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Melatonin Saliva Direct RIA
1. Introduction
1.1 Intended use and principle of the test
125I – Radioimmunoassay for the direct quantitative determination of melatonin in saliva.
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.
(12) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,
sample, standard and control.