Abcam - Serotonin ELISA Kit

ab133053 –
Serotonin ELISA Kit
Instructions for Use
For quantitative detection of Serotonin in Human Serum, Platelets,

Plasma and Urine.
This product is for research use only and is not intended for diagnostic
use.
Version 7 Last Updated 9 March 2017

Table of Contents
INTRODUCTION
1. BACKGROUND 2
2. ASSAY SUMMARY 3
GENERAL INFORMATION
3. PRECAUTIONS 4
4. STORAGE AND STABILITY 5
5. MATERIALS SUPPLIED 5
6. MATERIALS REQUIRED, NOT SUPPLIED 6
7. LIMITATIONS 6
8. TECHNICAL HINTS 7
ASSAY PREPARATION
9. REAGENT PREPARATION 8
10. STANDARD PREPARATIONS 9
11. SAMPLE COLLECTION AND STORAGE 11
12. PLATE PREPARATION 13
ASSAY PROCEDURE
13. ASSAY PROCEDURE 14
DATA ANALYSIS
14. CALCULATIONS 16
15. TYPICAL DATA 17
16. TYPICAL SAMPLE VALUES 18
17. ASSAY SPECIFICITY 21
RESOURCES
18. TROUBLESHOOTING 22
19. NOTES 23
Discover more at www.abcam.com 1

INTRODUCTION
1. BACKGROUND
Abcam’s Serotonin in vitro competitive ELISA (Enzyme-Linked
Immunosorbent Assay) kit is designed for the accurate quantitative
measurement of Serotonin in Human Serum, Platelets, Plasma and
Urine.
A goat anti-rabbit IgG antibody has been precoated onto 96-well

plates. Standards or test samples are added to the wells, along with an
alkaline phosphatase (AP) conjugated-serotonin antigen and a
polyclonal rabbit antibody specific to Serotonin. After incubation the
excess reagents are washed away. pNpp substrate is added and after
a short incubation the enzyme reaction is stopped and the yellow color
generated is read at 405 nm. The intensity of the yellow coloration is
inversely proportional to the amount of Serotonin captured in the plate.
Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine found in the

central nervous system, gastrointestinal tract, and blood with broad
physiological functions as a neurotransmitter, in gastric motility,
hemostasis, and cardiovascular integrity. Defects in serotonin signaling
have been linked to a large number of complex behavioural disorders
in humans, including anxiety and depression, autism, and eating
disorders, and to neurodegenerative disorders such as Parkinson’s.
Platelets serve as the major reservoir of serotonin in the bloodstream.
When activated, platelets release serotonin in to the bloodstream
where it acts as a powerful vasoconstrictor. Treatment with Selective
Serotonin Uptake Inhibitors (SSRIs) dramatically reduces platelet
serotonin concentrations, and altered levels of serotonin in the
circulatory system are implicated in such diverse conditions such as
asthma, liver cirrhosis and carcinoid tumors.

INTRODUCTION
2. ASSAY SUMMARY
Prepare all reagents and samples as

instructed.
Add standards and samples to

appropriate wells.
Add prepared labeled AP-conjugate to

appropriate wells.
Add Serotonin antibody to appropriate

wells. Incubate at room temperature.
Add pNpp substrate to each well.

Incubate at room temperature. Add
Stop Solution to each well. Read
immediately.

GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.
 Some kit components contain azide, which may react with lead or
copper plumbing. When disposing of reagents always flush with
large volumes of water to prevent azide build-up.
 Stop Solution is a solution of trisodium phosphate. This solution is
caustic; care should be taken in use.
 The activity of the alkaline phosphatase conjugate is dependent on
the presence of Mg2+ and Zn2+ ions. The activity of the conjugate is
affected by concentrations of chelators (>10 mM) such as EDTA
and EGTA.
 We test this kit’s performance with a variety of samples, however it
is possible that high levels of interfering substances may cause
variation in assay results.
 Care should be taken handling the standard and conjugate
material because of the known and unknown effects of Serotonin.

GENERAL INFORMATION
4. STORAGE AND STABILITY

Store kit at +4°C immediately upon receipt, apart from the Alkaline
Phosphatase Conjugate, Standard and Antibody, which should be
stored at -20°C. Avoid multiple freeze-thaw cycles.
Refer to list of materials supplied for storage conditions of individual

components.
5. MATERIALS SUPPLIED
Storage
Item Amount
Condition
Goat anti-rabbit IgG Microplate (12 x 8 wells) 96 Wells +4ºC
Serotonin Alkaline Phosphatase Conjugate 2 x 150 ng -20ºC
Serotonin Antibody 2 vials -20ºC
Serotonin Standard 2 x 250 ng -20ºC
Assay Buffer 27 mL +4ºC
20X Wash Buffer Concentrate 30 mL +4ºC
pNpp Substrate 20 mL +4ºC
Stop Solution 5 mL +4ºC
Plate Sealer 2 x 1 unit +4ºC

GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED

These materials are not included in the kit, but will be required to
successfully utilize this assay:
 Standard microplate reader - capable of reading at 405 nm,
preferably with correction between 570 and 590 nm
 Automated plate washer (optional)
 Adjustable pipettes and pipette tips. Multichannel pipettes are
recommended when large sample sets are being analyzed
 Eppendorf tubes
 Microplate Shaker
 Absorbent paper for blotting
 6 M hydrochloric acid
 Deionized water
7. LIMITATIONS
 Assay kit intended for research use only. Not for use in diagnostic
procedures
 Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted

GENERAL INFORMATION
8. TECHNICAL HINTS
 Standards can be made up in either glass or plastic tubes.
 Pre-rinse the pipette tip with the reagent, use fresh pipette tips for
each sample, standard and reagent.
 Pipette standards and samples to the bottom of the wells.
 Add the reagents to the side of the well to avoid contamination.
 This kit uses break-apart microtiter strips, which allow the user to
measure as many samples as desired. Unused wells must be kept
desiccated at 4°C in the sealed bag provided. The wells should be
used in the frame provided.
 Care must be taken to minimize contamination by endogenous
alkaline phosphatase. Contaminating alkaline phosphatase
activity, especially in the substrate solution, may lead to high
blanks. Care should be taken not to touch pipet tips and other
items that are used in the assay with bare hands.
 Prior to addition of substrate, ensure that there is no residual wash
buffer in the wells. Any remaining wash buffer may cause variation
in assay results.
 This kit is sold based on number of tests. A ‘test’ simply
refers to a single assay well. The number of wells that contain
sample, control or standard will vary by product. Review the
protocol completely to confirm this kit meets your
requirements. Please contact our Technical Support staff with
any questions.

ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents and samples to room temperature (18 - 25°C)
prior to use.
9.1 Serotonin Antibody
Reconstitute one vial of Serotonin antibody with 3 mL of the
assay buffer and vortex thoroughly. Unused reconstituted
antibody should be discarded.
9.2 Serotonin Conjugate
Allow the Serotonin Alkaline Phosphatase Conjugate to
equilibrate to room temperature. Reconstitute one vial of
serotonin conjugate with 3 mL of the assay buffer. Mix
thoroughly. Any unused conjugate should be aliquoted and
re-frozen at or below -20°C.
9.3 1X Wash Buffer
Prepare the 1X Wash Buffer by diluting 5 mL of the 20X
Wash Buffer Concentrate in 95 mL of deionized water. Mix
thoroughly and gently.
9.4 Conjugate 1:20 Dilution for Total Activity Measurement
Prepare the Conjugate 1:20 Dilution by diluting 5 µL of the
reconstituted conjugate with 95 µL of the assay buffer. The
dilution should be used within three hours of preparation.
This 1:20 dilution is intended for use in the Total Activity (TA)
wells only.

ASSAY PREPARATION
10. STANDARD PREPARATIONS

Prepare serially diluted standards immediately prior to use. Always
prepare a fresh set of standards for every use. Diluted standards
should be used within 60 minutes of preparation.
10.1 Reconstitute one vial of Serotonin standard with 500 µL

Assay Buffer. Vortex to ensure pellet is fully dissolved. This
solution is Standard 1.
10.2 Label 5 tubes #2 – #6.
10.3 Add 375 µL of Assay Buffer into all tubes.
10.4 Prepare a 125 ng/mL Standard 2 by adding 125 µL of the
500 ng/mL Standard 1 to tube #2. Mix thoroughly and
gently.
10.5 Prepare Standard 3 by transferring 125 μL from Standard 2
to tube #3. Mix thoroughly and gently.
10.6 Using the table below as a guide, repeat for tubes #4
through# 6.

ASSAY PREPARATION
Volume
Volume Starting Final
Sample to of
Standard to Dilute Conc. Conc.
Dilute Diluent
(µL) (ng/mL) (ng/mL)
(µL)
1 Stock - 500 - 500
2 Standard 1 125 375 500 125
3 Standard 2 125 375 125 31.25
4 Standard 3 125 375 31.25 7.81
5 Standard 4 125 375 7.81 1.95
6 Standard 5 125 375 1.95 0.49

ASSAY PREPARATION
11. SAMPLE COLLECTION AND STORAGE

The Serotonin ELISA kit is compatible with natural and recombinant
Serotonin samples in serum, platelets, plasma and urine.
11.1 Protocol for Platelets
Platelets are isolated from whole blood and should be counted, prior to
extracting the serotonin
11.1.1 Blood should be collected by venipuncture into plastic
tubes containing anticoagulant; either citrate or EDTA
may be used.
11.1.2 Centrifuge at 1,600 x g for 15 minutes at room
temperature.
11.1.3 Collect the white buffy coat layer with a plastic pipette
and transfer to a fresh plastic tube note the volume
collected.
11.1.4 Count platelets in a hemocytometer.
11.1.5 Wash the platelets with twice the original volume using
physiological saline and centrifugation at 2,000 x g for
10 minutes. Repeat wash for a total of two washes.
11.1.6 Resuspend the platelet pellet back to the original
volume using distilled water. This suspension is then
frozen followed by thawing shortly before assay. The
frozen suspension may be aliquoted and stored for up
to two weeks.
11.2 Protocol for Serum
11.2.1 Collect whole blood in appropriate serum tubes.
temperature.
11.2.3 Remove serum to a clean plastic tube.
11.2.4 Sample should be aliquoted and frozen within two
hours of collection.
11.2.5 Samples may be stored frozen for up to weeks.

ASSAY PREPARATION
11.3 Protocol for Plasma

11.3.1 Collect whole blood in appropriate tubes.
temperature.
11.3.3 Remove plasma to a clean plastic tube.
11.3.4 Sample should be aliquoted and frozen within two
hours of collection.
11.3.5 Samples may be stored frozen for up to two weeks.
11.4 Protocol for Urine
11.4.1 Collect spontaneous or 24 hour urine in a bottle
containing 10 -15 mL of 6N HCl as preservative.
temperature.
11.4.3 Remove supernatant to a clean plastic tube.
11.4.4 Sample should be aliquoted and frozen and stored for
up to two weeks.

ASSAY PREPARATION
12 PLATE PREPARATION
 The 96 well plate strips included with this kit are supplied ready to
use. It is not necessary to rinse the plate prior to adding reagents.
 Unused well strips should be returned to the plate packet and
stored at +4°C.
 For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).
 Well effects have not been observed with this assay.
Recommended plate layout

1 2 3 4
A Bs Std 1 Std 5 Sample 3
B Bs Std 1 Std 5 Sample 3
C TA Std 2 Std 6 etc
D TA Std 2 Std 6 etc
E NSB Std 3 Sample 1
F NSB Std 3 Sample 1
G B0 Std 4 Sample 2
H B0 Std 4 Sample 2
Key:
Bs = Blank; contains substrate only.
TA = Total Activity; contains conjugate (5 µL) and substrate.
NSB = Non-specific binding; contains assay buffer, conjugate and
substrate.
B0 = 0 pg/mL standard; contains assay buffer, conjugate, antibody and
substrate.

ASSAY PROCEDURE
13 ASSAY PROCEDURE
 Equilibrate all materials and reagents to room temperature
 It is recommended to assay all standards, controls and
samples in duplicate
 Refer to the recommended plate layout in Section 12 before
proceeding with the assay
13.1 Add 150 µL of the Assay Buffer into the NSB (non-specific
binding) wells.
13.2 Add 100 µL of the Assay Buffer into the B0 (0 pg/mL
standard) wells.
13.3 Add 100 µL of prepared standards and 100 µL diluted
samples to the appropriate wells.
13.4 Add 50 μL of Serotonin Alkaline Phosphatase Conjugate
into NSB, B0, standard and sample wells, i.e. not TA (Total
Activity) and Bs (blank) wells.
13.5 Add 50 μL of Serotonin antibody into B0, standard and
sample wells, i.e. not Bs, TA and NSB wells.
13.6 Incubate the plate at room temperature on a plate shaker
for 2 hours at ~500 rpm. The plate may be covered with the
plate sealer provided.
13.7 Empty the contents of the wells and wash by adding 400 µL
of 1X Wash Buffer to every well. Repeat the wash 2 more
times for a total of 3 Washes. After the final wash, empty or
aspirate the wells, and firmly tap the plate on a lint free
paper towel to remove any remaining wash buffer.
13.8 Add 5 μL of the diluted (1:20) Serotonin Alkaline
Phosphatase Conjugate to the TA well only.
13.9 Add 200 μL of the pNpp Substrate solution to every well.
Incubate at room temperature for 1 hour without shaking.
13.10 Add 50 μL Stop Solution into each well. The plate should be
read immediately.

ASSAY PROCEDURE
13.11 After blanking the plate reader against the blank (Bs) wells,
read optical density at 405 nm, preferably with correction
between 570 and 590 nm. If the plate reader is not able to
be blanked against the Bs wells, manually subtract the
mean optical density of the blank wells from all readings.
The optimal speed for each shaker will vary and may range from
120 – 700 rpm. The speed must be set to ensure adequate mixing
of the wells, but not so vigorously that the contents of the wells
splash and contaminate other wells.

DATA ANALYSIS
14 CALCULATIONS
A four parameter algorithm (4PL) provides the best fit, though other
equations can be examined to see which provides the most accurate
(e.g. linear, semi-log, log/log, 4 parameter logistic). Interpolate protein
concentrations for unknown samples from the standard curve plotted.
14.1 Calculate the average net absorbance measurement (Average

Net OD) for each standard and sample by subtracting the
average NSB absorbance measurement from the average
absorbance measurement (Average OD) for each standard
and sample.
Average Net OD = Average Bound OD - Average NSB OD
14.2 Calculate the binding of each pair of standard wells as a

percentage of the maximum binding wells (B0), using the
following formula:
Percent Bound = (Net OD/ Net Bo OD) x 100
14.3 Plot the Percent Bound (B/B0) versus concentration of

serotonin for the standards. Approximate a straight line
through the points. The concentration of serotonin in the
unknowns can be determined by interpolation.
Samples producing signals greater than that of the highest standard

should be further diluted and reanalyzed, then multiplying the
concentration found by the appropriate dilution factor.

DATA ANALYSIS
15 TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only. A new standard curve must be generated for each
assay performed.
Mean
% Serotonin
Sample OD
Bound ng/mL
(-Blank)
Bs (0.088) - -
TA 0.997 - -
NSB 0.008 0 -
Standard 1 0.063 9.4 500
Standard 2 0.147 21.8 125
Standard 3 0.277 41.3 31.3
Standard 4 0.455 66.2 7.8
Standard 5 0.562 81.8 1.9
Standard 6 0.637 91.6 0.5
B0 0.691 100 0

DATA ANALYSIS
16 TYPICAL SAMPLE VALUES
SENSITIVITY –
The sensitivity, defined as 2 standard deviations from the mean signal
at zero, was determined from 7 independent standard curves. The
standard deviation was determined from 14 zero standard replicates to
be 0.293 ng/mL.
SAMPLE RECOVERY –
Spike Average
Recommended
Sample Type Concentration %
Dilution
(ng/mL) Recovery
100 106
Human Platelets 20 109 1:16
5 107
100 95
Human Serum 20 95 1:16
5 83
100 102
Human Citrate Plasma 20 101 1:16
5 83
100 107
Human Urine 20 91 1:16
5 104

DATA ANALYSIS
LINEARITY OF DILUTION –
Human samples containing serotonin were serially diluted 1:2 in the
assay buffer and measured in the assay. Results are shown in the
table below.
Average % of Expected
Dilution Platelets Serum Plasma Urine
Neat 88 - - -
1:2 103 - - -
1:4 115 - - -
1:8 114 93 - 88
1:16 117 118 115 107
1:32 112 116 110 105
1:64 100 106 73 103
PARALLELISM –
Dose-response curves from Human platelets and Human serum
diluted into assay buffer were compared to the Serotonin standard
curve. The parallel response indicates the standard effectively mimics
the native protein.

DATA ANALYSIS
PRECISION –
Intra-Assay
Intra-Assay
Serotonin (ng/mL)
%CV
Low 13.5 11.0
Medium 53.8 5.8
High 346.1 4.2
Inter-Assay
Inter-Assay
Serotonin (ng/mL)
%CV
Low 13.2 12.7
Medium 53.9 18.4
High 358.0 16.2

DATA ANALYSIS
17 ASSAY SPECIFICITY
The Serotonin ELISA Kit recognizes natural and recombinant forms of
Serotonin.
Compound Cross Reactivity
N-Acetyl Serotonin 17%
5-Hydroxy-L-tryptophan 0.4%
Tryptamine 0.1%
5-Hydroxyindoleacetic acid 0.03%
Melatonin 0.01%
Tyramine <0.004%
Tryptophan <0.004%

RESOURCES
18 TROUBLESHOOTING
Problem Cause Solution
Inaccurate pipetting Check pipettes

Poor
standard Prior to opening, briefly spin the
curve Improper standards stock standard tube and dissolve
dilution the powder thoroughly by gentle
mixing
Ensure sufficient incubation times;

Incubation times too
change to overnight
brief
standard/sample incubation
Low Signal
Inadequate reagent
Check pipettes and ensure correct
volumes or improper
preparation
dilution
Samples
give higher Starting sample
Dilute the specimens and repeat
value than concentration is too
the assay
the highest high
standard
Review manual for proper wash
Plate is insufficiently
technique. If using a plate washer,
washed
check all ports for obstructions
Large CV
Contaminated wash
Prepare fresh wash buffer
buffer
Low Improper storage of Store the all components as

sensitivity the kit directed

RESOURCES
19 NOTES

RESOURCES

RESOURCES

RESOURCES

UK, EU and ROW
Email: [email protected] | Tel: +44-(0)1223-696000
Austria
Email: [email protected] | Tel: 019-288-259
France
Email: [email protected] | Tel: 01-46-94-62-96
Germany
Email: [email protected] | Tel: 030-896-779-154
Spain
Switzerland
Email: [email protected]
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
US and Latin America

Email: [email protected] | Tel: 888-77-ABCAM (22226)
Canada
China and Asia Pacific

Email: [email protected] | Tel: 108008523689 (中國聯通)
Japan
Email: [email protected] | Tel: +81-(0)3-6231-0940
www.abcam.com | www.abcam.cn | www.abcam.co.jp
Copyright © 2017 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.
RESOURCES 27

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ab133053 –

Serotonin ELISA Kit

Instructions for Use

For quantitative detection of Serotonin in Human Serum, Platelets,

Version 7 Last Updated 9 March 2017

Discover more at www.abcam.com 1

A goat anti-rabbit IgG antibody has been precoated onto 96-well

Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine found in the

Discover more at www.abcam.com 2

Prepare all reagents and samples as

Add standards and samples to

Add prepared labeled AP-conjugate to

Add Serotonin antibody to appropriate

Add pNpp substrate to each well.

Discover more at www.abcam.com 3

Discover more at www.abcam.com 4

4. STORAGE AND STABILITY

Refer to list of materials supplied for storage conditions of individual

Discover more at www.abcam.com 5

6. MATERIALS REQUIRED, NOT SUPPLIED

Discover more at www.abcam.com 6

Discover more at www.abcam.com 7

Discover more at www.abcam.com 8

10. STANDARD PREPARATIONS

10.1 Reconstitute one vial of Serotonin standard with 500 µL

Discover more at www.abcam.com 9

Discover more at www.abcam.com 10

11. SAMPLE COLLECTION AND STORAGE

Discover more at www.abcam.com 11

11.3 Protocol for Plasma

Discover more at www.abcam.com 12

Recommended plate layout

Discover more at www.abcam.com 13

Discover more at www.abcam.com 14

Discover more at www.abcam.com 15

14.1 Calculate the average net absorbance measurement (Average

14.2 Calculate the binding of each pair of standard wells as a

14.3 Plot the Percent Bound (B/B0) versus concentration of

Samples producing signals greater than that of the highest standard

Discover more at www.abcam.com 16

Discover more at www.abcam.com 17

16 TYPICAL SAMPLE VALUES

Discover more at www.abcam.com 18

Dilution Platelets Serum Plasma Urine

Discover more at www.abcam.com 19

Discover more at www.abcam.com 20

Compound Cross Reactivity

N-Acetyl Serotonin 17%

5-Hydroxyindoleacetic acid 0.03%

Discover more at www.abcam.com 21

Problem Cause Solution

Inaccurate pipetting Check pipettes

Ensure sufficient incubation times;

Low Improper storage of Store the all components as

Discover more at www.abcam.com 22

Discover more at www.abcam.com 23

Discover more at www.abcam.com 24

Discover more at www.abcam.com 25

Discover more at www.abcam.com 26