Enzymatic hydrolysis combined with chemiluminescence immunoassay: Foresight for automated and rapid urinary aldosterone detection.

BACKGROUND

The 24-hour urinary aldosterone test offers superior stability in assessing aldosterone secretion compared to single-point plasma measurements, suggesting its potential utility as a diagnostic tool for primary aldosteronism. However, its clinical utility is hampered by labor-intensive manual urine treatment and the lack of diagnostic cut-off, with the former demonstrating considerable inter-laboratory variability. There is an urgent need for an automated assay to facilitate rapid and standardized 24-hour urinary aldosterone testing, accompanied by method-specific diagnostic cut-offs.

RESULTS

The systematic comparison of hydrolysis methodologies (hydrochloric acid, biogenic enzymes, and transgenic enzymes) reveals that IMCS-RT transgenic β-glucuronidase exhibits the highest efficiency, achieving complete hydrolysis within 30 min at room temperature (∼25 °C). Based on this, an automated chemiluminescence system is developed, integrating rapid enzymatic hydrolysis, antibody-mediated aldosterone capture, magnetic separation, and luminous reactions. It demonstrates excellent linearity (0.030-10.00 ng/mL, R2 = 0.996) and a clinically reportable range of 0.030-40.00 ng/mL. The measured aldosterone levels strongly correlate with those obtained via liquid chromatography-tandem mass spectrometry following acid hydrolysis (r = 0.9836) and enzymatic hydrolysis (r = 0.9846). Using samples from 85 patients with primary aldosteronism and 187 controls, we establish a diagnostic cut-off of 15.28 μg/24h, yielding an area under the curve of 0.80, a sensitivity of 0.776, and a specificity of 0.706.

SIGNIFICANCE: This study presents an automated chemiluminescence immunoassay system incorporating transgenic β-glucuronidase-mediated hydrolysis for rapid and reliable quantification of 24-hour urinary aldosterone. Method-specific diagnostic cut-offs further augment the clinical utility of this assay, offering a non-invasive diagnostic approach to primary aldosteronism.