Development of a time-resolved fluorescence microsphere immunochromatographic assay for simultaneous quantitative...

Estrone-3-glucuronide (E1-3-G) and pregnanediol-3-glucuronide (PdG), the predominant urinary metabolites of estradiol and progesterone, serve as critical non-invasive biomarkers for predicting threatened miscarriage and evaluating ovarian luteal function. We developed a rapid quantitative multiplex immunoassay based on time-resolved fluorescence microsphere immunoassay (TRFIA) for their simultaneous measurement. Method validation utilizing certified reference standards and negative urine matrices demonstrated broad linear dynamic ranges of 50-10 000 ng mL^-1 for E1-3-G and 1.5-100 μg mL^-1 for PdG, with limits of detection (LODs) of 30 ng mL^-1 and 1.5 μg mL^-1, respectively. Cross-reactivity assessments against 12 structurally related hormones (e.g., estrone and pregnenolone) and 9 urinary interferents (e.g., urea and albumin) confirmed high specificity (<15% deviation). Recovery rates (93.66-104.99% for E1-3-G; 90.98-108.67% for PdG) and precision (intra-/inter-batch coefficients of variation < 10%) met predefined acceptance criteria. Clinical validation with human urine samples revealed strong concordance with ELISA results (adjusted R² = 0.96 for E1-3-G and 0.92 for PdG; Bland-Altman analysis indicated >95% agreement). By combining broad dynamic ranges, high specificity, and rapid processing (10 minutes), this TRFIA-based method enables reliable and simultaneous quantification of E1-3-G and PdG. Consequently, it serves as a robust clinical tool for early pregnancy risk stratification and real-time ovarian function monitoring.