N=54
r=0.995
y=0.97x+0.011
Control method: Approved in vitro diagnostic(fluorescence polarization immuno assay)
6. Standard Material
Methotrexate (U.S. Pharmacopoeia)＊＊

Storage and Shelf Life ＊1. Storage temperature: 2–8C
2. Shelf life: 18 months from the date ofmanufacture(The expiration date is printed on the outerpackage.)
Packaging
NamePackage
MTX Antibody
1 × 16 mL
Nanopia eTDM Solution 1
Methotrexate
MTX Enzyme
1 × 8 mL
Solution 2
Constituent reagents are available in otherconfigurations. For further details please contact
SEKISUI MEDICAL CO., LTD.

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 pipettingduring the test.
2) 2-Methylisoazolone is added as an antiseptic
agent in the MTX Antibody Solution 1 andMTX Enzyme Solution 2. Therefore, if thereagent comes in accidentally contact with eyes,mouth or skin, rinse immediately with ample
water as first aid, and consult the doctor ifrequired.
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) After completion of measurement, this productshould be tightly stoppered and stored in arefrigerator.
5) Do not mix materials from different kit lotnumbers.
6) 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 orautoclaved 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 inReferences
1) Package insert of Methotrexate Injection 200mg･1000mg.
2) In house data, SEKISUI MEDICAL CO., LTD.
ContactSEKISUI MEDICAL CO., LTD.
international@sekisui.com
Manufacturer ＊＊SEKISUI MEDICAL CO., LTD.
1-3, Nihonbashi 2-chome, Chuo-ku, Tokyo, Japan

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