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Development of Dissociation-Enhanced Lanthanide Fluoroimmunoassay for Measuring Leptin
1Research Group of Bioprocess Engineering, Korea Food Research Institute, Seongnam, 13539, Republic of Korea. k9130sen@hanmail.net.
Insights
A new dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) was developed to measure leptin, a key satiety hormone. This assay is effective for quantifying leptin in biological samples, showing competitive performance against ELISA.
Area of Science:
- Biochemistry
- Immunology
- Analytical Chemistry
Background:
- Leptin is a crucial satiety hormone regulating appetite control.
- Accurate measurement of leptin levels is essential for understanding metabolic and endocrine functions.
- Existing assay methods may have limitations in sensitivity or efficiency.
Purpose of the Study:
- To develop and optimize a novel dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) for sensitive leptin detection.
- To validate the performance characteristics of the developed leptin DELFIA assay.
- To compare the efficacy of the new assay with established methods like ELISA.
Main Methods:
- A sandwich assay format was employed using microplates immobilized with anti-leptin antibodies.
- A second anti-leptin antibody, tagged with an europium chelate, was used for signal detection.
- Optimization of antibody coating (0.5 μg/well) and bioconjugate amounts (200 ng/well) was performed.
Main Results:
- The leptin DELFIA demonstrated a linear relationship (r² = 0.9646) over a concentration range of 0.01–50 ng/mL.
- A low limit of detection (LOD) of 0.01 ng/mL was achieved.
- The assay showed good convergence with calibration curves and effective measurement in rat serum samples, comparable to ELISA.
Conclusions:
- The developed leptin DELFIA is a sensitive and effective method for quantifying leptin.
- This assay offers a viable alternative to commercial ELISA kits for leptin measurement.
- The optimized DELFIA provides reliable results for various sample types, including serum.
Abstract:
Development of a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) for measuring leptin, a satiety hormone of appetite control, was conducted in sandwich assay format exploiting a microplate immobilized with an anti-leptin antibody and another antibody raised against leptin and tagged with an europium chelate. In the leptin DELFIA of this study, amounts of antibody coated to the microplate and of the bioconjugate for the second immune reaction were optimized as 0.5 μg and 200 ng per well, respectively. When plotted in double-logarithmic scale, a linear relationship of y (log10 response signal) = 0.6023× (log10 leptin concentration) + 3.4084 (r(2) = 0.9646) was obtained at the leptin concentrations of 0.01─50 ng/mL with the limit of detection of 0.01 ng/mL. Individual leptin concentrations in various samples were well convergent to the calibration curve of the current assay. When applied to the measurement of leptin in a rat serum, the present assay was found quite effective and was competitive to a commercial sandwich-type ELISA.
