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Published on: March 23, 2018
Chemiluminescent enzyme immunoassay for measuring leptin
Satoshi Sekiguchi1, Hideki Kohno, Kiyoshi Yasukawa
1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Japan.
Insights
This study developed a sensitive chemiluminescent enzyme immunoassay (CLEIA) for measuring serum leptin. The assay demonstrates high sensitivity and clinical usefulness for disease prevention and treatment.
Area of Science:
- Biochemistry
- Immunology
- Assay Development
Background:
- Leptin, an adipocyte-derived hormone, is crucial for metabolic regulation.
- Serum leptin concentration is a key biomarker for diabetes mellitus and other diseases.
- Accurate leptin measurement is essential for clinical diagnostics and therapeutic monitoring.
Purpose of the Study:
- To develop and validate a sensitive chemiluminescent enzyme immunoassay (CLEIA) for quantifying serum leptin.
- To optimize blocking agents for minimizing non-specific binding in the assay.
- To evaluate the assay's sensitivity, measurable range, and clinical applicability.
Main Methods:
- Construction of a CLEIA using anti-leptin polyclonal antibody and alkaline phosphatase (ALP).
- Utilized IgG-conjugated ferrite particles for leptin capture and ALP-conjugated Fab fragments for detection.
- Evaluated bovine serum albumin (BSA), Block ace, and CE510 for blocking non-specific binding.
Main Results:
- Bovine serum albumin (BSA) was identified as the most effective blocking agent.
- The developed CLEIA achieved a measurable range of 0.1-1.0 pg/mL for leptin.
- The assay exhibited a low detection limit of 0.1 pg/mL, indicating high sensitivity.
Conclusions:
- The developed CLEIA is a sensitive and clinically useful method for measuring serum leptin concentrations.
- The assay's performance supports its application in preventing and treating leptin-related diseases.
- This study highlights the feasibility of constructing sensitive enzyme immunoassays using a single polyclonal antibody.
Abstract:
Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1-1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.
