Instructions for use

Normetanephrine Plasma ELISA Fast Track

BA E-8200R

Normetanephrine Plasma ELISA Fast Track
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of free Normetanephrine in plasma.
Related Products:
 Metanephrine Plasma ELISA Fast Track 2-MET Plasma ELISA Fast Track


2-MET Plasma RIA Fast Track

Normetanephrine (Normetadrenaline) is first extracted using an ion exchange matrix followed by anacylation process.
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 analytes
compete 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 standards.
The antibodies used in this test kit only recognise the biologically relevant L-forms of Metanephrines.
Commercially available synthetic Normetanephrine or Metanephrine is always a mixture of the D- and Lforms. The ratio between both forms differs widely from lot to lot. This has important implications if
synthetic Metanephrines are used to enrich native samples. As only about 50% of the synthetic
Metanephrines - the L-portion - will be detected by use of this kit, spiked samples will be underestimated.
Therefore native samples containing solely the L-form should be used.
1.2 Background
Metanephrine and Normetanephrine are the metabolites of the catecholamines Epinephrine andNorepinephrine, respectively. Cells derived from neuroendocrine tumors (e.g. pheochromcytoma) are known
to produce catecholamines which are secreted episodically via vesicles into the blood stream. But beside
this, a small portion of the catecholamines is metabolized inside the cells to the corresponding
catecholamines metabolites – namely Metanephrine, Normetanephrine and 3-Methoxytyramine – which are
secreted at low levels continuously into the blood stream.
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 used
to run the assay. Reliable performance will only be attained by strict and careful adherence to the
instructions provided.
(2) Reagents of this kit which contain human serum or plasma have been tested and confirmed negative for
HIV I/II, HBsAg and HCV by approved procedures. All reagents, however, should be treated as potential
biohazards in use and for disposal.
(3) The principles of Good Laboratory Practice (GLP) have to be followed.
(4) In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective
gloves and protective glasses where necessary.
(5) 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.
(6) For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water.
(7) 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. Microtiter strips which are removed from the frame
for usage should be marked accordingly to avoid any mix-up.
(8) Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
(9) 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.
(10) Incubation times do influence the results. All wells should be handled in the same order and timeintervals.
(11) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,
sample, standard and control.
(12) A standard curve must be established for each run.

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(13) The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report.
(14) 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.
(15) 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.
(16) 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.
(17) For information on hazardous substances included in the kit please refer to Safety Data Sheet (SDS). The
Safety Data Sheet for this product is made available directly on the website of the manufacturer or uponrequest.
(18) 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.
2.2.1 Interfering substances
Samples containing precipitates or fibrin strands might cause inaccurate results.
Hemolytic samples (up to 1 mg/ml hemoglobin), icteric samples (up to 25 mg/dl bilirubin) and lipemic
samples (up to 1700 mg/dl triglycerides) have no influence on the assay results.
2.2.2 Drug interferences
Please refer to point “Sample collection and storage”.
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
Adhesive Foil - Ready to use

BA D-0090
Content:

Adhesive Foils in a resealable pouch

Volume:1 x 4 foils

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-0040

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.
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BA E-0231

Normetanephrine Microtiter Strips - Ready to use

Content:

1 x 96 well (12x8) antigen precoated microwell plate in a resealable yellow pouch withdesiccantNormetanephrine Antiserum - Ready to use

BA E-8210
Content:

Anti- Normetanephrine rabbit antibody, yellow coloured

Volume:1 x 6 ml/vial, yellow cap

Standards and Controls - Ready to use
Cat. no.

Component

Colour/Cap

Concentration
pg/ml
NMN

Concentration
pmol/lNMN

Volume/Vial

BA E-8301

white

0

0

4 ml

BA E-8302

light yellow

72

393

4 ml

240

1 310

4 ml

BA E-8303

orangeBA E-8304

dark blue

720

3 931

4 ml

BA E-8305

light grey

2 400

13 1044 ml

BA E-8306

7 200

39 3124 ml

BA E-8351

black
light green

BA E-8352

dark red

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

Conversion:

Normetanephrine (pg/ml) x 5.46 = Normetanephrine (pmol/l)Content:

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

Content:

Tris-Buffer

Volume:1 x 10 ml/vial, yellow cap
Assay Buffer - Ready to use

Content:

25% organic solvent

Volume:1 x 30 ml/vial, orange cap

BA R-8312

4 ml

Adjustment Buffer - Ready to use

BA E-8327

BA R-8313

4 ml

Acylation Concentrate – Concentrated

Content:

Acylation reagent in DMSO

Volume:1 x 1.5 ml/vial, dark grey cap

Hazards
identification:
H302 Harmful if swallowed.
BA R-8318
Content:
BA R-8325

Extraction Plate - Ready to use
1 x 96 well plate, precoated with ion-exchanger in a resealable pouch
Cleaning Concentrate - Concentrated 25x

Content:

Buffer with sodium acetate

Volume:1 x 20 ml/vial, brown cap

BA R-8326

Elution Buffer - Ready to use

Content:

0.1 M Sodium hydroxide, dark purple coloured

Volume:1 x 14 ml/vial, dark green cap

BA R-8828

Equalizing-Reagent - Ready to use

Content:

Human serum, negative for HIV I/II, HBsAg and HCVVolume:1 x 14 ml/vial, white cap

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

Calibrated precision pipettes to dispense volumes between 20 - 350 μl; 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
Microtiter plate shaker (shaking amplitude 3 mm; approx. 600 rpm)
Absorbent material (paper towel)
Water (deionized, distilled, or ultra-pure)
Vortex mixer

5. Sample collection and storage
Medications like Serotonin-noradrenaline reuptake inhibitors, tryicyclic antidepressants, MAO inhibitors,
antihypertensive drugs and L-DOPA can influence Metanephrine and Normetanephrine level. People who aretaking such medication should consult with their doctor before specimen collection.
Sympathomimetic agents, sport and smoking can also influence Metanephrine and Normetanephrine level.
Alcohol and caffeinated drinks should be avoided the day before and including the day of sample collection.
EDTA- or Heparin-Plasma
Whole blood should be collected into centrifuge tubes (MonovetteTM or VacuetteTM) containing EDTA or
heparin as anti-coagulant and centrifuged (according to manufacturer’s instructions) immediately aftercollection.
Haemolytic, icteric and lipemic samples should not be used for the assay.
Storage: up to 6 hours at 2 - 8 °C, for longer period (up to 6 month) at -20 °C.
Repeated freezing and thawing should be avoided.
6. Test procedure
The ELISA can be run using an overnight incubation without shaking (results within approx. 24 hours) oralternatively as a fast version with shortened antiserum incubation times with shaking (results withinapprox. 6 hours).
Allow all reagents to reach room temperature and mix thoroughly by gentle inversion before use. Number
the Extraction Plate and microwell plate (microtiter strips which are removed from the frame for usage
should be marked accordingly to avoid any mix-up).
Duplicate determinations are recommended.
The binding of the antibodies, the enzyme conjugates, and the activity of the enzyme used are temperaturedependent, and the absorption values may vary if a thermostat is not used. The higher the temperature, thehigher 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.
In case of overflow, read the absorbance of the solution in the wells within 10 minutes, using a
microplate reader set to 405 nm.
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
Cleaning Buffer
Dilute the 20 ml Cleaning Concentrate with water (deionized, distilled, or ultra-pure) to a final volume of 500
ml.
Storage: 1 month at 2 – 8 °C
Acylation Solution
As the Acylation Solution is only stable for a maximum of 3 minutes, it should not be prepared before
starting the assay. Therefore its preparation is described in the protocol in chapter 6.3, step 3.
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.
Extraction Plate
In rare cases residues of the cation exchanger can be seen in the wells as small, black dots or lines. These
residues do not influence the quality of the product.

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6.2 Preparation of samples
Extraction
1.

Pipette 20 μl of standards and controls into the respective wells of the Extraction Plate.

2.

Add 20 µl Standard A to all wells containing plasma samples.

3.

Add 200 μl of Equalizing Reagent to the wells with standards and controls.

4.

Pipette 200 μl of plasma samples to the respective wells.

5.

Incubate plate for 2 hours at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

6.

Empty plate and blot dry by tapping the inverted plate on absorbent material.

7.

Pipette 250 µl of Assay Buffer into all wells. Incubate the plate for 5 min at RT (20 – 25 °C) on a
shaker (approx. 600 rpm). Empty plate and blot dry by tapping the inverted plate on absorbent
material.
Wash the plate 3 x by adding 350 μl of Cleaning Buffer, discarding the content and blotting dryeach time by tapping the inverted plate on absorbent material.

8.
9.
10.

Pipette 100 μl of Elution Buffer into all wells.
Please note: the colour changes caused by the elution buffer can vary between standards and samples.
Cover plate with adhesive foil. Incubate 15 min at RT (20 - 25 °C) on a shaker (approx. 600 rpm).
Remove the foil.
Do not decant the supernatant thereafter!
The following volumes of the supernatant are needed for the subsequent ELISA:
Normetanephrine 25 μl

6.3 Normetanephrine ELISA
1.

Pipette 25 µl of Adjustment Buffer into all wells of the Normetanephrine Microtiter Strips.

2.

Pipette 25 µl of the extracted standards, controls and samples into the respective wells.

3.

Preparation of Acylation Solution:
Pipette 80 µl Acylation Reagent Concentrate (BA R-8312) to 3 ml water (deionized, distilled, or
ultra-pure) and mix thoroughly.

4.

Pipette 25 µl of the freshly prepared Acylation Solution into all wells.

5.

Incubate for 15 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

6.

Pipette 50 µl of the Normetanephrine Antiserum into all wells.

7.

Cover the plate with Adhesive Foil, shake for 1 min at RT (20 – 25 °C) on a shaker and incubate for
15 - 20 h (overnight) at 2 – 8 °C without shaking.
Alternatively incubate for 2 h at RT (20 - 25 °C) on a shaker (approx. 600 rpm).

8.

Remove the foil. Discard or aspirate the contents of the wells. Wash the plate 4 x by adding 300 µl of
Wash Buffer, discarding the content and blotting dry each time by tapping the inverted plate on
absorbent material.

9.

Pipette 100 µl of the Enzyme Conjugate into all wells.

10.

Incubate for 30 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

11.

Discard or aspirate the contents of the wells. Wash the plate 4 x by adding 300 µl of Wash Buffer,discarding the content and blotting dry each time by tapping the inverted plate on absorbent
material.

12.

Pipette 100 µl of the Substrate into all wells and incubate for 20 - 30 min at RT (20 – 25 °C) on a
shaker (approx. 600 rpm). Avoid exposure to direct sunlight!

13.

Add 100 µl of the Stop Solution to all wells and shake the microtiter plate to ensure a homogeneous
distribution of the solution.

14.

Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to
450 nm (if available a reference wavelength between 620 nm and 650 nm is recommended).

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7. Calculation of results
Measuring range

Normetanephrine(overnight ELISA)

22.8 – 7 200 pg/ml

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
concentrations of the analyte. OD-values found below the standard curve correspond to highconcentrations of the analyte in 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.
Samples found with concentrations higher than the highest standard (Standard F) should be diluted
accordingly with the included Equalizing Reagent and have to be re-assayed.
Conversion
Normetanephrine (pg/ml) x 5.46 = Normetanephrine (pmol/l)
Expected reference value
It is strongly recommended that each laboratory should determine its own reference value.
The expected reference values indicated below are based on method comparison studies to LC-MS/MS (1)with blood samples taken in the sitting position.
Normetanephrine< 196 pg/ml
7.1 Quality control
The confidence limits of the kit controls are indicated in the QC-Report.
7.2 Typical standard curve
Example, do not use for calculation!

OD

Normetanephrine

pg/ml
8. Assay characteristics (overnight ELISA)

Analytical Sensitivity

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LOD (pg/ml)
LOQ (pg/ml)

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Normetanephrine
17.9
22.8

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Cross Reactivity (%)

Substance

Normetanephrine
Derivatized Metanephrine
0.72
Derivatized Normetanephrine
100
Analytical Specificity(Cross Reactivity)
3-Methoxytyramin
6.5*
Adrenaline< 0.01
Noradrenaline< 0.01
Dopamin< 0.01
Vanillic mandelic acid
< 0.01
Homovanillic acid
< 0.01
L-DOPA< 0.01
L-Tyrosin< 0.01
Tyramine< 0.01
Normetanephrine< 0.01
Acetaminophen< 0.01*Normetanephrine concentrations are not influenced by 3-Methoxytyramine in case of normal 3Methoxytyramine concentrations. Only very high 3-Methoxytyramine concentrations found in rare cases of
exclusively dopamine secreting tumours can cause false positive results.
Precision
Intra-Assay
Normetanephrine

Linearity

Recovery

Sample Mean (pg/ml)
1
149
2
282
3
734
4
1 956

Normetanephrine

Normetanephrine

Method Comparison
ELISA vs. LC-MS/MS(1)Inter-Assay
CV (%)
Normetanephrine
9.5
9.1
8.2
10.5

Serial dilution up to
1:64
Mean (%)
109
Normetanephrine

Sample Mean (pg/ml)
1
156
2
287
3
769
4
1 949

Mean (%)
98

CV (%)
10.6
5.0
5.1
5.9

Range (%)
92 - 102
Range (%)
105 - 114

y=0.93x + 13; r2 = 0.99; n = 48

9. References/Literature(1) De Jong et al. Plasma free metanephrine measurement using automated online solid phase extraction
HPLC-Tandem mass spectrometry. Clin Chem, 53(9): 1684-1693 (2007)(2) Eisenhofer et al. Laboratory evaluation of pheochromocytoma and paraganglioma. Clin Chem, 60:14861499 (2014)(3) Eisenhofer et al. Plasma metadrenalines: Do they provide useful information about sympatho-adrenal
function and catecholamine metabolism? Clin Sci (Lond),88:533-542 (1995)(4) Berkel et al. Diagnosis of endocrine disease: Biochemical diagnosis of pheochromocytoma andparaganglioma. Eur J Endocrinol, 170: R109-R119(5) Manz et al. Development of enantioselective immunoassays for free plasma metanephrines.
Ann.N.Y.Acad.Sci., 1018:582-587 (2004)(6) De Jong et al. Dietary Influences on Plasma and Urinary Metanephrines: Implications for Diagnosis of
Catecholamine-Producing Tumors. J Clin Endocrinol Metab, 94(8):2841-2849 (2009)(7) Deutschbein et al. Influences of Various Confounding Variable and Storage Conditions on Metanephrine
and Normetanephrine Levels in Plasma. Clin Endocrinol, 72(2):153-160 (2010)(8) Eisenhofer et al. Biochemical Diagnosis of Pheochromocytoma: How to Distinguish True- from FalsePositive Test Results. The Journal of Clinical Endocrinology & Metabolism, 88(6):2656-2666 (2003)

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

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective: 2019-12-10

Contains sufficient for<n> tests

For research use
only!

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