Instructions for use

Serotonin Research ELISA ™

BA E-5900

Serotonin Research ELISA
1.

Intended use and principle of the test
Ultra-sensitive Enzyme Immunoassay for the quantitative determination of Serotonin. Flexible test
system for various biological sample types and volumes.
Serotonin is acylated and detected by the subsequent competitive ELISA kit, which uses the microtiterplate format. Antigens are bound to the solid phase of the microtiter plate. The acylated standards,
controls and samples and the solid phase bound analytes compete for a fixed number of antibody bindingsites. After the system is in equilibrium, free antigen and free antigen-antibody complexes are removed
by washing. The antibody bound to the solid phase is detected 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: 12.0

Effective: 2016-03-30

2/7

4.

Materials

4.1

Contents of the kit
Standards and Controls - Concentrated*
Cat. no.

Component

Colour/Cap

Volume/Concentration
Concentration
ng/ml nmol/l pg/sample vol. pmol/sample vol.
Vial

BA R-8901

white00004 ml

BA R-8902

light yellow

0.015

0.085

1.5

8.5

4 ml

BA R-8903

orange

0.05

0.28

528.4

4 ml

BA R-8904

dark blue

0.15

0.85

1585

4 ml

BA R-8905

light grey

0.52.850284

4 ml

BA R-8906

2.514250

1 418

4 ml

BA R-8951

blacklight green
dark red

Refer to QC-Report for expected value andacceptable range!

4 ml

BA R-8952
Conversion:
Content:

4 ml
Serotonin (ng/ml) x 5.67 = Serotonin (nmol/l)Serotonin (pg/sample volume) x 5.67 = Serotonin (pmol/sample volume)
TRIS buffer with non-mercury preservative, spiked with defined quantity of serotonin*Standards and controls have to be diluted 1+1000 prior to use (please see chapter 6.1 for furtherinformation).
The shown concentrations apply to diluted standards and controls (1+1000) when 100 µl of undiluted
sample is used. Please refer to chapter 7 for the use of a correction factor in case of using diluted
samples and/or less sample volume.
BA E-5934
Content:

Acylation Plate - Ready to use
1 x 96 well plate, pre-coated with acylation reagent
Stabilizer - Ready to use

BA E-5937
Content:

Stabilizing agent, 10% solution

Volume:1 x 4 ml/vial, purple cap
Dilution Concentrate – Concentrated 20x

BA E-5941
Content:

TRIS buffer with 1% stabilizing agent and a non-mercury preservative

Volume:1 x 50 ml/vial, white cap

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

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

BA E-5910

Serotonin Antiserum - Ready to use

Content:

Rabbit anti-serotonin antibody, blue coloured

Volume:1 x 3 ml/vial, blue cap

Version: 12.0

Effective: 2016-03-30

3/7

BA E-5911

4.2

5.

Acylation Buffer - Ready to use

Content:

TRIS buffer with non-mercury preservative

Volume:1 x 4 ml/vial, yellow cap

BA E-0931

Serotonin Microtiter Strips - Ready to use

Content:

1 x 96 well (12x8) antigen precoated microwell plate in a resealable pouch withdesiccantAdditional materials and equipment required but not provided in the kit− Calibrated precision pipettes to dispense volumes between 1 – 100 µl; 10 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− Plate shaker (shaking amplitude 3 mm; approx. 600 rpm)
− Absorbent material (paper towel)− Water (deionized, distilled, or ultra-pure)− Vortex mixer
Sample collection and storage
In general this assay is dedicated for any biological sample such as serum, urine, platelets, platelet-poor
plasma, tissue homogenates, dialysates and other samples.
Storage: up to 6 hours at 2 – 8 °C, for longer periods (up to 6 months) at -20 °C. Avoid exposure todirect sunlight.
Serum
Collect blood by venipuncture (Monovette™ or Vacuette™ for serum), allow to clot, and separate serumby centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients
receiving anticoagulant therapy may require increased clotting time.
Haemolytic and especially lipemic samples should not be used for the assay.
Urine
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.
Platelets
More than 98 percent of the circulating serotonin is located in the platelets and is released during bloodclotting. Blood must be collected by venipuncture in plastic tubes (Monovette™ or Vacuette™) containing
EDTA or Citrate as anticoagulant.
To obtain platelet-rich plasma (PRP) the samples are centrifuged for 10 minutes at room temperature(200 x g). Transfer the supernatant to another tube and count the platelets.
The platelet pellet is obtained by adding 800 µl of physiological saline to 200 µl of PRP (containingbetween 350,000 – 500,000 platelets/µl) and centrifugation (4,500 x g, 10 minutes at 4 °C). The
supernatant is then discarded.
200 µl of water (deionized, distilled, or ultra-pure) is added to the pellet and mixed thoroughly on avortex mixer. This suspension can be stored frozen for several weeks at < -20 °C.
After thawing of the frozen samples, centrifuge at 10,000 x g for 2 minutes at room temperature.
To protect Serotonin against oxidative degradation the samples should contain 1% Stabilizer
BA E-5937.

6.

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

6.1

Sample preparation
The Serotonin Research ELISA is a flexible high sensitive test system for various biological sample types
and sizes. 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.
• Using poorly diluted or undiluted samples might lead to incorrect results due to a matrix effect. Therefore
it is advisable to perform a linearity experiment prior to the test. Make different dilutions of a sample with
the included diluent (e.g. 1:1, 1:4, 1:10, 1:20 and so on), spike each dilution with the same known
concentration and check the recovery. If the samples are found consistently correct and no matrix effectis detected, samples can be used undiluted.

Version: 12.0Effective: 2016-03-30

4/7

• If a matrix effect is detected, samples should be diluted with the included diluent prior to the test. It isalso possible to dilute the standards with the sample matrix instead of diluent, in order to create thesame matrix conditions for standards and samples. Currently the following buffers/solvents are evaluatedfor use: Ringer Buffer, PBS and 0.9% NaCl. If another substance is used, please check the compatibility
by a Proof of Principle prior to the measurement. Prepare a stock solution of serotonin or use standard F.
Add small amounts (to change the native sample matrix as little as possible) of the stock solution or
standard F to the sample matrix and check the recovery. Please take the correction factor into account(see chapter 7).
• If the expected sample concentrations are higher than the standard range (see chapter 4.1) samples
should be diluted accordingly with the included diluent. Please take the correction factor into account (seechapter 7).
• The measuring range and sensitivity of this test are defined by the correction factor, which is calculated
by sample volume and dilution (see chapter 7). If the expected concentrations are unknown, please test
different dilutions and amounts of sample volume, to make sure that the samples will fall into themeasuring range of this assay.
• Serotonin decomposes fast in acidic solution (< pH 3) and at higher temperatures.
• When acidic sample solutions are used, protect serotonin by keeping the temperature low (2 -8 °C). Use
pre-cooled buffers and materials. Adjust the pH to (6 – 7.4) as soon as possible.
• A pH 7 - 8.5 during acylation (see step 6.3.4) is mandatory.
• To protect serotonin against oxidative degradation add Stabilizer

BA E-5937 (refer to chapter 5).

If you need any support in establishing a protocol for your specific purposes, do not hesitate to contact
the manufacturer directly!
6.2

Preparation of reagents
Diluent
Dilute the 50 ml Dilution Concentrate with water (deionized, distilled, or ultra-pure) to a final volume of1000 ml.
The Diluent (Diluted Dilution Concentrate) contains 1% Stabilizer
BA E-5937.
Storage: 1 month at 2 – 8 °C
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
Standards and controls
The standards and controls have to be diluted freshly 1 + 1000 with Diluent or buffer*1, for example:
10 µl standard + 10 ml Diluent or buffer*1.
*1 The buffer used for the respective experiment, enriched with 1% Stabilizer

6.3

BA E-5937.

Acylation

1.

Pipette 100 µl of diluted standards, diluted controls, and 1 – 100 µl of samples into therespective wells of the Acylation Plate*2).

2.

Add Diluent or buffer* (refer to 6.1) to the wells containing the samples to a final volume of 100 µl.

3.

Add 25 µl Acylation Buffer to all tubes.

4.

Acylate for 30 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).
100 µl of the acylated standards, controls and samples are needed for the subsequent ELISA*2)

The wells of the Acylation Plate are covered by plastic bars which have to be removed prior to use.

Version: 12.0

Effective: 2016-03-30

5/7

6.4

Serotonin ELISA

1.

Pipette 100 µl of the acylated standards, controls and samples into the appropriate wells of theSerotonin/5-HIAA Microtiter Strips.

2.

Pipette 25 µl of the Serotonin Antiserum into all wells.

3.

Cover plate with Adhesive Foil and incubate for 15 - 20 h at 2 – 8 °C.

4.

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.

5.

Pipette 100 µl of the Enzyme Conjugate into all wells.

6.

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

7.

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.

8.

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

9.

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

10.

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

7.

Calculation of results
The calibration curve from which the concentrations of the samples can be read off, is obtained byplotting the absorbance readings (calculate the mean absorbance) measured for the standards (linear, yaxis) against the corresponding standard concentrations (logarithmic, x-axis).
The use of a non-linear regression for curve fitting (e.g. spline, 4- parameter, akima) is recommended.
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:
100 µl (volume of standards)
Correction factor =sample volume (µl)

Three different examples are shown to illustrate the calculation of possible correction factors:
Example 1)
10 µl of the undiluted sample are acylated and the concentration taken from the standard curve is 0.02
ng/ml serotonin.
Correction factor = 100/10 = 10
Final concentration of the sample = 0.02 ng/ml x 10 = 0.2 ng/ml serotonin
Example 2)
100 µl of the 1:100 prediluted sample are acylated and the concentration taken from the standard curveis 0.02 ng/ml serotonin.
Correction factor = 100
Final concentration of the sample = 0.02 ng/ml x 100 = 2.0 ng/ml serotonin
Example 3)
10 µl of the 1:100 prediluted sample are acylated and the concentration taken from the standard curve is
0.02 ng/ml serotonin.
Correction factor = 100 x (100/10) = 1000
Final concentration of the sample = 0.02 ng/ml x 1000 = 20 ng/ml serotonin
Conversion
Serotonin (pg/ml) x 5.67 = Serotonin (pmol/l)Serotonin (pg/sample volume) x 5.67 = Serotonin (pmol/sample volume)

Version: 12.0

Effective: 2016-03-30

6/77.1

Quality control
The confidence limits of the kit controls are listed in the QC-Report.

8.

Assay characteristics

8.1

Sensitivity
Serotonin
Sensitivity

0.005 ng/ml x C*

C* = correction factor (refer to 7.)
8.2

Specificity
CompoundCross-reactivity (%)

Serotonin

100

Tryptamine

0.19

Melatonin

0.03

5-Hydroxyindole acetic acid

<0.002

Phenylalanine<0.002

Histidine<0.002Tyramine<0.0025-Hydroxytryptophan<0.002For literature or any other information please contact your local supplier.
Symbols:

Version: 12.0Storage
temperature

Manufacturer

Contains sufficient for<n> tests

Expiry date

Batch code

For in-vitro diagnostic
use only!

Consult instructions
for useContent

CE labelled

CautionCatalogue
number

For research use
only!

Effective: 2016-03-30

7/7