Instructions for use

Serotonin ELISA Fast Track

BA E-8900R

Serotonin ELISA Fast Track
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of Serotonin in serum, urine and platelets.
In the first step, Serotonin is quantitatively acylated.
The subsequent competitive ELISA kit uses the microtiter plate format. The antigen is bound to the solid
phase of the microtiter plate. The acylated standards, controls and samples and the solid phase boundanalyte compete for a fixed number of antiserum binding sites. After the system is in equilibrium, free
antigen and free antigen-antiserum 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 ismonitored 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
Serotonin (5-hydroxytryptamine) is an intermediate product of tryptophan metabolism and is locatedprimarily in the enterochromaffin cells of intestine (EC-cells), serotonergic neurons of the brain, platelets ofthe blood and is well established as a neurotransmitter in the central nervous system. EC-cell production
accounts for 80% of the body's serotonin content. Serotonin is predominately metabolized to 5hydroxyindoleacetic acid (5-HIAA), which is excreted by the kidneys.
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 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 calibrator 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 Sheets (SDS).
The Safety Data Sheet for this product is made available directly on the website of the manufacturer orupon request.
(17) Kit reagents must be regarded as hazardous waste and disposed of 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
Serum/Plasma
Samples containing precipitates or fibrin strands might cause inaccurate results.
Hemolytic samples (up to 4 mg/ml hemoglobin), icteric samples (up to 50 mg/dl bilirubin) and lipemicsamples (up to 1700 mg/dl triglycerides) have no influence on the assay 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
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 Contents of the kit
BA D-0023
Contents:

Reaction Tubes *)- Ready to use
Reaction Tubes in a resealable pouch

Volume:2 x 50 tubes*) Instead of the Reaction Tubes, it is also possible to use 48 wells macrotiter plates for the sample
preparation and acylation (please refer to 6.2). These plates (BA D-0033) are available upon request.
BA E-0030

Wash Buffer Concentrate - Concentrated 50x

Contents:

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

Contents:

Goat anti-rabbit immunoglobulins conjugated with peroxidase

Volume:1 x 12 ml/vial, red cap

BA E-0055

Substrate - Ready to use

Contents:

Chromogenic substrate containing tetramethylbenzidine, substrate buffer andhydrogen peroxide

Volume:1 x 12 ml/vial, black capBA E-0080

Stop Solution - Ready to use

Contents:

0.25 M sulfuric acid

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

Hazards
identification:
H290 May be corrosive to metals.
BA E-0931

Serotonin Microtiter Strips - Ready to use

Contents:

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

BA E-8910

Serotonin Antiserum - Ready to use

Contents:

Rabbit anti-serotonin antibody, blue coloured

Volume:1 x 12 ml/vial, blue cap

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Acylation Reagent - Ready to use

BA E-8912Contents:

Acylation reagent in dimethylsulfoxide

Volume:1 x 3 ml/vial, green cap
Acylation Buffer - Ready to use

BA E-8911
Contents:

TRIS buffer with non-mercury preservative

Volume:1 x 55 ml/vial, light grey cap

Standards and Controls - Ready to use
Cat. no.

Component

Colour/Cap

Concentration
ng/mlConcentration
nmol/lVolume/Vial

BA R-8901

white004 ml

BA R-8902

light yellow

15

85.1

4 ml

BA R-8903

orange

50

284

4 ml

BA R-8904

dark blue

1508514 ml

BA R-8905

light grey500

2 840

4 ml

BA R-8906

2 500

14 1754 ml

BA R-8951

blacklight green

BA R-8952

dark red

Refer to vial labels for expected value andacceptable range!

4 ml
4 ml

Conversion:

Serotonin (ng/ml) x 5.67 = Serotonin (nmol/l)Contents:

TRIS buffer with non-mercury preservatives, spiked with defined quantity of serotonin

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

Calibrated precision pipettes to dispense volumes between 25 – 500 µl
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
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
Foods or liquids containing serotonin such as pineapple, eggplant, avocados, bananas, currants, kiwis,
melon, mirabelles, plums, peaches chocolate, gooseberries, tomatoes, or walnuts, should be avoided
2 days before and including the day of the sample collection (24-hour urine). Selective Serotonin Reuptake
Inhibitors (SSRIs) influence serotonin levels. People who are taking such medications should consult with
their doctor before specimen collection.
Repeated freezing and thawing of the samples should be avoided.
Serum
Collect blood by venipuncture (monovette or vacuette for serum), allow to clot, and separate serum bycentrifugation according to manufacturer’s instructions at room temperature. Do not centrifuge before
complete clotting has occurred.
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.
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.
Storage: up to 24 hours at 2 – 8 °C, for longer periods (up to 6 months) at -20 °C.
Avoid exposure to direct sunlight.

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More than 98 percent of the circulating serotonin is located in the platelets and is released during bloodclotting. Blood must be collected by venepuncture according to manufacturer’s instructions 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 xg). 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 (containing between
350,000 – 500,000 platelets/µl) and centrifugation (4,500 x g, 10 minutes at 4 C). The supernatant is thendiscarded.
200 µl of water (deionized, distilled, or ultra-pure) is added to the pellet and mixed thoroughly on a vortex
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.
25 µl of the supernatant is used for the acylation reaction.
For the determination of Serotonin in platelet-poor plasma and cerebrospinal fluid the Serotonin
Research™ ELISA (for details contact your local supplier) should be used.
6. Test procedure for serum, urine and platelets
Allow all reagents and samples to reach room temperature. The measurement in duplicates isrecommended. It is recommended to number the strips of the microwell plate before usage to avoid any
mix-up.
The binding of the antisera and the enzyme conjugates and the activity of the enzyme used are temperaturedependent, and the extinction values may vary if a thermostat is not used. The higher the temperature, thehigher the extinction values will be. Corresponding variations also apply to 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 Reagent
The Acylation Reagent (BA E-8912) has a freezing point of 18.5 °C. To ensure that the Acylation Reagent isliquid when being used, it must be ensured that the Acylation Reagent has reached room temperature andforms a homogeneous, crystal-free solution before being used.
Serotonin 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 of serum, urine and platelets
1.

Pipette 25 µl of standards, controls and serum, urine or platelets into the respective Reaction
Tubes.

2.

Add 500 µl of Acylation Buffer to all tubes.

3.

Add 25 µl of Acylation Reagent to all tubes.

4.

Mix thoroughly and incubate for 15 min at RT (20 – 25 °C).
Take 25 µl of the prepared standards, controls and samples for the Serotonin ELISA

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

Pipette 25 µl of the acylated standards, controls and samples into the appropriate wells of theSerotonin Microtiter Strips.

2.

Pipette 100 µl of the Serotonin Antiserum into all wells.

3.

Incubate 30 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).
Without usage of a shaker: shake the Serotonin Microtiter Strips shortly by hand and incubate for
1 h at RT (20 – 25 °C).

4.

Discard or aspirate the contents 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.

5.

Pipette 100 µl of the Conjugate into all wells.

6.

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).

7.

Discard or aspirate the contents 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.

9.

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 min at RT (20 – 25 °C).
Avoid exposure to direct sunlight!

10.
11.

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
Serotonin
10.2 – 2 500 ng/ml

Measuring range

The calibration 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 for urine and serum samples can be read directly from the calibration curve.
Calculation of serotonin in platelets
The content of serotonin in platelets is referred to 109 platelets.
Illustrative example:
Measured Serotonin concentration: 100 ng/ml
Number of the platelets in the PRP: 300 000 / µl = 0.3 x 109 platelets/ml with serotonin content of
100 ng.
The resulting serotonin content in the platelets is:
333 ng/ 109 platelets (100 ng serotonin x 1.0 x 109 /0.3 x 109)
Conversion
Serotonin (ng/ml) x 5.67 = Serotonin (nmol/l)
Expected reference values
It is strongly recommended that each laboratory should determine its own reference values.
Serotonin
70 – 270 ng/ml
50 - 250 µg/24h
500 - 950 ng/109 plateletsSerum24-hour urine
Serotonin in platelets

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7.1 Quality control
The confidence limits of the kit controls are printed on the QC-Report.
7.2 Typical standard curve:This example is a mean of 10 different runs; do not use for calculation!

OD

Serotonin

ng/ml
8. Assay characteristics
Limit of Detection (LOD)
Limit of Quantitation (LOQ)

Sensitivity

6.2 ng/ml
10.2 ng/ml

Substance

Cross Reactivity (%)

Tryptamine
Melatonin
5-Hydroxyindole acetic acidPhenylalanine
Histidine
Tyramine
5-HydroxytryptophaneAnalytical Specificity(Cross Reactivity)

Precision
Intra-Assay

Inter-Assay
SampleSerotonin
Urine (n = 40)
Serotonin
Serum (n = 20)

123123

Linearity
Urine
Serum

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0.05
0.08
< 0.014< 0.014< 0.019< 0.018< 0.014

Range (ng/ml)
mean ± SD
140.7 ± 16.3
421.2 ± 38.6
1560 ± 215.3
101.3 ± 9.6
246.8 ± 31.2
667.5 ± 71.6

CV (%)
11.6
9.2
13.8
9.7
12.6
10.8

SampleSerotonin
Urine (n = 15)
Serotonin
Serum (n = 7)

12312

Range (ng/ml)
CV (%)
mean ± SD
126.1 ± 14.2
11.3
414.5 ± 48.6
11.7
1343 ± 200.2
14.9
83.1 ± 10.3
12.4
244.3 ± 25.4
10.4

Range ng/ml

Serial dilution
up toMean Linearity(%)

Range (%)

30 - 3500
40 - 3000

1:65
1:33

100
96

88 - 118
80 -113

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Recovery

Mean (%)
96108

Urine
Serum

Method comparison versus
RIA*

Urine
Serum

Range (%)
74 - 105
89 - 126% Recovery
after spiking

y = 0.94x + 19.58; R2 = 0.98
y = 0.85x + 33.18; R2 = 0.97

*Commercial available RIA
9. References/Literature(1) Oliveira et al. Disturbances of W nt/β-catenin pathway and energy metabolism in early CKD: effect ofphosphate binders. Nephrol Dial Transplant, 28(10):2510-2517 (2013)(2) Shahin et al. Detection of Plasma and Urinary Monoamines and Their Metabolites in NonsegmentalVitiligo. Acta Dermatovenerol Croat, 20(1):14-20 (2012)(3) Ciprandi et al. Serotonin in Allergic Rhinitis: a Possible Role for Behavioural Symptoms, 10(3):183-188(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: 2019-07-24

Contains sufficient for<n> tests

For research use
only!

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