open access
Development of radioimmunoassay for the measurement of human leptin in serum
open access
Abstract
MATERIAL AND METHODS: Labelling of recombinant human leptin with 125I 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/µg 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.
Abstract
MATERIAL AND METHODS: Labelling of recombinant human leptin with 125I 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/µg 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.
Keywords
radioimmunoassay; leptin; 125I; obesity; BMI
Title
Development of radioimmunoassay for the measurement of human leptin in serum
Journal
Issue
Pages
105-109
Published online
2003-10-10
Page views
520
Article views/downloads
1444
Bibliographic record
Nucl. Med. Rev 2003;6(2):105-109.
Keywords
radioimmunoassay
leptin
125I
obesity
BMI
Authors
Ana R. Lagarde
Katalin Nagy
Tibor Forgách
Gyozo A. Jánoki