Development of Free Testosterone Chemiluminescence Detection Kit and Its Clinical Application.

Abstract

Background: Free testosterone (FT), the bioactive form comprising 1%-2% of total testosterone, directly enters cells. Unlike total testosterone, FT levels are less affected by sex hormone-binding globulin and better reflect biological activity. Accurate serum FT measurement is crucial for diagnosing conditions like male hypogonadism, PCOS, metabolic syndrome, osteoporosis, and Alzheimer's. However, existing methods suffer from inadequate sensitivity, complexity, and high cost, necessitating improved detection technologies. We developed and evaluated a chemiluminescence assay kit for FT.

Methods: FT was quantified using a competitive immunoassay where sample FT competes with acridinium ester-labeled testosterone derivatives for binding to biotinylated antitestosterone antibodies on magnetic beads. Key parameters (magnetic bead concentration, biotinylation, labeling, and antibody/derivative concentrations) were optimized. Kit performance was rigorously assessed for linearity, limit of blank (LoB), accuracy, precision, stability, specificity, and clinical relevance. FT levels were measured in 1615 male and 2035 female patient samples to analyze clinical significance.

Results: The assay demonstrated excellent linearity (r > 0.99), low LoB (0.021 pg/mL), high accuracy (deviation < 5%), precision (CV < 5%), and 12-month stability. Specificity testing showed no cross-reactivity. Method comparison with 392 clinical samples yielded a strong correlation (r = 0.9941). Analysis of patient samples revealed significant FT level differences among males with various diagnoses: lower levels in prostate cancer patients and higher levels in conditions like hair loss.

Conclusion: The developed chemiluminescence FT assay kit exhibits superior performance, low cost, and high automation, fully meeting clinical requirements. FT measurement provides a valuable reference for diagnosing and assessing specific diseases, aiding improved clinical management.