Instructions for use

Normetanephrine Urine ELISA Fast Track

BA E-8500R

Normetanephrine Urine ELISA Fast Track
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of Normetanephrine in urine
During the sample preparation Normetanephrine (Normetadrenaline) is quantitatively acylated.
The subsequent competitive ELISA uses the microtiter plate format. The antigen is bound to the solid phaseof the microtiter plate. The acylated standards, controls and samples and the solid phase bound analytecompete for a fixed number of antibody binding sites. After the system is in equilibrium, free antigen andfree antigen-antibody complexes are removed by washing. The antibody bound to the solid phase isdetected by an anti-rabbit IgG-peroxidase conjugate using TMB as a substrate. The reaction is monitored at
450 nm.
Quantification of unknown samples is achieved by comparing their absorbance with a reference curve
prepared with known standard concentrations.
1.2 Background
Metanephrine and Normetanephrine are the metabolites of the catecholamines Epinephrine andNorepinephrine, respectively. They are metabolized to Vanillylmandelic acid or excreted with the urine.
As catecholamine secretion from neuroendocrine cells might show high variations, urine samples collected
over a period of 24 hours are used to average these fluctuations.
2. Procedural cautions, guidelines, warnings and limitations
2.1 Procedural cautions, guidelines and warnings(1) This kit is intended for professional use only. Users should have a thorough understanding of this protocol
for the successful use of this kit. Only the test instruction provided with the kit is valid and has to be usedto run the assay. Reliable performance will only be attained by strict and careful adherence to theinstructions provided.
(2) The principles of Good Laboratory Practice (GLP) have to be followed.
(3) In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective
gloves and protective glasses where necessary.
(4) All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly
before use. Avoid repeated freezing and thawing of reagents and specimens.
(5) For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water.
(6) The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch with desiccant and used in the frame provided.
(7) Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
(8) Once the test has been started, all steps should be completed without interruption. Make sure that therequired reagents, materials and devices are prepared ready at the appropriate time.
(9) Incubation times do influence the results. All wells should be handled in the same order and timeintervals.
(10) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,
sample, standard and control.
(11) A standard curve must be established for each run.
(12) The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report.
(13) Do not mix kit components with different lot numbers within a test and do not use reagents beyond
expiry date as shown on the kit labels.
(14) Avoid contact with Stop Solution containing 0.25 M H2SO4. It may cause skin irritation and burns. In caseof contact with eyes or skin, rinse off immediately with water.
(15) TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them.
(16) For information on hazardous substances included in the kit please refer to Safety Data Sheet (SDS). TheSafety Data Sheet for this product is made available directly on the website of the manufacturer or uponrequest.
(17) Kit reagents must be regarded as hazardous waste and disposed according to national regulations.
2.2 Limitations
Any inappropriate handling of samples or modification of this test might influence the results.

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2.2.1 Interfering substances
24-hour urine
Please note the sample preparation! If the percentage of the final concentration of acid is too high, this willlead to incorrect results for the urine samples.
2.2.2 Drug interferences
There are no known substances (drugs)
Normetanephrine level in the sample.

whichingestioninterfereswith

themeasurementof

2.2.3 High-Dose-Hook effectNo hook effect was observed in this test.
3. Storage and stability
Store the unopened reagents at 2 - 8 C until expiration date. Do not use components beyond the expiry
date indicated on the kit labels. Once opened the reagents are stable for 1 month when stored at
2 – 8 °C. Once the resealable pouch has been opened, care should be taken to close it tightly with desiccantagain.
4. Materials
4.1 Content of the kit
BA D-0023

Reaction Tubes - Ready to use

Content:

Reaction Tubes in a resealable pouch

Volume:2 x 50 tubes

BA E-0030

Wash Buffer Concentrate - Concentrated 50x

Content:

Buffer with a non-ionic detergent and physiological pH

Volume:1 x 20 ml/vial, light purple cap

BA E-0045

Enzyme Conjugate - Ready to use

Content:

Goat anti-rabbit immunoglobulins conjugated with peroxidase

Volume:1 x 12 ml/vial, red cap

BA E-0055

Substrate - Ready to use

Content:

Chromogenic substrate containing tetramethylbenzidine, substrate buffer and hydrogen
peroxide

Volume:1 x 12 ml/vial, black cap

BA E-0080

Stop Solution - Ready to use

Content:

0.25 M sulfuric acid

Volume:1 x 12 ml/vial, light grey cap

Hazards
identification:
H290 May be corrosive to metals.
BA E-0231

Normetanephrine Microtiter Strips - Ready to use

Content:

1 x 96 well (12x8) antigen precoated microwell plate in a resealable yellow pouch withdesiccant

BA E-8510

Normetanephrine Antiserum - Ready to use

Content:

Rabbit anti-Normetanephrine antibody, yellow coloured

Volume:1 x 12 ml/vial, yellow cap

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Acylation Concentrate - Concentrated

BA R-0012
Content:

Concentrated acylation reagent

Volume:1 x 0.5 ml/vial, pink cap

Hazards
identification:

H314 Causes severe skin burns and eye damage.
Hydrochloric Acid - Ready to use

BA R-8619
Content:

0.25 M hydrochloric acid, yellow coloured

Volume:1 x 30 ml/vial, dark green cap

Standards and Controls - Ready to use
Cat. no.

Component

Colour/Cap

Concentration
ng/ml (= µg/l)
NMN

Concentration
nmol/lNMN

Volume/Vial

BA R-8601

white004 ml

BA R-8602

light yellow

301644 mlBA R-8603

orange

90491

4 ml

BA R-8604

dark blue

300

1 638

4 ml

BA R-8605

light grey

900

4 914

4 ml

BA R-8606

3 000

16 3804 ml

BA R-8651

black
light green

BA R-8652

dark red

Refer to QC-Report for expected value
and acceptable range!

Conversion:

Normetanephrine (ng/ml) x 5.46 = Normetanephrine (nmol/l)Content:

Acidic buffer with non-mercury preservatives, spiked with defined quantity of
Normetanephrine

4 ml
4 ml

Acylation Diluent - Ready to use

BA R-0075
Content:

Dimethylsulfoxide

Volume:1 x 4 ml/vial, dark grey cap
Acylation Buffer - Ready to use

BA R-8611
Content:

TRIS buffer

Volume:1 x 30 ml/vial, white cap

4.2 Additional materials and equipment required but not provided in the kit





Calibrated precision pipettes to dispense volumes between 10 – 600 µl; 1.2 – 3 ml
Microtiter plate washing device (manual, semi-automated or automated)
ELISA reader capable of reading absorbance at 450 nm and if possible 620 - 650 nm
Absorbent material (paper towel)
Water (deionized, distilled, or ultra-pure)
Vortex mixer
Temperature controlled water bath (90 °C) or similar heating device
The assay can be performed with or without shaking. If a microtiter plate shaker is used, it should havethe following characteristics: shaking amplitude 3 mm; approx. 600 rpm

5. Sample collection and storage
Spontaneous or 24-hour urine, collected in a bottle containing 10 - 15 ml of 6 M HCl, should be used.
Determine the total volume of urine excreted during a period of 24 h for calculation of the results.
Storage: up to 5 days at 2 – 8 °C, for longer periods (up to 6 months) at -20 °C.
Repeated freezing and thawing should be avoided.
Avoid exposure to direct sunlight.

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6. Test procedure
Allow all reagents to reach room temperature and mix thoroughly by gentle inversion before use. Number
the Reaction Tubes accordingly. Duplicate determinations are recommended. It is recommended to number
the strips of the microwell plate before usage to avoid any mix-up.
The binding of the antibodies and the enzyme conjugates and the activity of the enzyme used aretemperature dependent, and the absorption values may vary if a thermostat is not used. The higher thetemperature, the higher the absorption values will be. The absorption values also depend on the incubation
times. The optimal temperature during the Enzyme Immunoassay is between 20 – 25 °C.
6.1 Preparation of reagents
Wash Buffer
Dilute the 20 ml Wash Buffer Concentrate with water (deionized, distilled, or ultra-pure) to a final volume of1000 ml.
Storage: 1 month at 2 – 8 °C
Acylation Solution
Before preparing the Acylation Solution make sure that the Acylation Diluent (BA R-0075) has reached room
temperature (≥ 20°C) and forms a homogenous, crystal-free solution.
Dilute the Acylation Concentrate (BA R-0012) 1 + 60 with Acylation-Diluent in a glass or polypropylene-vial.
Acylation Concentrate
Acylation-Diluent

10 µl
600 µl20 µl
1.2 ml25 µl
1.5 ml

50 µl
3 ml

The Acylation Solution has to be prepared freshly prior to the assay (not longer than 60 minutes inadvance). Discard after use!
Normetanephrine Microtiter Strips
In rare cases residues of the blocking and stabilizing reagent can be seen in the wells as small, white dots orlines. These residues do not influence the quality of the product.
6.2 Sample preparation and acylation
Hydrolysis
1.

Pipette 25 µl of standards, controls, and urine samples into the respective Reaction Tubes.

2.

Add 250 µl Hydrochloric Acid to all tubes.

3.

Mix thoroughly (vortex) and hydrolyze for 30 min at 90 C.

4.

Cool down the tubes to room temperature.
For the measurement of the free Normetanephrine only, leave away step 3 and 4.

Acylation
1.

Pipette 250 µl of Acylation Buffer into all tubes.

2.

Add 25 µl of Acylation Solution (refer to 6.1) to all tubes.

3.

Mix thoroughly (vortex) and acylate for 15 min at RT (20 – 25 °C).

4.

Add 2.5 ml water (deionized, distilled, or ultra-pure) to all tubes.
Take 25 µl of the acylated standards, controls and urine samples for the Normetanephrine
ELISA.

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6.3 Normetanephrine ELISAThe usage of a shaker is not mandatory. The alternative protocol without shaker is highlighted in italic andshaded in grey.
1.
2.
3.

4.

5.
6.
7.

8.
9.

10.
11.

Pipette 25 µl of the acylated standards, controls and samples into the appropriate wells of theNormetanephrine Microtiter Strips.
Pipette 100 µl of the Normetanephrine Antiserum into all wells.
Incubate 30 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).
Without usage of a shaker: shake Normetanephrine Microtiter Strips shortly by hand and incubate
for 1 h at RT (20 – 25 °C).
Discard or aspirate the content of the wells. Wash the plate 3 x by adding 300 µl of Wash Buffer,discarding the content and blotting dry each time by tapping the inverted plate on absorbent
material.
Pipette 100 µl of the Enzyme Conjugate into all wells.
Incubate for 15 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).
Without usage of a shaker: incubate for 15 min at RT (20 – 25 °C).
Discard or aspirate the content of the wells. Wash the plate 3 x by adding 300 µl of Wash Buffer,discarding the content and blotting dry each time by tapping the inverted plate on absorbent
material.
Pipette 100 µl of the Substrate into all wells.
Incubate for 15 ± 2 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).
Without usage of a shaker: incubate for 15 min ± 2 at RT (20 – 25 °C).
Avoid exposure to direct sunlight!
Add 100 µl of the Stop Solution to each well and shake the microtiter plate to ensure a homogeneous
distribution of the solution.
Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to450 nm (if available a reference wavelength between 620 nm and 650 nm is recommended).

7. Calculation of results
Normetanephrine
16.2 – 3 000 ng/ml

Measuring range

The standard curve is obtained by plotting the absorbance readings (calculate the mean absorbance) of thestandards (linear, y-axis) against the corresponding standard concentrations (logarithmic, x-axis).
Use a non-linear regression for curve fitting (e.g. spline, 4- parameter, akima).
This assay is a competitive assay. This means: the OD-values are decreasing with increasing concentrationsof the analyte. OD-values found below the standard curve correspond to high concentrations of the analytein the sample and have to be reported as being positive.
The concentrations of the samples and controls can be read directly from the standard curve.
The amount of analyte excreted per day (µg/day) is calculated according to:concentration of the sample (in µg/l) x volume of urine excreted per day (in l/day)ExampleThe concentration of the sample read from the curve is 125 µg/l. The amount of urine collected during
24 hours is 1.3 l. Then the amount of analyte excreted during one day would be:
125 µg/l x 1.3 l/day = 162.5 µg/day
Samples found with concentrations higher than the highest standard (Standard F) should be diluted
accordingly with Standard A and have to be re-assayed.
Conversion
Normetanephrine (ng/ml) x 5.46 = Normetanephrine (nmol/l)
Expected reference value
It is strongly recommended that each laboratory should determine its own reference value.
24-hour urine

Normetanephrine< 600 µg/day

7.1 Quality control
The confidence limits of the kit controls are indicated on the QC-Report.

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7.2 Typical standard curve
Example, do not use for calculation!

OD

Normetanephrine

ng/ml
8. Assay characteristicsAnalytical Sensitivity

LOD

Normetanephrine
14.7 ng/ml

LOB

10.4 ng/ml

LOQ

16.2 ng/ml
Cross Reactivity (%)

Substance

Analytical Specificity(Cross Reactivity)

Precision
Intra-Assay< 0.01

Inter-Assay
SampleNormetanephrine

Normetanephrine
0.111000.19< 0.010.64< 0.01

Derivatized Metanephrine
Derivatized Normetanephrine
Derivatized 3-methoxytyramine
Adrenaline
Noradrenaline
Dopamine
Vanillic mandelic acid, L-Dopa,
Homovanillic acid, L-Tyrosin, Tyramin

1234Range(ng/ml)53.9 ± 7.1
116 ± 12.3
322 ± 28.1
1121 ± 128

CV (%)
1311911Sample
Normetanephrine

1234Range(ng/ml)
52.5 ± 10.7
103 ± 15.2
276 ± 38.7
738 ± 83.0

CV (%)
20151411Linearity

Normetanephrine

Range (ng/ml)
98.8 – 347

Serial dilution up to
1:64

Mean (%)
101

Recovery

Normetanephrine

Mean (%)
100

Range (%)
93 – 111

Range (ng/ml)
26.5 - 3124

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Method Comparison
Normetanephrine
HPLC = 0.9 ELISA + 0.6
r = 0.99; n = 40
versus HPLC**The concentrations were assessed using both the ELISA and the HPLC method (external QC samples
from UK NEQAS). The correlation between ELISA and HPLC is excellent. Please take in mind, that the UK
control values are the mean of about 40 different HPLC users, and contain always one pathologicalsample per sending.
9. References/Literature(1) Parrott et al. Urinary corticosterone and normetanephrine levels after voluntary wheel and forced
treadmill running in the db/db mouse. Journal of Diabetes Mellitus, 1(4):71-78 (2011)(2) Petramala et al. Multiple Catecholamine-Secreting Paragangliomas: Diagnosis after Hemorrhagic Strokein a Young Woman. Endocrine Practice, 14(3):340-346 (2008)(3) Sato et al. Central control of bone remodeling by neuromedin U. Nature Medicine, 13:1234-1240 (2007)

For updated literature or any other information please contact your local supplier.
Symbols:

Version: 14.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2019-05-24

Contains sufficient for<n> tests

For research use
only!
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