Instructions for use

Adrenaline Research ELISA ™

BA E-5100

Adrenaline Research ELISA
1.

Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of adrenaline (epinephrine). Flexible test
system for various biological sample types and volumes.
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 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 standard curve
prepared with known standard concentrations.

2.

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 latex 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 Material Safety Data Sheet(MSDS). The Material Safety Data Sheet for this product is made available directly on the website of themanufacturer or upon request.
(17) Kit reagents must be regarded as hazardous waste and disposed according to national regulations.
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 withdesiccant again.

Version: 11.0

Effective: 2015-07-07

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4.

Materials

4.1

Content of the kit
BA D-0032
Content:

Microtiter Plate - Ready to use
1 x 96 wells, empty in a resealable pouch

BA D-0090

Adhesive Foil - Ready to use

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
Substrate - Ready to use

BA E-0055
Content:

Chromogenic substrate containing tetramethylbenzidine, substrate buffer and hydrogen
peroxide

Volume:1 x 12 ml/black 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

BA E-0131
Content:

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

BA E-5110

Adrenaline Antiserum - Ready to use

Content:

Rabbit anti-adrenaline antibody, blue coloured

Volume:1 x 6 ml/vial, blue cap

BA R-0050

Adjustment Buffer - Ready to use

Content:

TRIS buffer

Volume:1 x 4 ml/vial, green cap
TE Buffer - Ready to use

BA R-4617
Content:

TRIS-EDTA buffer

Volume:1 x 4 ml/vial, brown cap

Standards and Controls - Ready to use
Cat. no.

Component

Colour/CapConcentration
ng/mlConcentration
nmol/lVolume/VialBA R-5601

white004 ml

BA R-5602

light yellow

0.5

2.74 ml

BA R-5603

orange1.5

8.2

4 ml

BA R-5604

dark blue

527

4 ml

BA R-5605

light grey

20109

4 ml

BA R-5606

80437

4 ml

BA R-5651

black
light green

BA R-5652

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

Version: 11.0

Effective: 2015-07-07

3/8BA R-6611

Acylation Buffer - Ready to use

Content:

Buffer with light alkaline pH for the acylation

Volume:1 x 20 ml/vial, white cap

BA R-6612

Acylation Reagent - Ready to use

Content:

Acylation reagent in DMF and DMSO

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

Hazards
identification:
H225 Highly flammable liquid and vapour.
H360 May damage fertility or the unborn child.
H319 Causes serious eye irritation.
BA R-6614

Coenzyme - Ready to use

Content:

S-adenosyl-L-methionine

Volume:1 x 4 ml/vial, purple cap

BA R-6615

Enzyme - LyophilizedContent:

Catechol-O-methyltransferase

Volume:4 vials, pink cap

BA R-6618
Content:

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

BA R-6619

4.2

5.

Hydrochloric Acid - Ready to use

Content:

0.025 M Hydrochloric Acid, yellow coloured

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

Additional materials and equipment required but not provided in the kit− Calibrated precision pipettes to dispense volumes between 1 – 750 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− Shaker (shaking amplitude 3 mm; approx. 600 rpm)
− Temperature controlled incubator (37 °C) or similar heating device− Absorbent material (paper towel)− Water (deionized, distilled, or ultra-pure)− Vortex mixer
Sample collection and storage
Storage: up to 6 hours at 2 – 8 °C; for longer periods (up to 6 months) at -20 °C or –80 °C.
Advice for the preservation of the biological sample: to prevent catecholamine degradation, add EDTA
(final concentration 1 mM) and sodium metabisulfite (final concentration 4 mM) to the sample.

6.

Test procedure
Allow reagents and samples to reach room temperature and mix thoroughly by gentle inversion before
use. Duplicate determinations are recommended.
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

Version: 11.0

Effective: 2015-07-07

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6.1

Preparation of reagents
Wash Buffer
Dilute the 20 ml Wash Buffer Concentrate with water (deionized, distilled, or ultra-pure) to a final volumeof 1000 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)and mix thoroughly. Add 0.3 ml of Coenzyme followed by 0.7 ml of Adjustment Buffer. The total volumeof the Enzyme 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!

6.2

Sample preparation
The Adrenaline Research ELISA is a flexible test system for various biological sample types and volumes.
It is not possible to give a general advice how to prepare the samples. However, the following basics
should help the researcher to fit the protocol to his specific needs.
• Avoid excess of acid: excess of acid might exceed the buffer capacity of the extraction buffer.
A pH > 7.0 during the extraction is mandatory.
• Prevent adrenaline degradation by adding preservatives to the sample (see Sample collection andstorage).
• Avoid chaotropic chemicals like perchloric acid. The high salt content might reduce the recovery ofadrenaline. If your samples already contain high amounts of perchloric acid, neutralize the sample prior
to the extraction step.
• Tissue samples can be homogenised in 0.01 N HCl in the presence of EDTA and sodium metabisulfite.
Under these conditions, adrenaline is positively charged which reduces binding to proteins and optimizes
solubility.
• Avoid samples that contain substances with a cis-diol structure. These will reduce the recovery of theadrenaline.
• It is advisable to perform a “Proof of Principle” to determine the recovery of the adrenaline in yoursamples. Prepare a stock solution of adrenaline. Add small amounts (to change the native sample matrix
as less as possible) of the stock solutions to the sample matrix and check the recovery.
• The used sample volume determines the sensitivity of this test. Determine the sample volume needed todetermine the adrenaline in your sample by testing different amounts of sample volume.
If you need any support in establishing a protocol for your specific purposes, do not hesitate to contact
the manufacturer directly!

6.3

Extraction and acylation
The Adrenaline Research ELISA offers a flexible test system for various biological sample types andvolumes. Step 1 of the extraction procedure depends on the sample volume:- in case you have sample volumes between 1 – 100 µl follow 1.1- in case you have sample volumes between 100 – 500 µl follow 1.2- in case you have sample volumes between 500 – 750 µl follow 1.3
Within a run it is only possible to measure samples with the same volume!

1.

1.1
Sample volume 1 – 100 µl

1.2
Sample volume 100 – 500 µl

1.3Sample volume 500 – 750 µl

2.

Pipette into the respective wells
Pipette into the respective wellsof the Extraction Plate:of the Extraction Plate:
10 µl standards, 10 µl
10 µl standards, 10 µlcontrols and 1 – 100 µl
controls and 100 – 500 µl
sample.
sample.
Fill up each well with water
Fill up each well with water(deionized, distilled, or ultra(deionized, distilled, or ultra-pure)pure) to a final volume of
to a final volume of 500 µl [e.g.
100 µl [e.g. 10 µl standard plus
10 µl standard plus 490 µl water
90 µl water (deionized, distilled,(deionized, distilled, or ultraor ultra-pure)].
pure)].
Pipette 25 µl of TE Buffer into all wells

3.

Cover the plate with Adhesive Foil. Shake 60 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

4.

Remove the foil and empty the plate. Blot dry by tapping the inverted plate on absorbent material.

Version: 11.0

Effective: 2015-07-07

Pipette into the respective wellsof the Extraction Plate:
10 µl standards, 10 µlcontrols and 500 – 750 µl
sample.
Fill up each well with water(deionized, distilled, or ultrapure) to a final volume of
750 µl [e.g. 10 µl standard plus
740 µl water (deionized, distilled,or ultra-pure)].

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5.

Pipette 1 ml of Wash Buffer into all wells.

6.

Shake 5 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

7.

Blot dry by tapping the inverted plate on absorbent material.

8.

Wash one more time as described (step 5, 6 and 7)!

9.

Pipette 150 µl of Acylation Buffer into all wells.

10.

Pipette 25 µl of Acylation Reagent into all wells.

11.

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

12.

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

13.

Pipette 1 ml of Wash Buffer into all wells.

14.

Shake 5 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

15.

Blot dry by tapping the inverted plate on absorbent material.

16.

Wash one more time as described (step 13, 14, 15).

17.

Pipette 100 µl of Hydrochloric Acid into all wells.

18.

Cover plate with Adhesive Foil. Shake 10 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).
Do not decant the supernatant thereafter!
90 µl of the supernatant is needed for the subsequent enzymatic conversion

6.4

Enzymatic Conversion

1.

Pipette 90 µl of the extracted standards, controls and samples into the respective wells of theMicrotiter Plate.

2.

Add 25 µl of Enzyme Solution (refer to 6.1) to all wells.

3.

Cover plate with Adhesive Foil. Shake 1 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm) to mix.

4.

Incubate for 2 h at 37°C. The following volumes of the supernatants are needed for the subsequent
ELISA:
Adrenaline

6.5

100 µlAdrenaline ELISA

1.

Pipette 100 µl of standards, controls and samples from the Enzyme Plate (refer to 6.4) into therespective pre-coated Adrenaline Microtiter Strips.

2.

Pipette 50 µl of the respective Adrenaline Antiserum into all wells.

3.

Cover the plate with Adhesive Foil. Shake 1 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

4.

Incubate for 15 – 20 h (overnight) at 2 – 8 °C.

5.

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

6.

Pipette 100 µl of Enzyme Conjugate into all wells.

7.

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

8.

Remove the foil. Discard or aspirate the content 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 Substrate into all wells.

10. Incubate 20 - 30 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).
Avoid exposure to direct sunlight!
11. Pipette 100 µl of Stop Solution into all wells.
12. 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).

Version: 11.0

Effective: 2015-07-07

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7.

Calculation of results
The standard curve from which the concentrations of the samples can be read off, is obtained by plotting
the absorbance readings (calculate the mean absorbance) measured for the standards (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 taken from the standard curve have to be multiplied by acorrection factor.
10 µl (volume of standards extracted)
Correction factor =sample volume (µl) extracted

Example750 µl of the sample is extracted and the concentration taken from the standard curve is 0.45 ng/ml
adrenaline.
Correction factor = 10/750 = 0.013
Concentration of the sample = 0.45 ng/ml x 0.013 = 0.006 ng/ml = 6 pg/ml adrenaline
Conversion
Adrenaline (ng/ml) x 5.46 = Adrenaline (nmol/l)
7.1

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

8.

Assay characteristics
Cross Reactivity (%)

Substance

Analytical Specificity(Cross Reactivity)

Adrenaline
1000.20< 0.0007
0.64
0.0009< 0.0007
0.03< 0.0007

Derivatized AdrenalineDerivatized Noradrenaline
Derivatized Dopamine
Metanephrine
Normetanephrine
3-Methoxytyramine
3-Methoxy-4-hydroxyphenylglycol
Tyramine
Phenylalanine, Caffeinic acid, L-Dopa,
Homovanillic acid, Tyrosine,
3-Methoxy-4-hydroxymandelic acid

Sensitivity(Limit of Detection)< 0.0007

Adrenaline
0.25 ng/ml x C*

C* = Correction factor (refer to 7.)
Analytical Sensitivity(750 µl undiluted sample)

Adrenaline
3.3 pg/ml

Functional Sensitivity(750 µl undiluted sample)

Adrenaline
5 pg/ml

Precision
Intra-Assay Human EDTA-Plasma

Adrenaline

Version: 11.0Sample

Mean ± 3 SD (pg/ml)

SD (pg/ml)

CV (%)

highmedium
low1329.3 ± 372.6
412.1 ± 129.6
37.9 ± 19.5

124.2
43.2
6.5

9.3
10.5
17.1

Effective: 2015-07-07

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Intra-Assay Cell Culture Medium (RPMI)
SampleMean ± 3 SD (pg/ml)

SD (pg/ml)

CV (%)

Adrenaline

highmediumlow

1649.6 ± 555.0
526.2 ± 186.6
38.7 ± 18.9

185
62.2
6.3

11.2
11.8
16.3

Recovery Adrenaline
Human EDTA-Plasma
Cell Culture Medium

Mean (%)
104.0
95.5

Range (%)
89.4 – 128.3
81.6 – 109.6

SD (%)
13.18.3

CV (%)
12.6
8.7

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

Version: 11.0

Storagetemperature

Manufacturer

Contains sufficient for<n> tests

Expiry date

Batch code

For in-vitro diagnostic
use only!

Consult instructions
for useContent

CE labelled

CautionCatalogue
numberFor research use
only!

Effective: 2015-07-07

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