Instructions for use

Adrenaline ELISA Fast Track

BA E-6100R

Adrenaline ELISA Fast Track
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of adrenaline (epinephrine) in plasma and urine.
Adrenaline (epinephrine) is extracted by using a cis-diol-specific affinity gel, acylated and then converted
enzymatically.
The competitive ELISA kit uses the microtiter plate format. The antigen is bound to the solid phase of themicrotiter plate. The derivatized 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 standard concentrations.
1.2 BackgroundIn humans the catecholamines Adrenaline (Epinephrine), Noradrenaline (Norepinephrine) and Dopamine areneurotransmitters of the sympathetic nervous system and are involved in many physiological processes. The
sympathetic nervous system sets the body to a heightened state of alert, also called as the body’s fight-orflight response.
In the human body the catecholamines and their metabolites indicate the adaptation of the body to acuteand chronic stress.
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 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 thoroughlybefore 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 provided with the kit.
(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). The
Safety 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.

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2.2 Limitations
Any inappropriate handling of samples or modification of this test might influence the results.
2.2.1 Interfering substances
PlasmaSamples containing precipitates or fibrin strands or which are haemolytic or lipemic might cause inaccurate
results.
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) which ingestion interferes with the measurement of adrenaline levelin the sample.
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.
BA E-0131
Content:
BA E-6110

Adrenaline Microtiter Strips- Ready to use
1 x 96 well (12x8) antigen precoated microwell plate in a resealable blue pouch withdesiccant
Adrenaline Antiserum - Ready to use

Content:

Rabbit anti-adrenaline antibody, blue coloured

Volume:1 x 6 ml/vial, blue cap

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Adjustment Buffer - Ready to use

BA R-0050
Content:

TRIS buffer

Volume:1 x 4 ml/vial, green cap

Standards and Controls - Ready to use
Cat. no.

Component

Colour/Cap

Concentration
ng/mlConcentration
nmol/lVolume/VialBA E-6601

white004 ml

15.54 ml

BA E-6602

light yellow

BA E-6603

orange4224 ml

BA E-6604

dark blue

1582

4 ml

BA E-6605

light grey

50273

4 ml

BA E-6606

black
light green

200

1 092

4 ml

BA E-6651
BA E-6652

dark red

Refer to QC-report for expected value andacceptable range!

4 ml
4 ml

Conversion:

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

Acidic buffer with non-mercury stabilizer, spiked with defined quantity of adrenaline
Acylation Buffer - Ready to use

BA R-6611
Content:

Buffer with light alkaline pH for the acylation

Volume:1 x 20 ml/vial, white capAcylation Reagent - Ready to use

BA R-6612
Content:

Acylation reagent in DMF and DMSO

Volume:1 x 3 ml/vial, light red cap

Hazards
identification:
H360D May damage the unborn child.
H226 Flammable liquid and vapour.
H312 + H332 Harmful in contact with skin or if inhaled.
H319 Causes serious eye irritation.
Assay Buffer - Ready to use

BA R-6613
Content:

1M hydrochloric acid and a non-mercury preservative

Volume:1 x 6 ml/vial, light grey cap
Coenzyme - Ready to use

BA R-6614
Content:

S-adenosyl-L-methionine

Volume:1 x 4 ml/vial, purple cap
Enzyme - Lyophilized

BA R-6615
Content:

Catechol-O-methyltransferase

Volume:2 vials, pink cap

BA R-6617

Extraction Buffer - Ready to use

Content:

Buffer containing carbonate

Volume:1 x 6 ml/vial, brown cap

BA R-6618
Content:
BA R-6619

Extraction Plate - Ready to use
2 x 48 well plates coated with boronate affinity gel in a resealable pouch
Hydrochloric Acid - Ready to use

Content:

0.025 M Hydrochloric Acid, yellow coloured

Volume:1 x 20 ml/vial, dark green cap

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4/84.2 Additional materials and equipment required but not provided in the kitCalibrated precision pipettes to dispense volumes between 10 – 700 µl; 1 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
PlasmaWhole blood should be collected into centrifuge tubes containing EDTA as anti-coagulant (Monovette™ orVacuette™ for plasma) and centrifuged according to manufacturer’s instructions immediately after collection.
Haemolytic 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.
Urine
Spontaneous urine or 24-hour urine, collected in a bottle containing 10 - 15 ml of 6 M HCl, can be used.
If 24-hour urine is used please record the total volume of the collected urine.
Storage: up to 48 hours at 2 - 8 °C, up to 24 hours at room temperature, for longer periods (up to6 month) at -20 °C. Repeated freezing and thawing should be avoided.
Avoid exposure to direct sunlight.
6. Test procedure
Allow all reagents to reach room temperature and mix thoroughly by gentle inversion before use. 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 antiserum and the enzyme conjugate and the activity of the enzyme are temperaturedependent, and the absorbance may vary if a thermostat is not used. The higher the temperature, thehigher the absorbance will be. Varying incubation times will have a similar influence on the absorbance. 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
Enzyme Solution
Reconstitute the content of the vial labelled ‘Enzyme’ with 1 ml water (deionized, distilled, or ultra-pure) andmix thoroughly. Add 0.3 ml of Coenzyme followed by 0.7 ml of Adjustment Buffer. The total volume of theEnzyme Solution is 2.0 ml.
The Enzyme Solution has to be prepared freshly prior to the assay (not longer than 10 - 15 minutes inadvance). Discard after use!
Adrenaline 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.

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6.2 Sample preparation, extraction and acylation
1.

Pipette 10 µl of standards, controls, urine samples and 300 µl of plasma samples into therespective wells of the Extraction Plate.

2.

Add 250 µl of water (deionized, distilled, or ultra-pure) to the wells with standards, controls andurine samples.

3.

Pipette 50 µl of Assay Buffer into all wells.

4.

Pipette 50 µl of Extraction Buffer into all wells.

5.

Cover plate with Adhesive Foil and incubate 30 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

6.

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

7.

Pipette 1 ml of Wash 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.

8.

Pipette another 1 ml of Wash Buffer into all wells. Incubate the plate for 5 min at RT (20 – 25 °C) on ashaker (approx. 600 rpm). Empty plate and blot dry by tapping the inverted plate on absorbent
material.

9.

Pipette 150 µl of Acylation Buffer into all wells.

10.

Pipette 25 µl of Acylation Reagent into all wells.

11.

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

12.

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

13.

Pipette 1 ml of Wash Buffer into all wells. Incubate the plate for 10 min at RT (20 – 25 °C) on ashaker (approx. 600 rpm). Empty plate and blot dry by tapping the inverted plate on absorbent
material.

14.

Pipette 150 µl of Hydrochloric Acid into all wells.

15.

Cover plate with Adhesive Foil. Incubate 10 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).
Remove the foil and discard.
Do not decant the supernatant thereafter!
The following volumes of the supernatant are needed for the subsequent ELISA:
Adrenaline

100 µl

6.3 Adrenaline ELISA
1.

Pipette 25 µl of the Enzyme Solution (refer to 6.1) into all wells of the Adrenaline Microtiter Strips.

2.

Pipette 100 µl of the extracted standards, controls and samples into the appropriate wells.

3.

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

4.

Pipette 50 µl of the respective Adrenaline Antiserum into all wells and cover plate with Adhesive Foil.

5.

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

6.

Remove the foil. Discard or aspirate the content of the wells. Wash the plate 3 x by adding 300 µl ofWash Buffer, discarding the content and blotting dry each time by tapping the inverted plate onabsorbent material.

7.

Pipette 100 µl of the Enzyme Conjugate into all wells.

8.

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

9.

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 absorbentmaterial.

10. Pipette 100 µl of the Substrate into all wells and incubate for 25 ± 5 min at RT (20 – 25 °C) on ashaker (approx. 600 rpm).
Avoid exposure to direct sunlight!
11. Add 100 µl of the Stop Solution to each well and shake the microtiter plate to ensure a homogeneous
distribution of the solution.
12. 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

Adrenaline
0.7 - 200 ng/ml
18 - 6667 pg/ml

Urine
PlasmaThe 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.
Urine samples and controls
The concentrations of the urine samples and the Controls can be read directly from the standard curve.
Calculate the 24 h excretion for each urine sample: µg/24h = µg/l x l/24h
Plasma samples
The read concentrations of the plasma samples have to be divided by 30.
Conversion
Adrenaline (ng/ml) x 5.46 = Adrenaline (nmol/l)
Expected reference values
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
Adrenaline< 20 µg/day(110 nmol/day)< 100 pg/ml

24-hour urinePlasma
7.1 Quality control

The confidence limits of the kit controls are printed on the QC-Report.
7.2 Typical standard curve
Example, do not use for calculation!

OD

Adrenaline

ng/ml
8. Assay characteristics

Analytical Sensitivity

LOD
LOQ

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Urine (ng/ml)
Plasma (pg/ml)
Urine (ng/ml)
Plasma (pg/ml)
Effective: 2019-07-22

Adrenaline
0.9
100.7
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Cross Reactivity (%)

Substance

Analytical Specificity(Cross Reactivity)

Adrenaline

Derivatized Adrenaline
Derivatized Noradrenaline
Derivatized Dopamine
Metanephrine
Normetanephrine
3-Methoxytyramine
3-Methoxy-4-hydroxyphenylglycol
TyraminePhenylalanine, Caffeinic acid, LDopa, Homovanillic acid, Tyrosine,3-Methoxy-4-hydroxymandelic acid

Precision
Intra-Assay Urine (n = 60)
Sample Range (ng/ml)

CV (%)

123

17.4
12.4
13.1

Adrenaline

6.2 ± 1.1
21.4 ± 2.7
59.4 ± 7.8Inter-Assay Urine (n = 33)
Sample Range (ng/ml)

CV (%)

123

17.9
11.7
12.1

Adrenaline

Linearity

Recovery

5.2 ± 0.9
17.8 ± 2.1
54.2 ± 6.6

Adrenaline

Adrenaline

1000.13< 0.010.18< 0.01< 0.01< 0.01< 0.01< 0.01

Intra-Assay Plasma (n = 60)
Sample Range (pg/ml)
Adrenaline

12364.7 ± 15.9
258 ± 32.5
948 ± 105

24.7
12.7
11.0

Inter-Assay Plasma (n = 18)
Sample Range (pg/ml)
Adrenaline

Urine
PlasmaSerial dilution up to
1:512
1:512

Urine
PlasmaMean (%)
106105

Range (%)
94 - 120
88 - 117

123

76.4 ± 11.1
247 ± 27.5771 ± 101
Range (%)
92 - 123
94 - 115

CV (%)CV (%)
14.5
11.1
13.1

Mean (%)
108105Range
4.5 – 53.5 ng/ml
9.1 – 4268 pg/ml

9. References/Literature(1) Kim et al. Vitamin C prevents stress-induced damage on the heart caused by the death ofcardiomyocytes, through the down-regulation of the excessive production of catecholamine, TNF-α, andROS production in GULO(-I-) Vit C-Insufficient mice. Free Radical Biology and Medicine, 65:573-583(2013)(2) Bada et al. Peripheral vasodilatation determines cardiac output in exercising humans: insight from atrial
pacing. The Journal of Physiology, 590(8):2051-2060 (2012)(3) Parks et al. Employment and work schedule are related to telomere length in women. Occupational &Environmental Medicine 68(8):582-589 (2011)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 useContentCaution

Catalogue
numberEffective: 2019-07-22

Contains sufficient for<n> tests

For research use
only!
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