Instructions for use

5-HIAA ELISABA E-1900R

5-HIAA ELISA
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of 5-Hydroxy-3-Indole Acetic Acid (5-HIAA) inurine.
First, 5-HIAA is derivatized by methylation. The subsequent competitive ELISA uses the microtiter plate
format. The antigen is bound to the solid phase of the microtiter plate. The methylated analyte in thestandards, controls and samples and the solid phase bound analyte compete for a fixed number of antibody
binding sites. After the system has reached 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 IgGperoxidase 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.
1.2 Background
5-HIAA (5-hydroxyindoleacetic acid) is the major urinary metabolite of serotonin, an ubiquitous bioactiveamine. Serotonin, and consequently 5-HIAA, is produced in excess by most carcinoid tumors, especiallythose associated with the carcinoid syndrome. The syndrome includes flushing and diarrhea, and, lessfrequently, heart failure and bronchoconstriction. Quantitation of urinary 5-HIAA is therefore intended to test
for carcinoid.
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 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 Safety Data Sheet (SDS). TheSafety 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.
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
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 5-HIAA level inthe 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. Make sure that the Methylation Reagent is recapped immediately after pipetting.
4. Materials
4.1 Content of the kit
Adhesive Foil - Ready to use

BA D-0090
Contents:

Adhesive Foils in a resealable pouch

Volume:1 x 4 foils

BA D-0023
Contents:
BA D-0024
Contents:
BA E-0030

Reaction Tubes - Ready to use
2 x 50 tubes in a resealable pouch
Reaction Plate - Ready to use
1 x 96 well plate, empty in a resealable pouch
Wash Buffer Concentrate - Concentrated 50x

Contents:

Buffer with a non-ionic detergent and physiological pHVolume:1 x 20 ml/vial, light purple cap

BA E-0040

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 and hydrogen
peroxide

Volume:1 x 12 ml/black vial, black cap

BA 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

5-HIAA Microtiter Strips - Ready to use

Contents:

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

BA E-1910

5-HIAA Antiserum - Ready to use

Contents:

Rabbit anti – 5-HIAA antibody, blue coloured

Volume:1 x 6 ml/vial, blue cap

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Standards and Controls - Ready to use
Cat. no.
BA E-1901

Component

Colour/Cap

Concentration
mg/l

Concentrationµmol/lVolume/Vial

004 mlwhiteBA E-1902

light yellow

0.5

2.63

4 ml

BA E-1903

orange

1.5

7.88

4 ml

BA E-1904dark blue

526.34 ml

1578.84 ml

50262.5

BA E-1905

light grey

BA E-1906

black

BA E-1951

light green

BA E-1952

dark red

Conversion:

5-HIAA (mg/l) x 5.25 = 5-HIAA (µmol/l)Contents:

Acidic buffer spiked with defined quantity of 5-HIAA

BA E-0041

Refer to QC-Report for expected value andacceptable range!

Acidic buffer with non-mercury preservatives

Volume:1 x 22 ml/vial, white cap
Assay Buffer – Ready to use

Contents:

TRIS containing buffer with non-mercury preservative

Volume:2 x 55 ml/vial, dark green cap

BA E-1937

4 ml

Diluent – Ready to use

Contents:

BA E-1913

4 ml
4 ml

Methylation Buffer - Ready to use

Contents:

Methanol and dimethylformamide

Volume:1 x 11 ml/vial, brown cap

Hazards
identification:
H226 Flammable liquid and vapour.
H301 + H311 + H331 Toxic if swallowed, in contact with skin or if inhaled
H360D May damage fertility or the unborn child.
H370 Causes damage to organs (eyes).
H319 Causes serious eye irritation.
H312 + H332 Harmful in contact with skin or if inhaled.
BA E-1939

Methylation Reagent – Ready to use

Contents:Methylation reagent in diethyl ether

Volume:1 x 2.25 ml, white capHazards
identification:
H225 Highly flammable liquid and vapour.
H302 Harmful if swallowed.
H370 Causes damage to organs.
H330 Fatal if inhaled.
H336 May cause drowsiness or dizziness.
H350 May cause cancer.
4.2 Additional materials and equipment required but not provided in the kit








Calibrated precision pipettes to dispense volumes between 20 - 300 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)
Ventilated hood
Water (deionized, distilled, or ultra-pure)
Vortex mixer

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5. Sample collection and storage
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: for longer periods (up to 6 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 antisera and the enzyme conjugates and the activity of the enzyme used are temperaturedependent, and the absorption values may vary if a thermostat is not used. The higher the temperature, thehigher the absorption values will be. Varying incubation times will have similar influences on the absorbance.
The optimal temperature during the Enzyme Immunoassay is between 20 – 25 °C.
The Methylation Reagent is highly volatile. If possible, please pipette the Methylation Reagent with arepetitive pipette and make sure that the vial is recapped immediately after pipetting.
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
5-HIAA 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 Predilution of the standards, controls and samples
1.

Pipette 50 µl of standards, controls and urine samples into the respective wells of the Reaction
Plate.

2.

Pipette 200 µl of the Diluent into all wells.

3.

Shake for 1 min at RT (20 - 25 °C) on a shaker (approx. 600 rpm).
20 µl are needed for the methylation.

6.3 Methylation
1.

Pipette 20 µl of the prediluted standards, controls and urine samples into the respective
Reaction Tubes.
The following steps 2-5 have to be performed in a ventilated hood!

2.

Pipette 100 µl of Methylation Buffer into all tubes.

3.

Add 20 µl of Methylation Reagent to each tube and mix each tube immediately after addition ofthe Methylation Reagent.

4.

Cover all tubes and methylate for 20 min at RT (approx. 20 C).

5.

Pipette 1000 µl of Assay Buffer into all tubes.
After this step the use of a ventilated hood is not necessary any more!
Proceed with the ELISA (Chapter 6.4) immediately as the methylated standards, controls andsamples are only stable for 1 h!

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6.4 5-HIAA ELISA
1.

Pipette 25 µl of the methylated standards, controls and samples into the appropriate wells of the5-HIAA Microtiter Strips.

2.

Pipette 50 µl of the 5-HIAA Antiserum into all wells.

3.

Cover plate with Adhesive Foil and incubate for 1 h at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

4.

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.

5.

Pipette 100 µl of the Enzyme Conjugate into all wells.

6.

Cover plate with Adhesive Foil and incubate for 1 h at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

7.

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 onabsorbent 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.
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).

10.

7. Calculation of results
5-HIAA
0.17 – 50 mg/l

Measuring range

The 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 concentrationsof the analyte. OD-values found below the standard curve correspond to high concentrations of the analytein 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.
The total amount of 5-HIAA excreted in urine during 24 h is calculated as following:mg/24h = mg/l x l/24h
Conversion
5-HIAA (mg/l) x 5.25 = 5-HIAA (µmol/l)
Expected reference value
It is strongly recommended that each laboratory should determine its own reference values.
5-HIAA< 15 mg/day

24-hour urine
7.1 Quality control

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

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7.2 Typical standard curve
Example, do not use for calculation!

OD

5-HIAAmg/l
8. Assay characteristics
Analytical Sensitivity(Limit of Detection)

5-HIAA
0.17 mg/l
Cross Reactivity (%)

Substance
5-HIAA
Serotonin
5-Hydroxy-DL-Tryptophan
Tryptamine
Melatonin
5-Hydroxytryptamin
Vanillic mandelic acid
Homovanillic Acid

Analytical Specificity(Cross Reactivity)

Precision
Intra-AssayInter-AssaySample
1 n = 402 n = 38
3 n = 40

Linearity

RecoveryRange (mg/l)
1.7 ± 0.2
6.6 ± 0.6
18.4 ± 1.9CV (%)
14.1
8.6
10.3Sample
1n=92n=93n=9Range (mg/l)
3.1 ± 0.3
7.3 ± 0.8
19 ± 2.2

CV (%)
8.6
10.8
11.4

5-HIAARange
2.4 – 24.3 mg/l

Serial dilution up to1:10Range (%)
98 - 112

5-HIAA

Mean (%)
101

Range (%)
93 - 111% Recovery
after spiking

Method Comparison versus
HPLC

Version: 15.0-r

5-HIAA
100
5.5
1.8
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1

5-HIAA

HPLC = 0.9 ELISA + 0.2Effective: 2019-12-11

r = 0.99; n = 47

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9. References/Literature(1) Beer et al. Acupuncture for Hot Flashes in Patients With Prostate Cancer Patients. Urology, 76(5):1182–1188 (2010)(2) Korse et al. Chromogranin A as an Alternative to 5-Hydroxyindoleacetic Acid in the Evaluation ofSymptoms during Treatment of Patients with Neuroendocrine Tumors. Neuroendocrinology, 89:296–301(2008)(3) van Tuyl et al. Detection of small-bowel neuroendocrine tumors by video capsule endoscopy.
Gastrointestinal Endoscopy, 64 (1):66-72 (2006)

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

Version: 15.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2019-12-11

Contains sufficient for<n> tests

For research use
only!

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