Abstract
A direct radioimmunoassay procedure for the determination of melatonin in the blood of blue fox has been validated and applied.
The assay required 50 μl of sample and standard, 100 ul of antiserum and 100 μl of (3H)melatonin. After overnight incubation at 4°C the antibody bound melatonin was separated from the free hormone with dextran-coated charcoal. Following centrifugation the antibody bound (3H)melatonin was determined in a beta scintillation counter.
The antiserum bound 30–35 % of the (3H)melatonin at a final dilution of 1:36000. The non specific binding represented less than 5% of the total radioactivity in all assays. The lowest detectable amount of melatonin was 2.6 fmol/tube, corresponding to 52.5 pmol/1. The inter-assay coefficient of variation at 178 and 510 pmol/1 was 15.6 and 8.8%, respectively. The precision profile, calculated from a 10-replicate standard curve, showed that the coefficient of variation decreased from 43% at 84 pmol/1 to 15% at 336 pmol/1, and remainded at or below 10% for concentrations exceeding 670 pmol/1.
Plasma was collected from 2 male blue foxes at about hourly intervals during a 24 h period in September and assayed for melatonin. Maximum (421 pmol/1) and minimum (97 pmol/1) concentrations of the hormone were inversely related to light intensity.
Keywords: reproduction
Sammendrag
Analysmetoden erfordrar 50 prov respektive standard, 100 μl antiserum och 100 μl tritierat melatonin. Efter inkubation över natt vid 4°C separeras det antikroppsbundna melatoninet frän det fria hormonet genom absorption till dextrankoi. Efter centrifugenng bestämms mängden antikroppsbundet tritierat melatonin i en scintillationsräknare.
Antiserat spätt 1:36000 band 30–35 % av det tritierade melatoninet. Den ospecifika bindningen (NSB) var lägre än 5 % av den totala radioaktiviteten. Metodens känslighet är 2.6 fmol per rör, vilket motsvarar 52.5 pmol/l.
Inomkörningsvariationen vid 178 och 510 pmol/l var 15.6 och 8.8%. Precisionsprofilen, kalkylerad från en standardkurva med 10 replikat, visade att variationskoefficienten minskade från 43 % vid 84 pmol/1 till under 10 % vid melatoninkoncent ral ioner överstigande 673 pmol/.
Blodprover togs med ungefär 1 timmes intervall under en 24 timmars period från två blårävshanar, och analyserades med avseende på koncentrationen av melatonin. De högsta koncentrationerna av melatonin uppmättes under dygnets mörka och de lägsta under dygnets ljusa del.
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Acknowledgements
We thank Dr. Gordon Niswender for supplying the melatonin antibody and Dr. Adrian Smith for supplying the blood samples.
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