Development of a fluorescent lateral flow test for the detection of the metabolic biomarker leptin in human serum

Abstract

Body mass index (BMI) measurement is the primary method for diagnosing and classifying obesity and metabolic syndrome, despite limitations and variable significance across populations. Metabolic biomarkers, such as adipokines, offer more precise insights and the potential for earlier diagnosis of conditions such as gestational diabetes. Leptin, an adipokine that regulates satiety and energy expenditure, directly correlates with adipose mass, and elevated leptin levels are associated with obesity. Despite its metabolic significance, no commercially available point-of-care (POC) method for leptin detection exists. We developed a sandwich Lateral Flow Immunoassay (LFA) for leptin, immobilizing a capture antibody on a nitrocellulose membrane and conjugating fluorescent europium(III) chelate polystyrene particles to a detection antibody. The LFA prototype successfully detected recombinant human leptin spiked in buffer and 50% calf serum across a range of 0.25-100 ng/mL, with an estimated limit of detection (blank + 3.3σ) of 0.25 ng/mL in a benchtop and a portable Europium fluorescence reader in both matrices. Additionally, the LFA prototype detected endogenous leptin in samples containing 25% human serum at concentrations as low as 0.21 ng/mL. Signal intensity ratios between test and control lines showed a strong correlation with leptin concentration measured by ELISA (Pearson correlation coefficients for the entire sample set (n=30) were r=0.96 and r=0.95 (both p <0.0001) for the benchtop and portable reader, respectively). The LFA’s range and sensitivity enable the detection of endogenous leptin at physiologically-relevant concentrations. These findings highlight the potential for developing robust LFAs for the detection of leptin and other metabolically-relevant biomarkers in point-of-care settings.