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Melatonin Direct RIA
1. Introduction
1.1 Intended use and principle of the test
125I – Radioimmunoassay for the direct quantitative determination of melatonin in human serum and
plasma.
The assay procedure follows the basic principle of radioimmunoassay, involving competition between a
radioactive and a non-radioactive antigen for a fixed number of antibody binding sites. When the system
is in equilibrium, the antibody bound radioactivity is precipitated with a second antibody in the presence
of polyethylene glycol. After centrifugation and decantation of the supernatant the precipitate is counted
in a gamma counter. The amount of 125I-labelled antigen bound to the antibody is inversely proportional
to the analyte concentration of the sample. Quantification of unknown samples is achieved by comparing
their activity with a reference curve prepared with known standards.
1.2 Clinical application
In mammals, melatonin is biosynthesized in the pineal gland from the essential dietary amino acid
tryptophan, with serotonin produced as an intermediate. In general melatonin acts as an endocrine
hormone playing a key role in the regulation of the biorhythm.
The serum and saliva melatonin levels in humans show a marked circadian rhythm characterized by very
low levels during day time and high levels during night time.
Altered patterns and/or levels of melatonin secretion have been reported to coincide with sleep disorders,
jet lag, depression, stress, schizophrenia, hypothalamic amenorrhea, pregnancy, anorexia nervosa, some
forms of cancer, immunological disorders as well as control of sexual maturation during puberty.
Many biological effects of melatonin are generated through activation of melatonin receptors, while others
are due to its role as a powerful scavenger for free radicals in the human body.
Therapeutic consequences should never be based on laboratory results alone even if all test results are in
agreement with the items as under point “Procedural cautions, guidelines and warnings”. Any laboratory
result is only a part of the total clinical picture of the patient.
Only in cases where the laboratory results are in an acceptable agreement with the overall clinical picture
of the patient it can be used for therapeutic consequences.
The test result itself should never be the sole determinant for deriving any therapeutic consequences.
2. Procedural cautions, guidelines, warnings and limitations
2.1 Precautions, 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 the
instructions provided.
(2) This assay was validated for certain types of samples as indicated in Intended Use (please refer to
Chapter 1). Any off-label use of this kit is in the responsibility of the user and the manufacturer cannot be
held liable.
(3) Reagents of this kit which contain human serum or plasma have been tested and confirmed negative for
HIV I/II, HBsAg and HCV by approved procedures. All reagents, however, should be treated as potential
biohazards in use and for disposal.
(4) The principles of Good Laboratory Practice (GLP) have to be followed.
(5) In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective
gloves and protective glasses where necessary.
(6) 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.
(7) For the dilution or reconstitution purposes use deionized, distilled or ultra-pure water.
(8) The radioactive material (125Iodine, half life 60 days, emitting ionizing X-radiation with 28 keV and Gradiation
with 35.5 keV) may be received, acquired, possessed and used only by physicians, laboratories
or hospitals. In compliance with regulations, a copy of the customer’s current radioisotope license must
be on file with the supplier. Orders cannot be shipped until the license is received by the supplier
(Radiation Protection Act of June 30, 1989).
(9) Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
(10) Once the test has been started, all steps should be completed without interruption. Make sure that the
required reagents, materials and devices are prepared ready at the appropriate time.
(11) Incubation times do influence the results. All tubes should be handled in the same order and time
intervals.
English
Version: 13.0 Effective: 2015-01-14 3/13
(12) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,
sample, standard and control.
(13) A standard curve must be established for each run.
(14) The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report.
(15) 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.
(16) Some reagents contain sodium azide (NaN3) as preservative. In case of contact with eyes or skin, rinse
off immediay with water. NaN3 may react with lead and copper plumbing to form explosive metal
azides. When disposing reagents, flush with a large volume of water to avoid azide build-up.
(17) 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 available directly on the website of the
manufacturer or upon request.
(18) The expected reference values reported in this test instruction are only indicative. It is recommended that
each laboratory establishes its own reference intervals.
(19) The results obtained with this test kit should not be taken as the sole reason for any therapeutic
consequence but have to be correlated to other diagnostic tests and clinical observations.
(20) 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.
2.2.1 Interfering substances
Plasma/Serum
Samples containing precipitates or fibrin strands or which are haemolytic or lipemic might cause
inaccurate results.
2.2.2 Drug interferences
There are no known substances (drugs) which ingestion interferes with the measurement of melatonin
level in the sample.
2.2.3 High-Dose-Hook effect
No 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.
Melatonin is sensitive to light-exposure. To avoid photo-oxidative reduction of melatonin, it is necessary
to keep it away from direct sunlight and from heat.
4. Materials
4.1 Content of the kit
BA R-0028 Equalizing Reagent - Lyophilized
Content: Human serum, negative for HIV I/II, HBsAg and HCV
Volume: 2 vials, dark green cap
BA R-0030 Precipitating Reagent - Ready to use
Content: Goat anti-rabbit serum in PEG phosphate buffer
Volume: 2 x 55 ml/vial, yellow cap
BA R-3310 Melatonin Antiserum - Ready to use
Content: Rabbit anti-melatonin antibody, blue coloured
Volume: 1 x 5.25 ml/vial, blue cap
Version: 13.0 Effective: 2015-01-14 4/13
Standards and Controls - Ready to use
Cat. no. Component Colour/Cap Concentration
pg/ml
Concentration
pmol/l
Volume/
Vial
BA R-3301 white 0 0 4 ml
BA R-3302 light yellow 3 12.9 4 ml
BA R-3303 orange 10 43 4 ml
BA R-3304 dark blue 30 129 4 ml
BA R-3305 light grey 100 430 4 ml
BA R-3306 black 300 1 290 4 ml
BA R-3307 brown 1 000 4 300 4 ml
BA R-3351 light green Refer to QC-Report for expected value and
acceptable range!
4 ml
BA R-3352 dark red 4 ml
Conversion: Melatonin (pg/ml) x 4.30 = Melatonin (pmol/l)
Content: TRIS buffer with non-mercury preservatives, spiked with defined quantity of melatonin
BA R-3313 Assay Buffer - Ready to use
Content: TRIS buffer
Volume: 1 x 15 ml/vial, light purple cap
Hazards
identification:
H315 Causes skin irritation.
H319 Causes serious eye irritation.
BA R-3315 Enzyme - Lyophilized
Content: Digestive enzyme
Volume: 4 vials, pink cap
BA R-3316 Enzyme Buffer - Ready to use
Content: 1 M hydrochloric acid
Volume: 1 x 15 ml/vial, orange cap
BA R-3320 125I – Melatonin - Ready to use
Content: 125I labeled Melatonin, red coloured
Volume: 1 x 3 ml/vial, red cap
Hazards
identification:
Radioactive, activity < 200 kBq
4.2 Additional materials and equipment required but not provided in the kit
? Calibrated precision pipettes to dispense volumes between 15 - 1000 μl; 3 ml, 10 ml
? Conical plastic tubes (polypropylene, polystyrene) and suitable rack
? Centrifuge (preferable refrigerated) capable of at least 3 000 x g
? Suitable device for aspirating or decanting the tubes
? Vortex mixer
? Gamma counter
? Water (deionized, distilled, or ultra-pure)
? Absorbent material (paper towel)
5. Sample collection and storage
The test should be performed with serum or EDTA-plasma samples.
Haemolytic and lipemic samples should not be used with this assay. The samples can be stored for up to
24 hours at 2 - 8 °C or for a longer period (up to 6 months) at -20 °C. Repeated freezing and thawing
should be avoided.
Version: 13.0 Effective: 2015-01-14 5/13
Serum
Collect blood by venipuncture (Monovette™ or Vacuette™ for serum), allow to clot, and separate serum
by centrifugation following manufacturer’s instructions . Do not centrifuge before complete clotting has
occurred. Patients receiving anticoagulant therapy may require increased clotting time.
Plasma
Whole blood should be collected into centrifuge tubes containing EDTA as anti-coagulant (Monovette™ or
Vacuette™) and centrifuged following manufacturer’s instructions immediay after collection.
6. Test procedure
Allow all reagents - with the exception of Precipitating Reagent - to reach room temperature and mix
thoroughly by gentle inversion before use. Number the assay tubes accordingly. Duplicate determinations
are recommended.
Pipetted liquids should not adhere to the wall of the RIA tubes. If necessary please centrifuge the tubes
for 1 minute at 500 x g to spin down adhering liquids.
For the assay the use of conical tubes is highly recommended.
6.1 Preparation of reagents
Enzyme Solution
Reconstitute the content of the vial with 3 ml of Enzyme Buffer prior to use. Mix by vortexing and leave it
30 minutes on a rotating mixer (please make sure that the lyophilisate is dissolved compley!). The
reconstituted enzyme solution cannot be stored and can only be used once. Upon request additional
Enzyme vials are provided.
Equalizing Reagent
The Equalizing Reagent has to be reconstituted with 10 ml water (deionized, distilled, or ultra-pure).
Reconstituted Equalizing Reagent which is not used immediay has to be frozen for max 1 month at
-20 ?C (in aliquots) and may be thawed only once.
6.2 Melatonin RIA
1. Pipette 15 μl of standards and controls into the respective tubes.
2. Pipette 150 μl of Equalizing Reagent into the tubes for NSB, standards and controls.
3. Pipette 150 μl of the samples into the respective tubes.
4. Add 50 μl of Enzyme Solution (refer to 6.1) to all tubes (except totals) and vortex.
5. Incubate for 1 h at RT (20 - 25 °C).
6. Pipette 100 μl of Assay Buffer into all tubes (except totals) and mix shortly.
7. Pipette 25 μl of the 125I Melatonin into all tubes.
8. Pipette 50 μl of Melatonin Antiserum into all tubes (except totals and NSB); mix thoroughly.
9. Cover tubes. Incubate for 20 - 24 h at RT (20 - 25 °C).
10. Mix the chilled (2 - 8 °C) Precipitating Reagent thoroughly, pipette each 1000 μl into all tubes
(except totals), and mix on a vortex.
11. Incubate for 20 min at 2 - 8 °C.
12. Centrifuge for 20 min at 3 000 x g, if possible in a refrigerated centrifuge.
13. Decant or aspirate the supernatant carefully (except totals). Blot the tubes dry and leave them
upside for 2 minutes.
14. Count all tubes for 1 min in a gamma counter.
Version: 13.0 Effective: 2015-01-14 6/13
7. Calculation of results
Measuring range
Melatonin
2.3 – 1 000 pg/ml
Subtract the mean cpm of the non-specific binding NSB from the mean cpm of standards, controls and
samples.
The standard curve from which the concentrations in the samples can be read off, is obtained by plotting
the percentage of (B-NSB)/ (B0-NSB) 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).
The concentrations of the samples and controls can be read directly from the calibration curve.
Samples found with concentrations higher than the highest standard (Standard G) should be diluted
accordingly with Equalizing Reagent (BA R-0028) and have to be re-assayed.
Conversion
Melatonin (pg/ml) x 4.30 = Melatonin (pmol/l)
Expected reference ranges
The reference concentration given below should be taken as a guideline only. It is recommended that
each laboratory should establish its own reference ranges.
The melatonin concentrations depend on age and on a circadian rhythm with a maximum at night
between 1.00 and 3.00 a.m. This maximum is usually clearly higher than the values during the daytime.
The melatonin levels in humans show a marked circadian rhythm characterized by very low levels during
day time (up to 30 pg/ml for serum) and high levels during night time (up to 150 pg/ml for serum). The
highest concentrations are found with infants up to the age of 3 years.
7.1 Quality control
It is recommended to use control samples according to national regulations. Use controls at both normal
and pathological levels. The kit or other commercial controls should fall within established confidence limits.
The confidence limits of the kit controls are indicated on the QC-Report.
7.2 Typical standard curve
Example, do not use for calculation!
Melatonin
B/B0 (%)
pg/ml
8. Assay characteristics
Analytical Sensitivity
(Limit of Detection) 2.3 pg/ml
Version: 13.0 Effective: 2015-01-14 7/13
Analytical Specificity
(Cross Reactivity)
Substance Cross Reactivity (%)
Melatonin
Melatonin 100
N-Acetylserotonin 0.98
5-Methoxytryptophol 0.11
5-Methoxytryptamine 0.07
6-Methoxytryptamine ? 0.01
5-Methoxyindol-3-acetic acid ? 0.01
Serotonin ? 0.01
DL-Tryptophan ? 0.01
DL-5-Methoxytryptophan ? 0.01
5-Hydroxy-L-Tryptophan ? 0.01
Precision
Intra-Assay Inter-Assay
Sample Range (pg/ml) CV (%) Sample Range (pg/ml) CV (%)
Melatonin 1 19.2 ± 1.9 9.8 Melatonin 1 29.4 ± 2.4 8.0
2 41.8 ± 4.0 9.7 2 73.9 ± 8.1 10.9
3 126 ± 16.9 13.4 3 154 ± 20.6 13.3
Linearity Range Mean (%) Range (%)
8.5 – 529 pg/ml 89 82 – 102
Recovery
Range Mean (%) Range (%)
Serum 19.7 – 808 pg/ml 90 70 - 107
Plasma 19.4 - 770 pg/ml 92 79 - 113
9. References/Literature
(1) Melatonin and omentin: influence factors in the obstructive sleep apnoea syndrome? Journal of
Physiology and Pharmacology, 64(3):353-360 (2014)
(2) Robeva et al. Decreased Daily Melatonin Levels in Women with Systemic Lupus Erythematosus - A Short
Report. Balkan Medical Journal, 30: 273-276 (2013)
(3) Gonciarz et al. Plasma insulin, leptin, adiponectin patients treated with melatonin, resistin, ghrelin, and
melatonin in nonalcoholic steatohepatitis. Journal of Pineal Research, 54(2):154-161 (2013)
For updated literature or any other information please contact your local supplier.
Symbols:
Storage
temperature Manufacturer
Contains sufficient for
<n> tests
Expiry date
Batch code
For in-vitro diagnostic
use only!
Consult instructions
for use Content
CE labelled
Caution
Catalogue
number
For research use
only!
Version: 13.0 Effective: 2015-01-14 8/13
Melatonin Direct RIA
1. Einleitung
1.1 Verwendungszweck und Testprinzip
125 I – Radioimmunoassay zur direkten quantitativen Bestimmung von Melatonin in humanem Serum und
Plasma.
Die Testdurchführung folgt den Grundprinzipien eines Radioimmunoassays. Radioaktiv markiertes Antigen
und nicht markiertes Antigen binden kompetitiv an eine definierte Anzahl von Antikörperbindungsslen.
Nach Gleichgewichtseinslung werden die Antigen-Antikörper-Komplexe mit einem zweiten Antikörper in
Anwesenheit von PEG gefällt. Das Präzipitat wird nach Zentrifugieren und Dekantieren oder Absaugen des
Überstands in einem Gamma-Counter gemessen. Die Menge an radioaktiv gebundenem Antigen ist
indirekt proportional zur Antigenkonzentration der Probe.
Die Konzentrationen der unbekannten Proben werden mit Hilfe einer Standardkurve und Abgleich der
gemessenen Aktivitäten ermitt.
1.2 Klinische Anwendung
Das Hormon Melatonin spielt eine Schlüsselrolle in der Regulation des Biorhythmus und wird in der
Zirbeldrüse aus dem Vorläufermolekül Serotonin produziert. Sowohl in Saliva als auch in Serum spiegelt
die Melatoninkonzentration den circadianen Rhythmus wider. Tagsüber ist der Melatoninspiegel niedrig
während er nachts um ein vielfaches ansteigt.
Der Einfluss von Melatonin im Menschen wird in der Literatur mit Schlafstörungen, Jetlag, Depression,
Stress, Schizophrenie, Ausbleiben der Regelblutung, Schwangerschaft, Magersucht, immunologischen
Erkrankungen und in der Kontrolle der sexuellen Reifung in der Pubertät beschrieben.
Viele biologische Effekte von Melatonin verlaufen über die Aktivierung des Melatoninrezeptors. Des
Weiteren dient es im menschlichen Körper als direkter Fänger von freien Radikalen.
Therapeutische Konsequenzen dürfen niemals allein auf Grund von Laborwerten herangezogen werden,
auch wenn diese Werte in Übereinstimmung mit den Qualitätskriterien der Methode beurteilt werden.
Jedes Laborergebnis trägt immer nur zu einem Teil des klinischen Bildes bei.
Nur wenn die Laborergebnisse in akzeptabler Übereinstimmung mit dem klinischen Gesamtbild stehen,
dürfen daraus therapeutische Konsequenzen abgeleitet werden.
Die Laborwerte selbst dürfen niemals der alleinige Grund für daraus abgeleitete therapeutische
Konsequenzen sein.
2. Verfahrenshinweise, Richtlinien, Warnungen und Anwendungsgrenzen
2.1 Verfahrenshinweise, Richtlinien und Warnungen
(1) Dieses Kit ist nur für den gewerblichen Gebrauch. Für eine erfolgreiche Anwendung dieses Kits benötigen
die Anwender ein umfassendes Verständnis dieses Protokolls. Einzig die im Kit enthaltene Testanleitung
ist gültig und bei der Durchführung des Assays zu verwenden. Für eine zuverlässige Leistung müssen die
mitgelieferten Anweisungen genau und sorgfältig befolgt werden.
(2) Dieser Assay wurde für die unter Verwendungszweck (siehe Kapi 1) angegebenen Probenarten
validiert. Jede nicht zugelassene Anwendung dieses Kits obliegt der Verantwortung des Anwenders und
entbindet den Hersler von jeglicher Haftung.
(3) Die humanes Serum oder Plasma enthaltenden Reagenzien des Kits wurden mit geprüften Verfahren auf
HIV I/II, HBsAg und HCV getestet und als negativ bestätigt. Dennoch sollten sämtliche Reagenzien bei
der Handhabung und Entsorgung als potenzielle biologische Gefahrstoffe behandelt werden.
(4) Die Grundsätze der Guten Laborpraxis (GLP) sind zu befolgen.
(5) Bei Bedarf Laborkit, geeignete Einweghandschuhe und Schutzbrille tragen, um die Exposition
gegenüber potenziell gesundheitsgefährdenden Stoffen zu reduzieren.
(6) Alle Reagenzien des Kits sowie die Proben sollten vor der Verwendung auf Raumtemperatur gebracht und
vorsichtig aber gründlich gemischt werden. Wiederholtes Einfrieren und Auftauen von Reag