This package insert follows the Pharmaceuticals, Medical devices and Other Therapeutic Products Act of Japan.
In Vitro Diagnostics
Marketing Approval No. 22400AMX01415000**Revised: September 2020 (5th edition)*Revised: January, 2017 (4th edition)

This package insert must be read carefully prior to use.
Everolimus assay kit(Classification No.: 83022000)

Calibrator A (CAL A)
Calibrator B (CAL B)
Calibrator C (CAL C)
Calibrator D (CAL D)
Calibrator E (CAL E)
Calibrator F (CAL F)

Nanopia TDM Everolimus
Important Precautions
When measurement is changed from a conventional
method (such as LC/MS/MS) to using this product,adequately assess differences from the values
obtained by the conventional method and establish
the appropriate target range that should be obtainedby using this product at each medical institution.

Intended Use
Measurement of the everolimus in whole blood
Assay Principle
1. Assay Principle
Pretreat the whole blood specimen with methanol
and the pretreatment solution, and subsequently
centrifuge. Use the supernatant thus obtained as
the specimen. When a certain amount of antieverolimus antibody is added to a specimen, theanti-everolimus antibody binds to everolimus
until it has reacted with all of the available
everolimus in the specimen. Next, everolimuscoated latex are added, which react with theresidual anti-everolimus antibody, leading to
agglutination. Since the extent of agglutination
depends on the concentration of everolimus in thespecimen, the everolimus concentration can be
determined by measuring the absorbance as anindicator of agglutination.

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, for
information 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 of
Assay Results" section, of this package insert.
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. 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.
8. EVER Antibody Solution 1 and Calibrators A to F
are prepared from materials of human origin andhuman blood that have been shown to be negativefor HBs antigen, HIV antibodies, and HCV
antibodies. However, these reagents (as well as the
specimens) should be considered potentially
infectious and handled with great care.

Specimen (everolimus) + Anti-everolimus antibody
Antigen-antibody reaction
Residual anti-everolimus antibody +Everolimus-coated latex
Agglutination via an antigen-antibody reaction
2. Features
1) Liquid reagents, ready-to-use.
2) Applicable to various automated analyzers.
＊＊

Procedural Precautions ＊1. Properties of Samples and Sampling Methods
1) Samples(1) Whole blood (EDTA) is used as the specimen.
(2) Pretreat specimens with methanol (HPLC
grade) and the pretreatment solution in the kit.
(3) Measure specimens promptly after
pretreatment, bearing the evaporation of
methanol particularly in mind.
2) Storage of samples1)(1) Specimens may be stored for up to 7 days in arefrigerator. If specimens cannot be measured
within 7 days of collection, store them at 20C and measure them within 28 days.
Bring samples to room temperature (15–30C)
before use.
(2) Specimens may be frozen and thawed up to
twice.
(3) Store specimens with protection from light,
because light may affect stability.
2. Interfering substances
1) Assay results are not affected by free bilirubin

Description (Kit Components)
Component: Ingredients
EVER Antibody Solution 1: Rabbit anti-everolimus
polyclonal antibody
EVER Latex Reagent 2: Everolimus-coated latex
Pretreatment Solution 31

(up to 18.7 mg/dL), conjugated bilirubin (up to
19.7 mg/dL), formazin turbidity (up to 1440
FTU), rheumatoid factors (up to 360 IU/mL), γglobulin (up to 12 g/dL), total protein (up to 12g/dL), or hematocrit (up to 60%).
2) Cross-reactivity
Cross-reactivity with everolimus analogs andvarious other agents is detailed in the tables
below.
(1)Everolimus metabolites
ConcenCrosstration
Substance
reactivity
tested(%)(ng/mL)
Seco acid of everolimus
57(RAD SA)
20
2
Precursor of seco acid of
5
12
everolimus (RAD PSA)
20
16
40–phosphatidylcholine
563
everolimus (RAD PC)
20
59
45–/46–hydroxy everolimus
5
ND(45/46 OH RAD)20
2
24–hydroxy everolimus (2459OH RAD)
20
5
25–hydroxy everolimus (25515
OH RAD)2022(2) Sirolimus and its metabolites
ConcenCrosstration
Substance
reactivity
tested(%)(ng/mL)
Sirolimus
10
46
Trihydroxy sirolimus; 7, 4190
4O-didesmethyl sirolimus
41-O-desmethyl hydroxy903sirolimus
41-O-desmethyl hydroxysirolimus; 7-O-desmethyl
902sirolimus
11-hydroxy sirolimus
9012Isomer of 11-hydroxy90
6sirolimus
Hydroxy sirolimus
90
2N-oxide sirolimus
907Isomer of hydroxyl
sirolimus or n-oxide901sirolimus
41-O-desmethyl sirolimus;
30
45
32-O-desmethyl sirolimus

Substance
Atenolol
Azathioprine
Caffeine
Captopril
Carbamazepine
Cefaclor
Chloramphenicol
Cimetidine
Ciprofloxacin
Cyclosporin A
Digoxin
Disopyramide
Erythromycin
Ethanol
LithiumMethicillin
Methotrexate
Morphine sulfate
Mycophenolic acid
Nadolol
Naproxen
NiacinNifedipine
Omeprazole
Penicillin G
Phenobarbital
Phenytoin
Piperacillin
Prazosin
Prednisone
Prednisolone
Primidone
Procainamide
Propranolol
Quinidine
Ranitidine
Rifampin
Salicylic acidSpectinomycin
Sulfamethoxazole
Tacrolimus
Theophylline
Tobramycin
Triamterene
Trimethoprim
Valproic acidVancomycin
Verapamil(3) Drugs commonly prescribed with everolimus
ConcenCrosstration
Substance
reactivity
tested(%)(μg/mL)
Acetaminophen
200
ND
N-acetylprocainamide
120
ND
Acyclovir
1000
0.0
Albuterol
0.18
ND
Allopurinol
60
ND
Amikacin
150
ND
Amphotericin B
100
ND
Ascorbic acid
30
ND

ConcenCrosstration
reactivity
tested(%)(μg/mL)
40
ND
10
ND
100
ND
50
0.0
120
0.0
230
ND
250
ND
100
ND
250
0.0
1
ND
0.01
-2
30
0.0
200
0.0
3500
ND
22.2
ND
240
ND
910
ND
6
ND
250
ND
333
ND
1000
0.0
800
ND
120
0.0
14
ND
100
0.0
150
ND
100
0.0
8
ND
25
ND
12
ND
12
ND
100
0.0
25
ND
0.5
ND
100
ND
200
ND
50
0.0
500
ND
100
ND
400
0.0
0.04
1
250
ND
20
ND
600
0.0
20
ND
1000
0.0
630
ND
10
ND
ND: Not detectable

3. Others
1) Use Calibrators A to F as the calibrationmaterials after carrying out pretreatment in thesame manner as for specimens.
2) For quality control of this product, use
Everolimus Control for Nanopia after2This product is compatible with various types ofautomated analyzer. An example of the assay
procedure is indicated below.

pretreatment in the same manner as for
specimens. Everolimus Control for Nanopia®
does not yield nominal values by other methods,
such as LC/MS/MS. Do not use Everolimus
Control for Nanopia for any purpose other thanquality control of Nanopia TDM Everolimus.
3) Measurement range
If the everolimus concentration in a specimen
exceeds the measurement range, dilute thespecimen with Calibrator A (0.0 ng/mL),
pretreat it, and perform re-measurement.

Sample
+
10 L

37C

37C

5 min

1 min

Reagent
Reagent
Measurement(1)(2)
Approx.
Approx. (Absorbance I※)
175 L
45 L

37C
Measurement

Approx.
(Absorbance II※)
5 min

Dosage/Administration (Assay Procedure) ＊1. Preparation of reagents
Reagent (1): EVER Antibody Solution 1 is readyto use.
Reagent (2): EVER Latex Reagent 2 is ready touse.
Pretreatment solution: Pretreatment Solution 3 is
supplied as a ready-to-use
solution.
Calibrator A: Calibrator A should be used after
being thawed at room temperature (15to 30C).
Calibrator B: Calibrator B should be used after
being thawed at room temperature (15to 30C).
Calibrator C: Calibrator C should be used after
being thawed at room temperature (15to 30C).
Calibrator D: Calibrator D should be used after
being thawed at room temperature (15to 30C).
Calibrator E: Calibrator E should be used after
being thawed at room temperature (15to 30C).
Calibrator F: Calibrator F should be used after
being thawed at room temperature (15to 30C).
Before using this product, gently invert the EVER
Latex Reagent 2 bottle to mix it thoroughly andcheck that there are no bubbles.
2. Instruments and Reagents
1) Micropipettes (10 to 100 μL and 100 to 500
μL)2) Pipette tip3) Methanol (HPLC grade)4) Microcentrifuge tube with a cap
5) Vortex mixer
6) High-speed shaker (if used)
7) Microcentrifuge
3. Pretreatment of Specimens
1) Pipette 300 μL of a specimen into a
microcentrifuge tube with a cap.
2) Add 350 μL of methanol (HPLC grade), cap thetube, and stir the contents with a Vortex mixer.
3) Add 50 μL of the pretreatment solution, cap thetube, and stir the contents for 35 seconds with a
Vortex mixer or for 10 minutes with a highspeed shaker.
4) Place the capped microcentrifuge tube
mentioned in section 3) above in the centrifuge(13400  g for 10 minutes).
5) Pipette 350 μL of the supernatant thus obtained
and use it as a specimen.
2. Assay Procedure

Calculation ofconcentration※

Absorbance I and II: Absorbance at 700 nm
Calibration material: Calibrators A to F
(Manufacture's assigned value)

Assessment of Assay Results
1. Reference standard range
Based on the results of clinical trials, therecommended whole blood trough concentrationof everolimus is 3 to 8 ng/mL (LC/MS/MS) for
heart or renal transplant recipients, while it is 5 to
15 ng/mL (LC/MS/MS) for patients with
subependymal giant cell astrocytoma associatedwith tuberous sclerosis complex (TSC-SEGA).
However, therapeutic and toxic concentrations of
this agent may overlap. Therefore, interpretationof assay results should also be based on thepatient's clinical findings and other examination
results.
It is recommended that the therapeutic
concentration range of everolimus be determinedat each institution by measuring the whole bloodconcentration in a statistically adequate numberof specimens.
2. Precautions for Assessment(1) There may be reactions or interfering reactions
with non-target substances. If assay resultsappear to be unreliable, repeat themeasurement (if necessary, after dilution) or
try another analytical methods.
(2) This product cross-reacts with metabolites of
everolimus, and with sirolimus and its
metabolites.
(3) Clinicians should make a comprehensive
clinical decision based on assay results inconjunction with patients' clinical symptoms
and other examination results.
(4) The recommended trough concentration rangeof everolimus (3 to 8 ng/mL) for heart or renaltransplant recipients was based on the resultsof a non-Japanese phase III study (in patientswith heart and renal transplantation) and aJapanese phase III study (in patients with renaltransplantation) using LC/MS/MS formeasurement.1) The recommended troughconcentration range of this drug (5 to 15
ng/mL) for patients with TSC-SEGA wasbased on the results of a non-Japanese phase II
study using LC/MS/MS for measurement.2),3)

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Clinical Significance ＊＊Everolimus is a macrolide immunosuppressant thatwas developed from sirolimus (also called rapamycin
and known to inhibit the immune system) in 1992. In
Japan, everolimus was approved (brand name,
Certican Tablets) in January 2007 for the preventionof graft rejection in heart transplant recipients and in
December 2011 for the prevention of graft rejection
in renal transplant recipients, and it is used to control
immunosuppression after heart or renaltransplantation. Common adverse reactions toeverolimus include leukopenia, thrombocytopenia,
and hyperlipidemia. The package insert states that thedose of this drug should be adjusted by monitoring
the trough blood concentration because the blood
everolimus concentration is influenced by food andthe patient's condition. The package insert also statesthat the whole blood concentration of everolimus
should be measured regularly when it is used in
combination with ciclosporin. Furthermore, it statesthat the recommended trough blood concentration
range of everolimus is 3 to 8 ng/mL, based on
efficacy and safety analyses, and that neither efficacy
nor safety has been evaluated at blood concentrations
above12 ng/mL.
Everolimus was also approved for the treatment of
TSC-SEGA in November 2012 (as Afinitor Tablets)
and in December 2012 (as Afinitor Dispersible
Tablets). The recommended trough bloodconcentration range is 5 to 15 ng/mL based on
efficacy and safety analyses performed during a nonJapanese phase II study. Many patients with TSCSEGA are relatively young children, who show
marked individual differences of the blood
everolimus concentration or also have epilepsy orother neurologic symptoms. Concomitant drugs may
thus affect the blood concentration of everolimus insuch patients. Therefore, the package insert states thatthe trough blood concentration of everolimus shouldbe measured about 2 weeks after the start ofadministration or after changing the dose, and that itshould be measured at appropriate times according to
the patient's conditions that may affect the bloodconcentration of everolimus.
For other information including indications, effects,
and dosage of everolimus drugs (e.g. Certican andAfinitor), please refer to the package insert of eachdrug.

Nanopia TDM Everolimus

product and those obtained by LC/MS/MS wasassessed using specimens from transplant
recipients at more than one institution after thisproduct was marketed. The slope was 0.82 to
1.03, while it was 0.87 when the correlation wasassessed using specimens from patients with
TSC-SEGA. This product yields slightly lower
values than those obtained by LC/MS/MS.
The correlation shown below is between values
obtained with this product and values obtained by
LC/MS/MS using specimens from transplant
recipients and from patients with TSC-SEGA.
1) Whole blood (EDTA, specimens from hearttransplant recipients)
N = 91, r = 0.946, y (obtained with Nanopia
TDM Everolimus) = 0.96x (obtained by thereference method) + 0.31
Reference method: LC/MS/MS

LC/MS/MS

Nanopia TDM Everolimus

2) Whole blood (EDTA, specimens from patientswith TSC-SEGA)
N = 50, r = 0.943, y (Nanopia TDM Everolimus)= 0.87 x (LC/MS/MS) + 1.33

Performance
1. Sensitivity
1) Absorbance of standard solution (0.0 ng/mL) is 
900 (Abs.  10000).
2) The difference of absorbance between standard
solution (0.0 ng/mL) and another standard
solution (1.5 ng/mL) is  90 (Abs.  10000).
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 Range4): (On Hitachi 7180 automatedanalyzer)
2.0–20.0 ng /mL
5. Correlation4)
The correlation between values obtained with thisLC/MS/MS

6. Standard Calibration Materials
Everolimus (in-house reference standard)
The calibration materials for this product are
prepared by weighing everolimus (in-housereference standard) and adding it to whole humanblood. Values of the calibration materials are
obtained after correction using specimens fromrenal transplant recipients, the values of which
are measured by LC/MS/MS at the reference
institution.

4

Precautions for Use or Handling ＊1. Precautions for Handling (to Ensure Safety)
1) All samples used in the test and Calibrators A to
F should be handled as a material possibly
infected with HIV, HBV, HCV, or other viruses.
To prevent infection, use disposable gloves andavoid mouth pipetting during the test.
2) Sodium azide is added as an antiseptic agent in
the EVER Antibody Solution 1, EVER Latex
Reagent 2, and Pretreatment Solution 3.
Therefore, if the reagent comes in accidentallycontact with eyes, mouth or skin, rinse
immediately with ample water as first aid, andconsult the doctor if required.
3) Proclin 300, which possesses skin-irritative
potential, is added as an antiseptic agent in the
Calibrators A to F. Therefore, if the reagent
comes in contact with skin or clothes, rinse
immediately with ample water, and consult thedoctor if skin irritation develops.
4) Methanol (HPLC grade), which is used for
pretreatment, is highly flammable and is known
to severely irritate the eyes. Handle it withgreat care after reading the safety data sheet.
2. Precautions for use
1) EVER Antibody Solution 1, EVER Latex
Reagent 2, and Pretreatment Solution 3 should
be stored as directed in a tightly-stopped
container. Avoid freezing. Do not use theproduct if it has been frozen because freezing
can cause deterioration of the reagents, leading
to inaccurate results.
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, reagentsshould be tightly stoppered and stored in arefrigerator.
5) Before use, Calibrators A to F should be thawed
at room temperature (15 to 30C), mixed byinversion, and pretreated.
6) After thawing, Calibrators A to F remain stablefor 6 weeks when refrigerated (2 to 8C) intightly stoppered containers with protection
from light.
7) Do not mix materials from different kit lot
numbers.
8) Do not perform the assay under direct sunlight
3. Precautions for Disposal
1) Before disposal, used samples, Calibrators A toF, specimen containers, and calibrator
containers must be immersed in sodium
hypochlorite solution at a concentration ofgreater than 0.1 % for longer than 1 hour or
autoclaved at 121 ºC for 20 minutes.
2) To prevent infections from spilled samples,
Calibrators A to F, or solutions containing
Calibrators A to F or samples, wipe the spill area
thoroughly with disinfectants such as sodium
hypochlorite solution at a concentration greaterthan 0.1%.
3) The reagents and treated samples, and residual
Calibrators A to F should be discarded as
medical waste or industrial waste according to
the waste disposal regulations.

4) Pretreatment Solution 3 contains copper (II)sulfate. It should be disposed of in accordancewith the Water Pollution Control Act or related
regulations.
5) EVER Antibody Solution 1, EVER Latex
Reagent 2, and Pretreatment Solution 3 containsodium azide as a preservative. Sodium azide
may react with lead or copper pipes and produce
highly explosive metallic azide. When disposingof this product, flush it away with copious
amounts of water.
4. Other precautions
Do not use the containers for other purposes.
Storage and Shelf Life ＊The storage conditions and shelf-life of each kit
component are as follows:
Components
Storage
Shelf life
conditions
EVER Antibody
1 year from
Solution 1
2–8ºC
the date of
EVER Latex Reagent 2
manufacture
Pretreatment Solution 3
Calibrator A (CAL A)
Calibrator B (CAL B)-25 to -15ºC
1 year from
Calibrator C (CAL C)(withthe date ofprotection
Calibrator D (CAL D)
manufacturefrom light)
Calibrator E (CAL E)
Calibrator F (CAL F)(The expiration date is printed on the outer package.)

Packaging
Nanopia TDM
Everolimus

Everolimus
Calibrator for
Nanopia(sold separately)

Name
EVER Antibody
Solution 1
EVER Latex Reagent 2
Pretreatment Solution 3
Calibrator A (CAL A)
Calibrator B (CAL B)
Calibrator C (CAL C)
Calibrator D (CAL D)
Calibrator E (CAL E)
Calibrator F (CAL F)

Packaging1  18 mL
1  5 mL1  5 mL1  3.0 mL6concentrati
ons

References
1) Novartis Pharma K.K. Japan, Certican Tablets
0.25 mg, 0.5 mg and 0.75 mg. [Instructions forUse]. Japanese.
2) Novartis Pharma K.K. Japan, Afinitor Tablets 2.5
mg and 5 mg. [Instructions for Use]. Japanese.
3) Novartis Pharma K.K. Japan, Afinitor Dispersible
Tablets 2 mg and 3 mg. [Instructions for Use].
Japanese.
4) SEKISUI MEDICAL CO., LDT. In house date.
Japanese.
Contact
SEKISUI MEDICAL CO., LTD.
international@sekisui.com

5Manufacturer ＊SEKISUI MEDICAL CO., LTD.
1-3, Nihonbashi 2-chome, Chuo-ku, Tokyo, Japan"Nanopia" is trademark or registered trademarks of
SEKISUI MEDICAL CO., LTD. in Japan and/or
other countries.
All other brands, product names and service names
are trademarks or registered trademarks of theirrespective companies.

6