Development of Free Testosterone Chemiluminescence Detection Kit and Its Clinical Application

Background: Free testosterone makes up only 1–2% of the total testosterone in the blood, but it is the active form of testosterone and enters cells directly to work. The level of total testosterone is substantially affected by sex hormone-binding globulin, whereas free testosterone can better reflect the actual biological activity. The clinical detection of free testosterone levels in serum is important for the diagnosis of related diseases, such as male hypogonadism, polycystic ovary syndrome, metabolic syndrome, osteoporosis, and Alzheimer's disease, and male hypogonadism. However, existing detection methods have problems, such as insufficient sensitivity, complex operation, and high cost, and there is an urgent need to develop efficient and accurate detection technologies. We aimed to develop a chemiluminescence detection kit for free testosterone and conducted a comprehensive performance evaluation. Methods: The content of free testosterone was quantified using a competitive immunoassay method. Free testosterone and testosterone derivatives competed for binding to biotinylated testosterone-specific antibodies. The reaction procedure was determined by the concentration of magnetic beads was optimized, antibodies were biotinylated, testosterone derivatives were labeled with acridinium ester, and the working concentrations of both the labeled antibodies and testosterone derivatives were fine-tuned. Following the determination of the reaction process, the performance of the assay kit was evaluated through a series of tests assessing linearity, the limit of blank, accuracy, precision, stability, specificity, and clinical relevance. Samples were collected from 1615 male and 2035 female patients, and their free testosterone levels were measured. Finally, the clinical significance of free testosterone levels in diagnostic applications was analyzed. Results: The linear r was > 0.99, limit of the blank was 0.021 pg/mL, accuracy deviation was < 5%, imprecision was < 5%, and stability lasted for 12 months. There was no cross-reactivity with similar substances. In total, 392 clinical free testosterone samples were collected for analysis and a methodological comparison was conducted. The correlation coefficient r was 0.9941, meeting clinical requirements. In total, 1615 male and 2035 female patient samples were collected for testosterone detection. Free testosterone levels showed significant differences among male patients with different clinical diagnoses. Prostate malignancy was associated with lower testosterone levels, whereas other diagnoses (such as those related to hair loss) were associated with higher testosterone levels. Therefore, free testosterone levels could serve as an important reference indicator for the diagnosis and assessment of certain diseases. Conclusion: The free testosterone chemiluminescence assay kit developed in this study exhibited excellent performance, low cost, and a high degree of automation. It fully meets the clinical requirements for detection. When tested on clinical patient samples, the level of free testosterone can be regarded as an important reference indicator for the diagnosis and assessment of certain diseases, which can help doctors make better diagnoses and treatments for related diseases.