Development of radioimmunoassay for the measurement of human leptin in serum.
BACKGROUND
Leptin is a 16 kDa polypeptide hormone encoded by the obese gene (ob) and secreted by adipose tissue. This hormone plays a major role in energy homeostasis and regulation of food intake and body weight. It also affects the metabolic, neuroendocrine and reproductive systems.


METHODS
Labelling of recombinant human leptin with (125)I was best performed by the Chloramine-T method. New Zealand white rabbits were immunised with recombinant human leptin, cross-reaction of obtained antisera was analyzed with 10 different antigens. The separation of bound and free fractions was performed using the second antibody - PEG method.


RESULTS
The obtained tracer had specific activities of 2.8-3.3 kBq/ microg and had a stability of 5 weeks. A highly specific polyclonal antibody was obtained without measurable cross-reaction against the analysed antigens. Concentrations of human leptin were measured by a single overnight incubation assay with a sensitivity of 0.5 ng/ml and a measuring range of 0.5-100 ng/ml. The intra-assay and inter-assay coefficient of variation was under 6% and 8%, respectively. Recovery ranged from 88% to 106%.


CONCLUSIONS
Serum human leptin concentrations can be accurately and precisely measured by this new radioimmunoassay. Preliminary results obtained from the measurement of serum leptin in lean, overweight and obese patients are presented. Serum leptin concentrations correlated with body mass index and were significantly higher in women than in men, except for obese patients.
Lagarde
Ana R
AR
"Fodor Jozsef" National Centre of Public Health - "Frederic Joliot-Curie" National Research Institute of Radiobiology and Radiohygiene Budapest, Hungary.
Nagy
Katalin
K
Forgách
Tibor
T
Jánoki
Gyozo A
GA
D000818:Animals; D006801:Humans; D007457:Iodine Radioisotopes / Q000097:blood*; D007553:Isotope Labeling / Q000379:methods*; D020738:Leptin / Q000097:blood*; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D019275:Radiopharmaceuticals / Q000097:blood; D011994:Recombinant Proteins / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods*
D007457:Iodine Radioisotopes; D020738:Leptin; D019275:Radiopharmaceuticals; D011994:Recombinant Proteins