Development of Free Testosterone Chemiluminescence Detection Kit and Its Clinical Application

Development of Free Testosterone Chemiluminescence Detection Kit and Its Clinical Application | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of Free Testosterone Chemiluminescence Detection Kit and Its Clinical Application Shuang Han, Min Liu, Simin Zhao, Wenjun Yan, Zhixian Lu, Xuan Huang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5969090/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Free testosterone Performance evaluation Hormone markers Chemiluminescence kit development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Testosterone, also known as androsterone or androstenone, with the molecular formula C 19 H 28 O 2 , is a steroid hormone secreted by the testes of men and ovaries of women. The adrenal glands also secrete small amounts of testosterone. It plays a role in maintaining muscle strength and mass, bone density, and strength, and enhances energy and physical performance. Testosterone was first identified as a secretory glycoprotein in the culture medium of mouse testicular support cells and is a relatively small cytoskeletal-associated protein that can bind tightly to the cell surface through receptor-associated proteins [ 1 , 2 ]. In clinical tests, serum testosterone levels in patients with type 1 and 2 diabetes are significantly reduced, and serum testosterone levels are negatively correlated with blood glucose concentration and disease duration. Testosterone has a significant protective effect on pancreatic islet cells and promotes insulin synthesis [ 3 ]. For male patients, elevated serum testosterone levels is observed in the following diseases: Leydig cell tumors, true precocious puberty, familial incomplete pseudohermaphroditism type II, complete or incomplete testicular feminization, male infertility syndrome, Reifenstein syndrome, and the use of drugs with androgenic effects, such as phenytoin and danazol [ 4 ]. Decreased serum testosterone levels is observed in the following diseases: congenital dysgenesis of the seminiferous tubules, anorchism, cryptorchidism, primary hypogonadism due to underdeveloped or non-developed interstitial cells, hypogonadism due to low secretion of gonadotropins, isolated LH deficiency syndrome, obesity-related hypogonadism, hyperprolactinemia, Cushing's syndrome, congenital adrenal hyperplasia due to deficiency of enzymes, such as 3β-hydroxysteroid dehydrogenase and 17-hydroxylase, and the use of drugs that inhibit testosterone synthesis, such as aminoglutethimide, spironolactone, marijuana, ketoconazole and dichlorodiphenyltrichloroethane [ 5 – 10 ]. In female patients, elevated serum testosterone levels have been observed in some cases of polycystic ovary syndrome (PCOS), theca cell hyperplasia, ovarian masculinizing tumors, and adrenal cortical hyperplasia [ 11 , 12 ]. After testosterone is secreted into the blood, 98% is bound to sex hormone-binding globulin (SHBG), and a small amount is bound to albumin and cortisol-binding globulin. Approximately 1–2% of total testosterone is in the free state. The level of SHBG in the blood almost determines the amount of free testosterone. The ratio of total testosterone to SHBG (T/SHBG) is known as the free testosterone index. Determination of free testosterone can overcome the influence of changes in the concentration of testosterone-binding proteins and directly reflect the biological activity of hormones. In many women with hypertrichosis or low SHBG levels, serum total testosterone is often within the normal range and the measured free testosterone level is significantly higher than 90% [ 13 , 14 ]. Free testosterone can reflect the characteristics of some diseases more accurately than total testosterone and has more significance in clinical diagnosis. Measuring free testosterone levels is also a way to indirectly measure SHBG levels in the blood. Measurement of free testosterone can be used to monitor Alzheimer's disease in older men and low levels of free testosterone indicate a high risk of Alzheimer's disease [ 15 , 16 ]. The clinical detection of free testosterone levels in the serum is crucial for diagnosing related diseases. The primary methods for detecting free testosterone can be classified into indirect and direct measurements. Indirect determination methods fall into two categories: (1) Simulated immunoassay: Although this method is operationally simple, its specificity is relatively low. Fluctuations in sex hormone-binding globulin (SHBG) levels significantly affect the results, rendering it unsuitable for clinical diagnosis. (2) Formula calculation method: This approach estimates free testosterone concentration based on measurements of total testosterone, SHBG, albumin concentrations, and dissociation constants. However, the accuracy of the results may be compromised when albumin concentration is abnormal. Furthermore, significant variations in algorithms among laboratories limit its reliability, making it unsuitable for clinical application until further validation confirms its consistency and accuracy. Direct determination methods include three main approaches: (1) Equilibrium dialysis: Recognized as a reliable method for determining free testosterone, this technique separates free testosterone from protein-bound testosterone using a semi-permeable membrane over a specified period. Despite its high accuracy, its clinical application is limited due to challenges such as large sample volume requirements, complex pretreatment procedures, difficulty achieving low detection limits, low throughput, high consumable costs, potential contamination during dialysis, poor precision, and insufficient specificity. (2) Ultrafiltration: This method pre-separates free testosterone from serum using ultrafiltration tubes and subsequently detects the bound fraction via reagent strips and liquid chromatography-tandem mass spectrometry (LC-MS/MS). While the procedure is relatively straightforward to perform, discrepancies in results may arise due to variations in ultrafiltration membrane performance and operational conditions. (3) Immuno-mass spectrometry: By integrating the strengths of immunoassay and mass spectrometry, this method utilizes kits to conjugate antibodies with free and total testosterone in serum samples for magnetic micro-enrichment, automated pretreatment, and LC-MS/MS detection. This approach provides enhanced accuracy and stability but entails higher equipment costs [ 17 ]. In summary, indirect determination methods are easy to implement but lack sufficient accuracy. Among direct determination methods, equilibrium dialysis provides accurate results but is time-consuming; ultrafiltration is simpler to operate but may yield inconsistent results; and immuno-mass spectrometry delivers efficient and precise outcomes at the expense of higher equipment investment.Compared with the current methods for detecting free testosterone, chemiluminescence immunoassays have the advantages of high sensitivity, good specificity, simple operation, rapidity, strong anti-interference ability, and low cost, making them suitable for large-scale clinical detection and research applications. However, there are currently few chemiluminescence immunoassay kits for detecting free testosterone content, both at home and abroad. Therefore, it is necessary to develop a more sensitive, specific, and safe chemiluminescence immunoassay for the detection of free testosterone. Materials and Methods Research population In total, 4, 042 patient samples were collected from the Affiliated Hospital of Jiangnan University. The research protocol was approved by the Human Ethics Review Committee of the Affiliated Hospital of Jiangnan University, indicating that we have adhered to ethical standards. Main reagents and materials Free testosterone antigen (Catalog No.: FT2011), free testosterone antibody (Catalog No.: FT3001), and testosterone analogs (Catalog No.: FT4001) were provided by Zhenjiang Airenake Biotechnology Co., Ltd. (Zhenjiang, China); magnetic bead reagents were purchased from Thermo Fisher Scientific (China), Catalog No.: 88817. Chemiluminescent and immunoluminescence instruments were purchased from Faponbiotech (Guangzhou, China). The pH meter was purchased from Mettler Toledo (Switzerland). Reagent preparation Magnetic bead reagent preparation: First, 2.8 µm magnetic beads were selected and activated with 0.1 M hydrochloric acid solution, then rinsed with deionized water until neutral. Next, streptavidin solution (concentration of 10 µg/mL) was mixed with the magnetic beads and incubated at 37 ℃ for 2 h to coat the streptavidin on the surface of the magnetic beads. Finally, the magnetic beads were suspended in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4). Calibrators: A set of free testosterone solutions of known concentrations was used to construct a standard curve. Calibrator concentrations were 0, 5, 10, 20, 50, 100, 200, and 400 pg/ml. Calibrators were prepared in a human serum matrix to better simulate a clinical sample environment. Quality control (QC) products: Free testosterone was diluted in a human serum matrix to prepare QC products at concentrations of 5 and 150 pg/mL. Labeled solution: A solution of testosterone analogs labeled with acridinium ester. The concentration of the testosterone analogs in the labeled solution was 1 µmol/L and the labeling efficiency of acridinium ester was over 90%, ensuring that the labeled substances have high chemiluminescence activity. Biotin antibody solution: The antibodies specifically bind to testosterone, which is labeled with biotin. The antibody concentration was 5 µg/mL and the molar ratio of biotin to antibody was 1:5. This solution was prepared in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4). Substrate solution: A specific substrate solution was used to trigger a chemiluminescence signal from the labeled substances. The main components of the substrate solution are hydrogen peroxide and sodium hydroxide, with concentrations of 0.1 and 0.25 mol/L, respectively. When the substrate came into contact with labeled substances, it triggered a strong chemiluminescent reaction. Washing solution: The magnetic beads were washed and unbound substances were removed to reduce the impact of non-specific binding on the test results. The main components of the washing solution were 0.05 M Tris-HCl buffer (pH 7.4) and 0.1% Tween-20 surfactant. Reaction process Free testosterone content was measured using a competitive method. Free testosterone and testosterone derivatives compete for binding to biotinylated testosterone antibodies. The sample was incubated at 37 ℃ for 30 min, then incubated with magnetic bead solution for 5 min. Magnetic separation is achieved by applying a magnetic field. The sample was washed thrice with a washing solution to form an antibody-antigen acridinium ester complex. The unbound reactants on the magnetic beads were washed away. Hydrogen peroxide and alkaline solutions were added to detect the luminescence. The luminescence value was input into the calibration curve to calculate free testosterone concentration (Fig. 1 ). Data analysis Data were analyzed using SPSS 25.0. Normally distributed continuous variables are expressed as mean ± standard deviation and non-normally distributed ones are expressed as median. The performance of the method is assessed in accordance with the EP guidelines established by the Clinical and Laboratory Standards Institute (CLSI). Linearity The calibrated diluent with antigen served as the high-concentration sample, and the diluent alone as the low-concentration sample. High-concentration samples near the upper linear range limit were serially diluted to various concentrations, including low-concentration samples near the lower limit. Each concentration was tested in triplicate, and the mean calculated. The analyte concentration (x-axis) and average test result (y-axis) were fitted by least-squares regression. The correlation coefficient (r) within the linear range was determined, requiring r > 0.99. Limit of blank The zero-concentration calibrator, after dilution, was used as the test sample. Twenty measurements were performed to obtain optical signal values. The mean (M) and standard deviation (SD) were calculated, and M-2SD was determined. A primary equation was derived from two-point regression fitting using the zero-concentration reference and the nearest adjacent concentration calibrator's data. The optical signal value corresponding to M-2SD was substituted. Accuracy Samples with high and low concentrations (5 pg/mL and 200 pg/mL) of free testosterone were prepared. The relative deviation (B) between the mean value (M) and the reference value was calculated using Equation B=(M-T)/T×100%(T - mark value) after three replicate measurements. The reagent was tested on samples at two concentration levels (5 pg/mL and 200 pg/mL), each measured in triplicate. Imprecision Prepare three batches of reagents and perform ten replicate detections for high-value (100 pg/mL) and low-value control (5.1 pg/mL) items in each batch. Calculate the mean value (M) and standard deviation (SD) of the results, and subsequently determine the coefficient of variation (CV) using the formula CV(%) = (SD/M) × 100%. In this context, CV denotes the coefficient of variation, M represents the mean concentration value, and SD signifies the standard deviation. Thermal stability test The quality controls (10.78 pg/mL, 100.87 pg/mL) of the configured free testosterone kit is placed at 4 ℃ and 37 ℃ respectively for 1, 3, 6, 10, 20, 25, and 30 days to test the luminescence value, The experimental results showed that the deviation of the concentration of the quality controls was less than 10% when the kit was placed at 37°C for different times compared to storage at 4°C. Kit specific test The specific substance was prepared at the corresponding concentration, and one batch of the free testosterone detection kit was employed to quantify the specific substance and calculate the cross-reactivity rate. The cross-reactivity rate (%) was determined using the formula: (free testosterone concentration / specific substance concentration) × 100%. Clinical relevance assessment To validate the detection efficacy of the kit developed in this study, a clinical performance evaluation was conducted. A total of 392 plasma samples from patients provided by the Affiliated Hospital of Jiangnan University were analyzed using both a self-developed chemiluminescence-based free testosterone detection kit and a commercially available kit. The results were compared, with values measured using the self-developed kit plotted on the x-axis and those from the commercial kit on the y-axis. Results Optimization of the working concentration of magnetic beads The magnetic bead concentration was selected based on the optimized antibody concentration. Specifically, magnetic bead concentrations of 15 mg/mL, 10 mg/mL, 7 mg/mL and 5 mg/mL were evaluated. According to the experimental results, the optimal luminescence effect was achieved at a magnetic bead concentration of 10 mg/mL. Consequently, the concentration of 10 mg/mL was chosen for subsequent experiments (Table 1 ). Table 1 Comparison of luminescence values in different magnetic bead concentrations of free testosterone kits Free testosterone calibrator Calibration concentration (pg/mL) Beads concentration 15 mg/mL Beads concentration 10 mg/mL Beads concentration 7 mg/mL Beads concentration 5 mg/mL S1 0 7328391 7612893 7427162 7213465 S2 5 1324212 1524681 1125361 1231461 S3 10 712361 763752 714535 709869 S4 25 293812 303452 271829 234265 S5 50 112831 152496 113728 109281 S6 100 70121 76843 73212 71231 S7 200 33238 38754 32142 31271 S8 400 10293 19376 14321 12131 Optimization of free testosterone antibody working concentration Biotin-antibody solution preparation: Initially, antibodies with specific binding affinity for testosterone were biotinylated. During the biotinylation process, the molar ratio of control biotin to antibody was maintained at 1:5. Subsequently, the biotinylated antibody was dissolved in a diluent solution at predetermined concentrations of 3 µg/mL, 5 µg/mL, and 7 µg/mL. Preparation of marker-antibody diluent: A stock solution was prepared by dissolving 12.05 g of Tris, 10 g of salicylic acid, and 2.0 g of NaN₃ in 1 L of purified water. The mixture was stirred at room temperature for 1 hour. Following this, 0.998 mL of MgCl₂·6H₂O (1 M), 1.027 mL of ZnCl₂ (0.1 M), and 1 g of BSA were added to the solution. The pH of the solution was adjusted to 7.0 ± 0.05. The solution was then filtered and stored at 4 ℃. Biotinylated testosterone analogs were subsequently dissolved in the diluent at concentrations of 0.01 ng/mL, 0.05 ng/mL, and 0.1 ng/mL. Aliquots of 30 µL of each concentration were used to perform the checkerboard titration method. The final working concentrations were set at 5 µg/mL for the biotinylated antibody and 0.05 ng/mL for the labeled testosterone analog (Table 2 ). Table 2 Checkerboard test data Biotin-T antibody 3 µg/mL Biotin-T antibody 5 µg/mL Biotin-T antibody 7 µg/mL pg/mL T-AE 0.01 ng/mL T-AE 0.05 ng/mL T-AE 0.1 ng/mL T-AE 0.01 ng/mL T-AE 0.05 ng/mL T-AE 0.1 ng/mL T-AE 0.01 ng/mL T-AE 0.05 ng/mL T-AE 0.1 ng/mL 0 3231421 2340123 20441234 50231231 76123123 9785188 20132312 30384736 34256882 25 2002049 1832032 17424123 4012312 3141232 592948 5235829 6374852 5313152 100 873231 741241 6984241 100413 76141 304294 702065 749958 770922 D/A 61.96% 78.29% 85.24% 7.99% 4.13% 6.06% 26.01% 20.98% 15.51% F/A 43.62% 40.46% 40.08% 0.20% 0.10% 3.11% 3.49% 2.47% 2.25% Method performance evaluation Linearity Figure 2 presents a linear analysis of free testosterone. The fitted linear regression equation is Y = 0.9990X + 0.6843, r = 0.9999, demonstrating an excellent linear relationship between the theoretical and actual sample concentrations. Limit of blank The results are summarized in Table 3 . The concentrations of free testosterone were 0.013, 0.021, and 0.016 pg/mL, and the limit of the free testosterone blank was 0.021 pg/mL. Table 3 Verification results of limit of blank of free testosterone Test 1 Test 2 Test 3 M 9898443.05 9230547.10 9779625.65 SD 54762.74 65679.14 161610.73 M-2SD 9788917.56 9099188.83 9456404.19 LOB (pg/mL) 0.013 0.021 0.016 Accuracy The experimental results are listed in Table 4 . The relative deviation of the free testosterone kit was within 5%, indicating that the kit had good accuracy. Table 4 Accuracy test of free testosterone kit Concentration (pg/mL) Test 1 Test 2 Test 3 Mean value (pg/mL) Relative deviation (%) 5.00 4.98 5.11 4.91 5.01 0.20% 200.00 202.10 199.10 201.60 200.35 0.18% Imprecision The results are summarized in Table 5 , with a precision criterion of 3% or lower . Table 5 Comparison of imprecision measurement results of free testosterone detection kits Number of tests Low value quality control High value quality control 1 5.12 100.38 2 5.29 101.23 3 4.98 100.98 4 5.38 99.98 5 5.02 100.21 6 5.11 100.13 7 5.24 99.87 8 5.12 102.34 9 5.29 100.42 10 4.99 100.71 Fitting concentration M 5.154 100.625 Fitting concentration SD 0.139379578 0.741308902 Fitting concentration CV 2.70% 0.74% Thermal stability test The relative error in the accelerated stability test was maintained within ± 5%, demonstrating excellent stability of the kit (Table 6 ). Table 6 Accelerated stability test results of free testosterone kit Days Nominal concentration Test concentration Deviation Test1 Test2 Test3 Test1 Test2 Test3 1 10.78 10.89 10.67 10.99 1.02% -1.02% 1.95% 100.87 102.23 98.82 101.89 1.35% -2.03% 1.01% 3 10.78 10.55 10.98 10.59 -2.13% 1.86% -1.76% 100.87 101.23 98.89 101.23 0.36% -1.96% 0.36% 6 10.78 10.45 10.99 10.48 -3.06% 1.95% -2.78% 100.87 101.23 100.21 102.89 0.36% -0.65% 2.00% 10 10.78 10.93 10.69 10.45 1.39% -0.83% -3.06% 100.87 103.23 101.78 98.23 2.34% 0.90% -2.62% 20 10.78 10.45 10.63 10.99 -3.06% -1.39% 1.95% 100.87 99.76 100.98 100.12 -1.10% 0.11% -0.74% 25 10.78 10.92 10.59 10.87 1.30% -1.76% 0.83% 100.87 99.34 96.91 99.23 -1.52% -3.93% -1.63% 30 10.78 10.43 10.83 10.69 -3.25% 0.46% -0.83% 100.87 99.29 98.91 97.17 -1.57% -1.94% -3.67% Free testosterone detection kits were evaluated at 3, 6, 9, and 12 months, respectively. The coefficient of variation (CV) values for quality control samples S2, S4, and S6 were consistently below 10%, thereby meeting the specified criteria. These findings indicate that the free testosterone detection kits can be effectively stored at room temperature for up to 12 months. Detailed results are presented in Table 7 . Table 7 Real-time stability evaluation results of free testosterone kit Test time (month) Imprecision(CV%) S2 S4 S6 3 2.91% 2.17% 5.46% 6 2.67% 4.95% 3.73% 9 4.52% 2.46% 4.21% 12 3.61% 1.35% 4.68% Kit specific test The specificity was found to be less than 0.1%, thereby meeting the specified requirements. The results are shown in Table 8 . Table 8 Specific test results of free testosterone kit Cross reactant Experimental concentration (pg/mL) Determination of concentration (pg/mL) Cross reactivity rate Dhea sulfate 5000 0.13 0.0026% Estradiol 1000 0.29 0.029% Cortisol 1000 0.07 0.007% Progesterone 5000 0.25 0.005% Prednisone 1000 0.11 0.011% Corticosterone 1000 0.08 0.008% Clinical relevance assessment Linear regression analysis revealed a strong linear correlation (r = 0.9941), as shown in Fig. 3 . These findings confirm that the newly developed kit meets clinical detection requirements. We performed a Bland–Altman analysis of the data, and the mean difference between the two measurements was − 0.03668 pg/mL, indicating that the kit measurements were very close to the hospital test results with little systematic bias. The 95% limit of agreement was calculated to be -18.56 to 18.49 pg per milliliter (Fig. 4 ). This indicates that most of the sample measurement differences were within this range. In summary, after many repeated tests, the detection range of the free testosterone chemiluminescence detection kit was 0–400 pg/mL. The limit of the blank was 0.021 pg/mL and linear test r was 0.9999, which is better accuracy and stability than the same type of kit on the market. By comparing multiple markers, a cheaper acridone ester was selected to ensure labeling efficiency and reduce the production cost, thus reducing the financial pressure on patients when using the free testosterone chemiluminescence detection kit developed in this study. We tested 1615 male and 2035 female patient samples using the free testosterone chemiluminescence kit (Figs. 5 and 6 ). Among the males, the free testosterone levels of most patients with prostate malignancy were relatively low, with a median of 2.24 pg/mL and a relatively narrow interquartile range (IQR of 1.82 pg/mL), indicating that the testosterone levels of patients in this diagnostic category were generally low and concentrated. However, there were some substantially high outlier values, suggesting that individual patients may have significantly different pathological features. Patients with diagnoses related to hair loss (such as androgenetic and seborrheic alopecia) generally had higher testosterone levels, with a median of approximately 28 pg/mL and a relatively wide IQR, especially for androgenetic alopecia, with an IQR of 7.56 pg/mL, indicating a large difference in testosterone levels among patients. The maximum values of these diagnostic categories were relatively high, further supporting the association between high testosterone levels and hair loss. The median was lower for diagnoses related to precocious puberty and rapid progression of puberty, but the IQR was relatively wide, especially for precocious puberty with an IQR of 16.03 pg/mL, indicating a large difference in testosterone levels among patients. Some patients had extremely high values, suggesting that these diagnoses may be related to early or rapid changes in sex hormone levels. The median testosterone level was higher in patients with benign prostatic hyperplasia (23.77 pg/mL), and the IQR was relatively wide (17.36 pg/mL), indicating a large difference in testosterone levels among patients. The maximum value was 81.41 pg/mL, indicating that testosterone levels in some patients were significantly elevated, possibly related to the severity of the disease or other factors. Patients in the different clinical diagnostic categories showed significant differences in free testosterone levels. Some diagnoses (such as prostate malignancy) were associated with lower testosterone levels, whereas others (such as diagnoses related to hair loss) were associated with higher testosterone levels. These results suggest that free testosterone can be an important reference indicator for the diagnosis and assessment of certain diseases. Free testosterone levels in female patients with depression vary significantly. Some patients have significantly elevated hormone levels, which may suggest a significant role of hormone levels in this disease. The free testosterone levels of patients with depression were significantly different and some patients had higher hormone levels, which may be related to disease severity. Discussion Recently, numerous studies have explored the relationship between free testosterone and cognitive function in men, particularly elderly men, and significant progress has been made in this area. Testosterone exerts its neuroprotective effects by improving synaptic transmission, preventing neuronal death, and enhancing synaptic plasticity. These protective effects may be crucial for maintaining cognitive function and delaying cognitive decline [ 18 ]. A research team from Huashan Hospital Affiliated to Fudan University and the School of Public Health of Fudan University found that elderly men with low levels of free testosterone had a significantly increased risk of cognitive decline. Specifically, the risk of cognitive decline in men with low free testosterone levels increased by 429%. This finding suggests that free testosterone may be an important biomarker of cognitive decline in elderly men [ 15 ]. The detection of free testosterone levels has significant clinical implications. Free testosterone is the biologically active form of testosterone in the body and directly reflects the effects of androgens. In men, a decline in free testosterone levels may lead to decreased libido, sexual dysfunction, reduced sperm production, and other issues affect fertility [ 19 – 21 ]. In women, abnormally elevated free testosterone levels may be associated with PCOS, which presents with symptoms, such as irregular menstruation, hirsutism, and acne. Free testosterone is an important indicator of hyperandrogenism [ 22 , 23 ]. For example, free testosterone levels are significantly elevated in patients with PCOS and the free androgen index is considered more diagnostically valuable than total testosterone. Additionally, changes in free testosterone levels are associated with metabolic disorders, such as thyroid dysfunction, obesity, and insulin resistance. As men age, free testosterone levels gradually decline, which may be related to symptoms, such as osteoporosis, muscle loss, and fatigue. The detection of free testosterone helps assess the risk of age-related diseases and guides testosterone replacement therapy. Changes in free testosterone levels may indicate the presence of other diseases. For example, adrenal cortical and testicular tumors may cause elevated free testosterone levels, whereas hypogonadism and liver dysfunction may lead to decreased free testosterone levels. Free testosterone testing is not only used for diagnosis, but also for monitoring treatment effects. For example, in patients with PCOS, reducing free testosterone levels through treatment can improve symptoms and reproductive outcomes. In men, free testosterone levels can be measured after testosterone replacement therapy to evaluate treatment effect [ 24 , 25 ]. In summary, free testosterone testing is an important means of assessing androgen levels and related diseases and provides more accurate biological information than total testosterone. Numerous studies have demonstrated that low testosterone levels in male patients with sepsis are associated with higher mortality rates, longer hospital stay, and poorer clinical outcomes. This may be related to the weakened protective effects of testosterone on immunity, metabolism, and muscle function [ 26 ]. Some studies have proposed that testosterone levels may serve as prognostic biomarkers in male patients with sepsis, with lower testosterone levels indicating a higher risk of death. Patients with sepsis often experience significant changes in sex hormone levels, with a notable decrease in testosterone levels in male patients. This decline may be associated with the systemic inflammatory response, stress state, and hypothalamic-pituitary-gonadal axis dysfunction caused by sepsis. Unlike males, estrogen levels in female patients with sepsis are relatively stable or even elevated, which may be related to the lower mortality rate in females with sepsis [ 27 , 28 ]. In our study, we found that testosterone levels in patients with malignant tumors were relatively low and low testosterone levels were associated with poor prognosis. Future studies should explore the mechanism of action of testosterone in sepsis and its potential therapeutic value. The developed kit uses a chemiluminescence immunoassay and is highly sensitive and capable of detecting free testosterone at the picogram level. Its wide detection and linear ranges cover multiple orders of magnitude to ensure the accuracy of the detection results. This kit specifically recognizes free testosterone and reduces nonspecific binding by optimizing the selection of antibodies and chemiluminescent markers. The operation process is simple, suitable for fully automated operation, reduces manual errors, and improves detection efficiency. This design is advantageous for clinical testing. Compared with traditional methods, the chemiluminescence method can effectively avoid matrix interference and ensure the accuracy of quantitative results. This is particularly important for detecting low-concentration samples. The preparation method of the kit is simple, low-cost, and reliable, and the cost is only one-tenth that of mass spectrometry, making it cost-effective for large-scale clinical detection. Conclusion In summary, the free testosterone chemiluminescence detection kit has become an ideal choice for the clinical detection of free testosterone owing to its high sensitivity, good specificity, and ease of operation. Abbreviations CV coefficient of variation QC quality control PCOS polycystic ovary syndrome SHBG sex hormone-binding globulin Declarations Ethics approval and consent to participate All procedures were performed in accordance with the ethical standards of the institutional and/or national research committees and the Declaration of Helsinki. This study was approved by the Medical Ethics Committee of the Affiliated Hospital of Jiangnan University (Wuxi, Jiangsu, China) (no. LS2024332), and written informed consent was obtained from all participants before enrolment in the study. Consent for publication Not applicable. Availability of data and materials Data used to support the findings of this study are available from the corresponding author upon request.The original data for this manuscript are available at https://pan.baidu.com/s/1klkbGT7LLXphoRp9xkGZ5w . Code: yngm. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Clinical Medical Research Project of the Affiliated Hospital of Jiangnan University and the People's Hospital of Donghai County for the construction of a regional medical center, Wuxi Institute of Translational Medicine (YJZ202304), Wuxi Municipal Science and Technology Bureau, "Taihu Light" Technology Research Project (Medical and Health Technology Research Project) (Y20242201), the Science and Technology Program of Wuxi Municipal Health and Family Planning Commission (Z202420), the Science and Technology Program of Wuxi Municipal Health and Family Planning Commission (T202237) and the Wuxi Medical Innovation Team (No. CXTDPY2021003), and General Project of Open Subjects of the Shanghai Key Laboratory of Molecular Imaging (No. KFKT-2023-35). Authors’ contributions Shuang Han was responsible for the study design, writing, and revision. Simin Zhao and Wenjun Yan collected samples. Min Liu was involved in the sample testing and data analysis. Lu et al. analyzed the relationship between clinical diagnosis and free testosterone levels. Huang provided guidance on the overall structure of the study. All the authors have read and approved the final version of this manuscript. Acknowledgements We are grateful for the cooperation of our medical colleagues and researchers at the Affiliated Hospital of Jiangnan University, as well as for the equipment and technical support provided by Jiangsu Baiming Biological Co., Ltd. References Olsson M, Wapstra E, Madsen T, Silverin B. Testosterone, ticks and travels: a test of the immunocompetence-handicap hypothesis in free-ranging male sand lizards. P Roy Soc B-Biol Sci. 2000;267(1459):2339–43. Dorizzi RM, De Vita F, Caruso B, Meneghelli S, Rocca M, Melloni N, Massocco A, Rizzotti P. Free testosterone/cortisol ratio; A highly individualized test for overtraining syndrome. Clin Chem. 2006;52(6):A89–89. Bello AK, Stenvinkel P, Lin M, Hemmelgarn B, Thadhani R, Klarenbach S, Chan C, Zimmerman D, Cembrowski G, Strippoli G, et al. Serum Testosterone Levels and Clinical Outcomes in Male Hemodialysis Patients. Am J Kidney Dis. 2014;63(2):268–75. Mooradian AD, Morley JE, Korenman SG. Biological Actions of Androgens. Endocr Reviews 1987, 8(1):1–28. Yu MW, Chen CJ. Elevated Serum Testosterone Levels and Risk of Hepatocellular-Carcinoma. Cancer Res. 1993;53(4):790–4. Gomes GK, de Branco FMS, Santos HO, Pereira JL, Orsatti FL, de Oliveira EP. Cholesterol intake and serum total cholesterol levels are not associated with total testosterone levels in men: a cross-sectional study from NHANES 2013–2014. Lipids Health Dis. 2023;22(1):168. Cheng LL, Wang SY. Lower serum testosterone is associated with increased likelihood of arthritis. Sci Rep-Uk 2023, 13(1). Cheng HY, Zhang XY, Li YH, Cao DZ, Luo CL, Zhang Q, Zhang SZ, Jiao YZ. Age-related testosterone decline: mechanisms and intervention strategies. Reprod Biol Endocrin 2024, 22(1). Ding EL, Song YQ, Malik VS, Liu SM. Sex differences of endogenous sex hormones and risk of type 2 diabetes - A systematic review and meta-analysis. Jama-J Am Med Assoc. 2006;295(11):1288–99. Shigehara K, Izumi K, Kadono Y, Mizokami A. Testosterone and Bone Health in Men: A Narrative Review. J Clin Med 2021, 10(3). Karakas M, Schäfer S, Appelbaum S, Ojeda F, Kuulasmaa K, Brückmann B, Berisha F, Schulte-Steinberg B, Jousilahti P, Blankenberg S et al. Testosterone Levels and Type 2 Diabetes-No Correlation with Age, Differential Predictive Value in Men and Women. Biomolecules : 2018, 8(3). Wang M, Zhang SH, He J, Zhang TQ, Zhu HJ, Sun RB, Yang N. Biochemical classification diagnosis of polycystic ovary syndrome based on serum steroid hormones. J Steroid Biochem 2025, 245. Walravens J, Sleumer B, Vos MJ, Snaterse G, Narinx N, Antonio L, Reyns T, Fiers T, Kema IP, Kaufman JM et al. SHBG Gene Polymorphisms and Their Influence on Serum SHBG, Total and Free Testosterone Concentrations in Men. J Clin Endocr Metab 2024. Ramachandran S, Hackett GI, Strange RC. Sex Hormone Binding Globulin: A Review of its Interactions With Testosterone and Age, and its Impact on Mortality in Men With Type 2 Diabetes. Sex Med Rev. 2019;7(4):669–78. Tang SN, Xiao ZX, Lin FT, Liang XN, Ma XX, Wu J, Zhou XW, Zhao QH, Gao JL, Xiao QY, et al. Joint effect of testosterone and neurofilament light chain on cognitive decline in men: The Shanghai Aging Study. Alzheimers Dement. 2024;20(8):5290–8. Cimino N, Kang MS, Honig LS, Rutherford BR. Blood-Based Biomarkers for Alzheimer's Disease in Older Adults with Posttraumatic Stress Disorder. Journal of Alzheimer's disease reports; 2022. Huang RM, Hong Y, Wu YK, Li WF, Liu WL. Simultaneous quantification of total and free testosterone in human serum by LC-MS/MS. Anal Bioanal Chem 2023, 415(27):6851–61. Moffat SD, Zonderman AB, Metter EJ, Kawas C, Blackman MR, Harman SM, Resnick SM. Free testosterone and risk for Alzheimer disease in older men. Neurology. 2004;62(2):188–93. Chandrasekaran N, Bustillos P, Starke N. Utility of Directly Measured Free Testosterone in Predicting Benefit of Testosterone Therapy in Men with Hypogonadal Symptoms and Normal Total Testosterone. J Sex Med. 2022;19(4):S17–8. Pyo Y, Kwon KH. Aging, testosterone and male fertility therapy: a review. J Mens Health. 2024;20(8):1–10. Galansky LB, Levy JA, Burnett AL. Testosterone and Male Sexual Function. Urol Clin N Am. 2022;49(4):627–35. Abdelazim IA, Alanwar A, AbuFaza M, Amer OO, Bekmukhambetov Y, Zhurabekovas G, Shikanova S, Karimova B. Elevated and diagnostic androgens of polycystic ovary syndrome. Menopause Rev. 2020;19(1):1–5. Hussein RS, Dayel SB, Abahussein O. Polycystic Ovary Syndrome and Reproductive Health: A Comprehensive Review. Clin Exp Obstet Gyn 2024, 51(12). Sukhapure M, Eggleston K, Douglas K, Fenton A, Frampton C, Porter RJ. Free testosterone is related to aspects of cognitive function in women with and without polycystic ovary syndrome. Arch Women Ment Health. 2022;25(1):87–94. Whittaker J, Harris M. Low-carbohydrate diets and men's cortisol and testosterone: Systematic review and meta-analysis. Nutr Health-Uk. 2022;28(4):543–54. Angele MK, Pratschke S, Hubbard WJ, Chaudry IH. Gender differences in sepsis Cardiovascular and immunological aspects. Virulence. 2014;5(1):12–9. Cao M, Wang GZ, Xie JF. Immune dysregulation in sepsis: experiences, lessons and perspectives. Cell Death Discov 2023, 9(1). Di Nisio A, Sabovic I, De Toni L, Santa Rocca M, Dall'Acqua S, Azzena B, Ponce MD, Foresta C. Testosterone is sequestered in dysfunctional adipose tissue, modifying androgen-responsive genes. Int J Obes. 2020;44(7):1617–25. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5969090","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":437860237,"identity":"bc6a6972-afb9-4979-86a7-6e7935a99356","order_by":0,"name":"Shuang Han","email":"","orcid":"","institution":"Affiliated Hospital of Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Han","suffix":""},{"id":437860238,"identity":"ccdb58dc-45d9-4794-b7e3-118cc64f3896","order_by":1,"name":"Min Liu","email":"","orcid":"","institution":"Affiliated Hospital of Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Liu","suffix":""},{"id":437860239,"identity":"4821cee6-0163-488c-b404-92d29dd4b663","order_by":2,"name":"Simin Zhao","email":"","orcid":"","institution":"Affiliated Hospital of Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Simin","middleName":"","lastName":"Zhao","suffix":""},{"id":437860240,"identity":"d72098db-a681-4133-8d44-8aa16064be7d","order_by":3,"name":"Wenjun Yan","email":"","orcid":"","institution":"Affiliated Hospital of Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Wenjun","middleName":"","lastName":"Yan","suffix":""},{"id":437860241,"identity":"ca04417a-e2ae-446e-8899-38b4d278a455","order_by":4,"name":"Zhixian Lu","email":"","orcid":"","institution":"Affiliated Hospital of Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Zhixian","middleName":"","lastName":"Lu","suffix":""},{"id":437860242,"identity":"81783ee9-ff95-4830-9fc1-15c537a66751","order_by":5,"name":"Xuan Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIie3QMQrCMBSA4eeiS6COrxR6A+FJwKnUqzQU6urY0VKISw+gh3B1jgTsEujq2F5A9AJinRyTUTA/ZMv3Qh6Az/eTsfGUyNZtVQ0Pd2KSGIyuOTqTiSw43DZyzlwAdacrwlSL6jhIQEjjxc5CwsM9J2Ba1JGQ/RZyvlIWEqBZ9oBayEjsCUGJs41M0ZAC0qIJLxKZCwmCZnwlKzjixJGEB8YpU0lMTIxLJoe/UGdW+Hwho1k7DI8yja0EMAPIvhNs1z8F1qE+n8/3970BlVtBGBjnC9wAAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Hospital of Jiangnan University","correspondingAuthor":true,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-02-06 01:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5969090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5969090/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80048099,"identity":"68b20422-ed99-417b-a82b-d24bdfbe0fad","added_by":"auto","created_at":"2025-04-07 10:00:48","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":208736,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the detection process of the free testosterone test kit\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5969090/v1/e448ef06f00f332546756f40.jpeg"},{"id":80043841,"identity":"cf30f9ae-1f30-4c0f-91b4-fb3c8d020b3e","added_by":"auto","created_at":"2025-04-07 09:36:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37194,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the linearity results of the free testosterone detection kit\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5969090/v1/0f805fd86d8f2623f1feb160.png"},{"id":80043840,"identity":"5a965d19-252f-477a-97d5-17d1bc4cb69d","added_by":"auto","created_at":"2025-04-07 09:36:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47450,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation result diagram of the test kit for free testosterone\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5969090/v1/b28fcc0e946635f36f6ee042.png"},{"id":80043855,"identity":"2cece245-4fc6-4881-8445-d7bd27ce8b25","added_by":"auto","created_at":"2025-04-07 09:36:48","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":186874,"visible":true,"origin":"","legend":"\u003cp\u003eThe mean of the measured values of the two methods-the difference between the measured values of the two methods (deviation: -0.03668; SD: 9.452; 95% limits of agreement were -18.56 and 18.49 pg/mL)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5969090/v1/70a1cd9747832120d52dfb3c.jpeg"},{"id":80046633,"identity":"275e38c0-8155-4e71-a005-2e41b6b01b6a","added_by":"auto","created_at":"2025-04-07 09:52:48","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176780,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the relationship between male free testosterone levels and clinical diagnosis\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5969090/v1/6adb2a1babc86f4ba572abde.jpeg"},{"id":80049215,"identity":"66f7ad8c-8972-4ba0-854b-0b1fb512daf4","added_by":"auto","created_at":"2025-04-07 10:08:48","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":435187,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the relationship between female free testosterone levels and clinical diagnosis\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5969090/v1/b8a8e94e2daaff2e40725183.jpeg"},{"id":80513345,"identity":"9a1c1aee-d1b1-4b9e-9de5-cc4b34fba92d","added_by":"auto","created_at":"2025-04-14 07:32:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2192102,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5969090/v1/23e71643-4819-4c57-99da-7b8fa7e2a0a1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Free Testosterone Chemiluminescence Detection Kit and Its Clinical Application","fulltext":[{"header":"Background","content":"\u003cp\u003eTestosterone, also known as androsterone or androstenone, with the molecular formula C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, is a steroid hormone secreted by the testes of men and ovaries of women. The adrenal glands also secrete small amounts of testosterone. It plays a role in maintaining muscle strength and mass, bone density, and strength, and enhances energy and physical performance. Testosterone was first identified as a secretory glycoprotein in the culture medium of mouse testicular support cells and is a relatively small cytoskeletal-associated protein that can bind tightly to the cell surface through receptor-associated proteins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn clinical tests, serum testosterone levels in patients with type 1 and 2 diabetes are significantly reduced, and serum testosterone levels are negatively correlated with blood glucose concentration and disease duration. Testosterone has a significant protective effect on pancreatic islet cells and promotes insulin synthesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. For male patients, elevated serum testosterone levels is observed in the following diseases: Leydig cell tumors, true precocious puberty, familial incomplete pseudohermaphroditism type II, complete or incomplete testicular feminization, male infertility syndrome, Reifenstein syndrome, and the use of drugs with androgenic effects, such as phenytoin and danazol [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Decreased serum testosterone levels is observed in the following diseases: congenital dysgenesis of the seminiferous tubules, anorchism, cryptorchidism, primary hypogonadism due to underdeveloped or non-developed interstitial cells, hypogonadism due to low secretion of gonadotropins, isolated LH deficiency syndrome, obesity-related hypogonadism, hyperprolactinemia, Cushing's syndrome, congenital adrenal hyperplasia due to deficiency of enzymes, such as 3β-hydroxysteroid dehydrogenase and 17-hydroxylase, and the use of drugs that inhibit testosterone synthesis, such as aminoglutethimide, spironolactone, marijuana, ketoconazole and dichlorodiphenyltrichloroethane [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In female patients, elevated serum testosterone levels have been observed in some cases of polycystic ovary syndrome (PCOS), theca cell hyperplasia, ovarian masculinizing tumors, and adrenal cortical hyperplasia [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. After testosterone is secreted into the blood, 98% is bound to sex hormone-binding globulin (SHBG), and a small amount is bound to albumin and cortisol-binding globulin. Approximately 1\u0026ndash;2% of total testosterone is in the free state. The level of SHBG in the blood almost determines the amount of free testosterone. The ratio of total testosterone to SHBG (T/SHBG) is known as the free testosterone index. Determination of free testosterone can overcome the influence of changes in the concentration of testosterone-binding proteins and directly reflect the biological activity of hormones. In many women with hypertrichosis or low SHBG levels, serum total testosterone is often within the normal range and the measured free testosterone level is significantly higher than 90% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Free testosterone can reflect the characteristics of some diseases more accurately than total testosterone and has more significance in clinical diagnosis. Measuring free testosterone levels is also a way to indirectly measure SHBG levels in the blood. Measurement of free testosterone can be used to monitor Alzheimer's disease in older men and low levels of free testosterone indicate a high risk of Alzheimer's disease [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe clinical detection of free testosterone levels in the serum is crucial for diagnosing related diseases. The primary methods for detecting free testosterone can be classified into indirect and direct measurements. Indirect determination methods fall into two categories: (1) Simulated immunoassay: Although this method is operationally simple, its specificity is relatively low. Fluctuations in sex hormone-binding globulin (SHBG) levels significantly affect the results, rendering it unsuitable for clinical diagnosis. (2) Formula calculation method: This approach estimates free testosterone concentration based on measurements of total testosterone, SHBG, albumin concentrations, and dissociation constants. However, the accuracy of the results may be compromised when albumin concentration is abnormal. Furthermore, significant variations in algorithms among laboratories limit its reliability, making it unsuitable for clinical application until further validation confirms its consistency and accuracy. Direct determination methods include three main approaches: (1) Equilibrium dialysis: Recognized as a reliable method for determining free testosterone, this technique separates free testosterone from protein-bound testosterone using a semi-permeable membrane over a specified period. Despite its high accuracy, its clinical application is limited due to challenges such as large sample volume requirements, complex pretreatment procedures, difficulty achieving low detection limits, low throughput, high consumable costs, potential contamination during dialysis, poor precision, and insufficient specificity. (2) Ultrafiltration: This method pre-separates free testosterone from serum using ultrafiltration tubes and subsequently detects the bound fraction via reagent strips and liquid chromatography-tandem mass spectrometry (LC-MS/MS). While the procedure is relatively straightforward to perform, discrepancies in results may arise due to variations in ultrafiltration membrane performance and operational conditions. (3) Immuno-mass spectrometry: By integrating the strengths of immunoassay and mass spectrometry, this method utilizes kits to conjugate antibodies with free and total testosterone in serum samples for magnetic micro-enrichment, automated pretreatment, and LC-MS/MS detection. This approach provides enhanced accuracy and stability but entails higher equipment costs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In summary, indirect determination methods are easy to implement but lack sufficient accuracy. Among direct determination methods, equilibrium dialysis provides accurate results but is time-consuming; ultrafiltration is simpler to operate but may yield inconsistent results; and immuno-mass spectrometry delivers efficient and precise outcomes at the expense of higher equipment investment.Compared with the current methods for detecting free testosterone, chemiluminescence immunoassays have the advantages of high sensitivity, good specificity, simple operation, rapidity, strong anti-interference ability, and low cost, making them suitable for large-scale clinical detection and research applications. However, there are currently few chemiluminescence immunoassay kits for detecting free testosterone content, both at home and abroad. Therefore, it is necessary to develop a more sensitive, specific, and safe chemiluminescence immunoassay for the detection of free testosterone.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eResearch population\u003c/h2\u003e \u003cp\u003eIn total, 4, 042 patient samples were collected from the Affiliated Hospital of Jiangnan University. The research protocol was approved by the Human Ethics Review Committee of the Affiliated Hospital of Jiangnan University, indicating that we have adhered to ethical standards.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMain reagents and materials\u003c/h3\u003e\n\u003cp\u003eFree testosterone antigen (Catalog No.: FT2011), free testosterone antibody (Catalog No.: FT3001), and testosterone analogs (Catalog No.: FT4001) were provided by Zhenjiang Airenake Biotechnology Co., Ltd. (Zhenjiang, China); magnetic bead reagents were purchased from Thermo Fisher Scientific (China), Catalog No.: 88817. Chemiluminescent and immunoluminescence instruments were purchased from Faponbiotech (Guangzhou, China). The pH meter was purchased from Mettler Toledo (Switzerland).\u003c/p\u003e\n\u003ch3\u003eReagent preparation\u003c/h3\u003e\n\u003cp\u003eMagnetic bead reagent preparation: First, 2.8 \u0026micro;m magnetic beads were selected and activated with 0.1 M hydrochloric acid solution, then rinsed with deionized water until neutral. Next, streptavidin solution (concentration of 10 \u0026micro;g/mL) was mixed with the magnetic beads and incubated at 37 ℃ for 2 h to coat the streptavidin on the surface of the magnetic beads. Finally, the magnetic beads were suspended in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4).\u003c/p\u003e \u003cp\u003eCalibrators: A set of free testosterone solutions of known concentrations was used to construct a standard curve. Calibrator concentrations were 0, 5, 10, 20, 50, 100, 200, and 400 pg/ml. Calibrators were prepared in a human serum matrix to better simulate a clinical sample environment.\u003c/p\u003e \u003cp\u003eQuality control (QC) products: Free testosterone was diluted in a human serum matrix to prepare QC products at concentrations of 5 and 150 pg/mL.\u003c/p\u003e \u003cp\u003eLabeled solution: A solution of testosterone analogs labeled with acridinium ester. The concentration of the testosterone analogs in the labeled solution was 1 \u0026micro;mol/L and the labeling efficiency of acridinium ester was over 90%, ensuring that the labeled substances have high chemiluminescence activity.\u003c/p\u003e \u003cp\u003eBiotin antibody solution: The antibodies specifically bind to testosterone, which is labeled with biotin. The antibody concentration was 5 \u0026micro;g/mL and the molar ratio of biotin to antibody was 1:5. This solution was prepared in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4).\u003c/p\u003e \u003cp\u003eSubstrate solution: A specific substrate solution was used to trigger a chemiluminescence signal from the labeled substances. The main components of the substrate solution are hydrogen peroxide and sodium hydroxide, with concentrations of 0.1 and 0.25 mol/L, respectively. When the substrate came into contact with labeled substances, it triggered a strong chemiluminescent reaction.\u003c/p\u003e \u003cp\u003eWashing solution: The magnetic beads were washed and unbound substances were removed to reduce the impact of non-specific binding on the test results. The main components of the washing solution were 0.05 M Tris-HCl buffer (pH 7.4) and 0.1% Tween-20 surfactant.\u003c/p\u003e\n\u003ch3\u003eReaction process\u003c/h3\u003e\n\u003cp\u003eFree testosterone content was measured using a competitive method. Free testosterone and testosterone derivatives compete for binding to biotinylated testosterone antibodies. The sample was incubated at 37 ℃ for 30 min, then incubated with magnetic bead solution for 5 min. Magnetic separation is achieved by applying a magnetic field. The sample was washed thrice with a washing solution to form an antibody-antigen acridinium ester complex. The unbound reactants on the magnetic beads were washed away. Hydrogen peroxide and alkaline solutions were added to detect the luminescence. The luminescence value was input into the calibration curve to calculate free testosterone concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS 25.0. Normally distributed continuous variables are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and non-normally distributed ones are expressed as median. The performance of the method is assessed in accordance with the EP guidelines established by the Clinical and Laboratory Standards Institute (CLSI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLinearity\u003c/h2\u003e \u003cp\u003eThe calibrated diluent with antigen served as the high-concentration sample, and the diluent alone as the low-concentration sample. High-concentration samples near the upper linear range limit were serially diluted to various concentrations, including low-concentration samples near the lower limit. Each concentration was tested in triplicate, and the mean calculated. The analyte concentration (x-axis) and average test result (y-axis) were fitted by least-squares regression. The correlation coefficient (r) within the linear range was determined, requiring r\u0026thinsp;\u0026gt;\u0026thinsp;0.99.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLimit of blank\u003c/h3\u003e\n\u003cp\u003eThe zero-concentration calibrator, after dilution, was used as the test sample. Twenty measurements were performed to obtain optical signal values. The mean (M) and standard deviation (SD) were calculated, and M-2SD was determined. A primary equation was derived from two-point regression fitting using the zero-concentration reference and the nearest adjacent concentration calibrator's data. The optical signal value corresponding to M-2SD was substituted.\u003c/p\u003e\n\u003ch3\u003eAccuracy\u003c/h3\u003e\n\u003cp\u003eSamples with high and low concentrations (5 pg/mL and 200 pg/mL) of free testosterone were prepared. The relative deviation (B) between the mean value (M) and the reference value was calculated using Equation B=(M-T)/T\u0026times;100%(T - mark value) after three replicate measurements. The reagent was tested on samples at two concentration levels (5 pg/mL and 200 pg/mL), each measured in triplicate.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImprecision\u003c/h2\u003e \u003cp\u003ePrepare three batches of reagents and perform ten replicate detections for high-value (100 pg/mL) and low-value control (5.1 pg/mL) items in each batch. Calculate the mean value (M) and standard deviation (SD) of the results, and subsequently determine the coefficient of variation (CV) using the formula CV(%) = (SD/M) \u0026times; 100%. In this context, CV denotes the coefficient of variation, M represents the mean concentration value, and SD signifies the standard deviation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThermal stability test\u003c/h2\u003e \u003cp\u003eThe quality controls (10.78 pg/mL, 100.87 pg/mL) of the configured free testosterone kit is placed at 4 ℃ and 37 ℃ respectively for 1, 3, 6, 10, 20, 25, and 30 days to test the luminescence value, The experimental results showed that the deviation of the concentration of the quality controls was less than 10% when the kit was placed at 37\u0026deg;C for different times compared to storage at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eKit specific test\u003c/h2\u003e \u003cp\u003eThe specific substance was prepared at the corresponding concentration, and one batch of the free testosterone detection kit was employed to quantify the specific substance and calculate the cross-reactivity rate. The cross-reactivity rate (%) was determined using the formula: (free testosterone concentration / specific substance concentration) \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical relevance assessment\u003c/h2\u003e \u003cp\u003eTo validate the detection efficacy of the kit developed in this study, a clinical performance evaluation was conducted. A total of 392 plasma samples from patients provided by the Affiliated Hospital of Jiangnan University were analyzed using both a self-developed chemiluminescence-based free testosterone detection kit and a commercially available kit. The results were compared, with values measured using the self-developed kit plotted on the x-axis and those from the commercial kit on the y-axis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of the working concentration of magnetic beads\u003c/h2\u003e \u003cp\u003eThe magnetic bead concentration was selected based on the optimized antibody concentration. Specifically, magnetic bead concentrations of 15 mg/mL, 10 mg/mL, 7 mg/mL and 5 mg/mL were evaluated. According to the experimental results, the optimal luminescence effect was achieved at a magnetic bead concentration of 10 mg/mL. Consequently, the concentration of 10 mg/mL was chosen for subsequent experiments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of luminescence values in different magnetic bead concentrations of free testosterone kits\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFree testosterone calibrator\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalibration concentration (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBeads concentration\u003c/p\u003e \u003cp\u003e15 mg/mL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeads concentration\u003c/p\u003e \u003cp\u003e10 mg/mL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBeads concentration\u003c/p\u003e \u003cp\u003e7 mg/mL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBeads concentration\u003c/p\u003e \u003cp\u003e5 mg/mL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7328391\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7612893\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7427162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7213465\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1324212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1524681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1125361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1231461\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e712361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e763752\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e714535\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e709869\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e293812\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e303452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e271829\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e234265\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e112831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e152496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e113728\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e109281\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e70121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e76843\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e73212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e71231\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38754\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31271\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14321\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12131\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of free testosterone antibody working concentration\u003c/h2\u003e \u003cp\u003eBiotin-antibody solution preparation: Initially, antibodies with specific binding affinity for testosterone were biotinylated. During the biotinylation process, the molar ratio of control biotin to antibody was maintained at 1:5. Subsequently, the biotinylated antibody was dissolved in a diluent solution at predetermined concentrations of 3 \u0026micro;g/mL, 5 \u0026micro;g/mL, and 7 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003ePreparation of marker-antibody diluent: A stock solution was prepared by dissolving 12.05 g of Tris, 10 g of salicylic acid, and 2.0 g of NaN₃ in 1 L of purified water. The mixture was stirred at room temperature for 1 hour. Following this, 0.998 mL of MgCl₂\u0026middot;6H₂O (1 M), 1.027 mL of ZnCl₂ (0.1 M), and 1 g of BSA were added to the solution. The pH of the solution was adjusted to 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05. The solution was then filtered and stored at 4 ℃. Biotinylated testosterone analogs were subsequently dissolved in the diluent at concentrations of 0.01 ng/mL, 0.05 ng/mL, and 0.1 ng/mL. Aliquots of 30 \u0026micro;L of each concentration were used to perform the checkerboard titration method. The final working concentrations were set at 5 \u0026micro;g/mL for the biotinylated antibody and 0.05 ng/mL for the labeled testosterone analog (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCheckerboard test data\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eBiotin-T antibody 3 \u0026micro;g/mL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eBiotin-T antibody 5 \u0026micro;g/mL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eBiotin-T antibody 7 \u0026micro;g/mL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT-AE 0.01 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT-AE 0.05 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT-AE 0.1 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT-AE 0.01 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eT-AE 0.05 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eT-AE 0.1 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT-AE 0.01 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eT-AE 0.05 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eT-AE 0.1 ng/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3231421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2340123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20441234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50231231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e76123123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9785188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20132312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e30384736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e34256882\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2002049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1832032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17424123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4012312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3141232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e592948\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5235829\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6374852\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5313152\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e873231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e741241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6984241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e76141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e304294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e702065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e749958\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e770922\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.96%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.29%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.24%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.13%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.06%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26.01%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20.98%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15.51%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.62%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.46%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.08%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.11%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.49%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.47%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.25%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMethod performance evaluation\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eLinearity\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents a linear analysis of free testosterone. The fitted linear regression equation is Y\u0026thinsp;=\u0026thinsp;0.9990X\u0026thinsp;+\u0026thinsp;0.6843, r\u0026thinsp;=\u0026thinsp;0.9999, demonstrating an excellent linear relationship between the theoretical and actual sample concentrations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLimit of blank\u003c/h2\u003e \u003cp\u003eThe results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The concentrations of free testosterone were 0.013, 0.021, and 0.016 pg/mL, and the limit of the free testosterone blank was 0.021 pg/mL.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVerification results of limit of blank of free testosterone\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTest 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTest 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTest 3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9898443.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9230547.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9779625.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54762.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65679.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e161610.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-2SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9788917.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9099188.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9456404.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOB (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAccuracy\u003c/h2\u003e \u003cp\u003eThe experimental results are listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The relative deviation of the free testosterone kit was within 5%, indicating that the kit had good accuracy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAccuracy test of free testosterone kit\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTest 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTest 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTest 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean value (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRelative deviation (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.20%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e202.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e199.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e201.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e200.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.18%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eImprecision\u003c/h2\u003e \u003cp\u003eThe results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, with a precision criterion of 3% or lower .\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of imprecision measurement results of free testosterone detection kits\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of tests\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow value quality control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh value quality control\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e101.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e102.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFitting concentration M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.625\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFitting concentration SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.139379578\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.741308902\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFitting concentration CV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.70%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.74%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eThermal stability test\u003c/h2\u003e \u003cp\u003eThe relative error in the accelerated stability test was maintained within \u0026plusmn;\u0026thinsp;5%, demonstrating excellent stability of the kit (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAccelerated stability test results of free testosterone kit\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDays\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNominal concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eTest concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eDeviation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTest1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTest2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTest3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTest1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTest2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTest3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.02%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.02%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.95%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e102.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e101.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-2.03%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.01%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2.13%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.86%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-1.76%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e101.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.36%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.96%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.36%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.06%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-2.78%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e102.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.36%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.65%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.39%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.83%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-3.06%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.34%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.90%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-2.62%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.06%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.39%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.95%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.11%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.74%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.76%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.83%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-3.93%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-1.63%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.46%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.83%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.57%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.94%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-3.67%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFree testosterone detection kits were evaluated at 3, 6, 9, and 12 months, respectively. The coefficient of variation (CV) values for quality control samples S2, S4, and S6 were consistently below 10%, thereby meeting the specified criteria. These findings indicate that the free testosterone detection kits can be effectively stored at room temperature for up to 12 months. Detailed results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eReal-time stability evaluation results of free testosterone kit\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTest time (month)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eImprecision(CV%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.91%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.17%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.46%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.67%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.73%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.46%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.21%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.61%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.68%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eKit specific test\u003c/h2\u003e \u003cp\u003eThe specificity was found to be less than 0.1%, thereby meeting the specified requirements. The results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecific test results of free testosterone kit\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCross reactant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExperimental concentration (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetermination of concentration (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCross reactivity rate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDhea sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0026%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstradiol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.029%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCortisol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProgesterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrednisone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.011%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorticosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.008%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eClinical relevance assessment\u003c/h2\u003e \u003cp\u003eLinear regression analysis revealed a strong linear correlation (r\u0026thinsp;=\u0026thinsp;0.9941), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. These findings confirm that the newly developed kit meets clinical detection requirements.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe performed a Bland\u0026ndash;Altman analysis of the data, and the mean difference between the two measurements was \u0026minus;\u0026thinsp;0.03668 pg/mL, indicating that the kit measurements were very close to the hospital test results with little systematic bias. The 95% limit of agreement was calculated to be -18.56 to 18.49 pg per milliliter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This indicates that most of the sample measurement differences were within this range.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, after many repeated tests, the detection range of the free testosterone chemiluminescence detection kit was 0\u0026ndash;400 pg/mL. The limit of the blank was 0.021 pg/mL and linear test r was 0.9999, which is better accuracy and stability than the same type of kit on the market. By comparing multiple markers, a cheaper acridone ester was selected to ensure labeling efficiency and reduce the production cost, thus reducing the financial pressure on patients when using the free testosterone chemiluminescence detection kit developed in this study.\u003c/p\u003e \u003cp\u003eWe tested 1615 male and 2035 female patient samples using the free testosterone chemiluminescence kit (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Among the males, the free testosterone levels of most patients with prostate malignancy were relatively low, with a median of 2.24 pg/mL and a relatively narrow interquartile range (IQR of 1.82 pg/mL), indicating that the testosterone levels of patients in this diagnostic category were generally low and concentrated. However, there were some substantially high outlier values, suggesting that individual patients may have significantly different pathological features. Patients with diagnoses related to hair loss (such as androgenetic and seborrheic alopecia) generally had higher testosterone levels, with a median of approximately 28 pg/mL and a relatively wide IQR, especially for androgenetic alopecia, with an IQR of 7.56 pg/mL, indicating a large difference in testosterone levels among patients. The maximum values of these diagnostic categories were relatively high, further supporting the association between high testosterone levels and hair loss. The median was lower for diagnoses related to precocious puberty and rapid progression of puberty, but the IQR was relatively wide, especially for precocious puberty with an IQR of 16.03 pg/mL, indicating a large difference in testosterone levels among patients. Some patients had extremely high values, suggesting that these diagnoses may be related to early or rapid changes in sex hormone levels. The median testosterone level was higher in patients with benign prostatic hyperplasia (23.77 pg/mL), and the IQR was relatively wide (17.36 pg/mL), indicating a large difference in testosterone levels among patients. The maximum value was 81.41 pg/mL, indicating that testosterone levels in some patients were significantly elevated, possibly related to the severity of the disease or other factors.\u003c/p\u003e \u003cp\u003ePatients in the different clinical diagnostic categories showed significant differences in free testosterone levels. Some diagnoses (such as prostate malignancy) were associated with lower testosterone levels, whereas others (such as diagnoses related to hair loss) were associated with higher testosterone levels. These results suggest that free testosterone can be an important reference indicator for the diagnosis and assessment of certain diseases.\u003c/p\u003e \u003cp\u003eFree testosterone levels in female patients with depression vary significantly. Some patients have significantly elevated hormone levels, which may suggest a significant role of hormone levels in this disease. The free testosterone levels of patients with depression were significantly different and some patients had higher hormone levels, which may be related to disease severity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecently, numerous studies have explored the relationship between free testosterone and cognitive function in men, particularly elderly men, and significant progress has been made in this area. Testosterone exerts its neuroprotective effects by improving synaptic transmission, preventing neuronal death, and enhancing synaptic plasticity. These protective effects may be crucial for maintaining cognitive function and delaying cognitive decline [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A research team from Huashan Hospital Affiliated to Fudan University and the School of Public Health of Fudan University found that elderly men with low levels of free testosterone had a significantly increased risk of cognitive decline. Specifically, the risk of cognitive decline in men with low free testosterone levels increased by 429%. This finding suggests that free testosterone may be an important biomarker of cognitive decline in elderly men [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe detection of free testosterone levels has significant clinical implications. Free testosterone is the biologically active form of testosterone in the body and directly reflects the effects of androgens. In men, a decline in free testosterone levels may lead to decreased libido, sexual dysfunction, reduced sperm production, and other issues affect fertility [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In women, abnormally elevated free testosterone levels may be associated with PCOS, which presents with symptoms, such as irregular menstruation, hirsutism, and acne. Free testosterone is an important indicator of hyperandrogenism [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For example, free testosterone levels are significantly elevated in patients with PCOS and the free androgen index is considered more diagnostically valuable than total testosterone. Additionally, changes in free testosterone levels are associated with metabolic disorders, such as thyroid dysfunction, obesity, and insulin resistance. As men age, free testosterone levels gradually decline, which may be related to symptoms, such as osteoporosis, muscle loss, and fatigue. The detection of free testosterone helps assess the risk of age-related diseases and guides testosterone replacement therapy. Changes in free testosterone levels may indicate the presence of other diseases. For example, adrenal cortical and testicular tumors may cause elevated free testosterone levels, whereas hypogonadism and liver dysfunction may lead to decreased free testosterone levels. Free testosterone testing is not only used for diagnosis, but also for monitoring treatment effects. For example, in patients with PCOS, reducing free testosterone levels through treatment can improve symptoms and reproductive outcomes. In men, free testosterone levels can be measured after testosterone replacement therapy to evaluate treatment effect [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In summary, free testosterone testing is an important means of assessing androgen levels and related diseases and provides more accurate biological information than total testosterone.\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated that low testosterone levels in male patients with sepsis are associated with higher mortality rates, longer hospital stay, and poorer clinical outcomes. This may be related to the weakened protective effects of testosterone on immunity, metabolism, and muscle function [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Some studies have proposed that testosterone levels may serve as prognostic biomarkers in male patients with sepsis, with lower testosterone levels indicating a higher risk of death. Patients with sepsis often experience significant changes in sex hormone levels, with a notable decrease in testosterone levels in male patients. This decline may be associated with the systemic inflammatory response, stress state, and hypothalamic-pituitary-gonadal axis dysfunction caused by sepsis. Unlike males, estrogen levels in female patients with sepsis are relatively stable or even elevated, which may be related to the lower mortality rate in females with sepsis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our study, we found that testosterone levels in patients with malignant tumors were relatively low and low testosterone levels were associated with poor prognosis. Future studies should explore the mechanism of action of testosterone in sepsis and its potential therapeutic value.\u003c/p\u003e \u003cp\u003eThe developed kit uses a chemiluminescence immunoassay and is highly sensitive and capable of detecting free testosterone at the picogram level. Its wide detection and linear ranges cover multiple orders of magnitude to ensure the accuracy of the detection results. This kit specifically recognizes free testosterone and reduces nonspecific binding by optimizing the selection of antibodies and chemiluminescent markers. The operation process is simple, suitable for fully automated operation, reduces manual errors, and improves detection efficiency. This design is advantageous for clinical testing. Compared with traditional methods, the chemiluminescence method can effectively avoid matrix interference and ensure the accuracy of quantitative results. This is particularly important for detecting low-concentration samples. The preparation method of the kit is simple, low-cost, and reliable, and the cost is only one-tenth that of mass spectrometry, making it cost-effective for large-scale clinical detection.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the free testosterone chemiluminescence detection kit has become an ideal choice for the clinical detection of free testosterone owing to its high sensitivity, good specificity, and ease of operation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCV coefficient of variation\u003c/p\u003e\u003cp\u003eQC quality control\u003c/p\u003e\u003cp\u003ePCOS polycystic ovary syndrome\u003c/p\u003e\u003cp\u003eSHBG sex hormone-binding globulin\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were performed in accordance with the ethical standards of the institutional and/or national research committees and the Declaration of Helsinki. This study was approved by the Medical Ethics Committee of the Affiliated Hospital of Jiangnan University (Wuxi, Jiangsu, China) (no. LS2024332), and written informed consent was obtained from all participants before enrolment in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData used to support the findings of this study are available from the corresponding author upon request.The original data for this manuscript are available at https://pan.baidu.com/s/1klkbGT7LLXphoRp9xkGZ5w\u003ca href=\"https://pan.baidu.com/s/1klkbGT7LLXphoRp9xkGZ5w.\"\u003e.\u003c/a\u003e Code: yngm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Clinical Medical Research Project of the Affiliated Hospital of Jiangnan University and the People\u0026apos;s Hospital of Donghai County for the construction of a regional medical center, Wuxi Institute of Translational Medicine (YJZ202304), Wuxi Municipal Science and Technology Bureau, \u0026quot;Taihu Light\u0026quot; Technology Research Project (Medical and Health Technology Research Project) (Y20242201), the Science and Technology Program of Wuxi Municipal Health and Family Planning Commission (Z202420), the Science and Technology Program of Wuxi Municipal Health and Family Planning Commission (T202237) and the Wuxi Medical Innovation Team (No. CXTDPY2021003), and General Project of Open Subjects of the Shanghai Key Laboratory of Molecular Imaging (No. KFKT-2023-35).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShuang Han was responsible for the study design, writing, and revision. Simin Zhao and Wenjun Yan collected samples. Min Liu was involved in the sample testing and data analysis. Lu et al. analyzed the relationship between clinical diagnosis and free testosterone levels. Huang provided guidance on the overall structure of the study. All the authors have read and approved the final version of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe are grateful for the cooperation of our medical colleagues and researchers at the Affiliated Hospital of Jiangnan University, as well as for the equipment and technical support provided by Jiangsu Baiming Biological Co., Ltd.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOlsson M, Wapstra E, Madsen T, Silverin B. Testosterone, ticks and travels: a test of the immunocompetence-handicap hypothesis in free-ranging male sand lizards. P Roy Soc B-Biol Sci. 2000;267(1459):2339\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDorizzi RM, De Vita F, Caruso B, Meneghelli S, Rocca M, Melloni N, Massocco A, Rizzotti P. Free testosterone/cortisol ratio; A highly individualized test for overtraining syndrome. Clin Chem. 2006;52(6):A89\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBello AK, Stenvinkel P, Lin M, Hemmelgarn B, Thadhani R, Klarenbach S, Chan C, Zimmerman D, Cembrowski G, Strippoli G, et al. Serum Testosterone Levels and Clinical Outcomes in Male Hemodialysis Patients. Am J Kidney Dis. 2014;63(2):268\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMooradian AD, Morley JE, Korenman SG. Biological Actions of Androgens. Endocr Reviews 1987, 8(1):1\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu MW, Chen CJ. Elevated Serum Testosterone Levels and Risk of Hepatocellular-Carcinoma. Cancer Res. 1993;53(4):790\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomes GK, de Branco FMS, Santos HO, Pereira JL, Orsatti FL, de Oliveira EP. Cholesterol intake and serum total cholesterol levels are not associated with total testosterone levels in men: a cross-sectional study from NHANES 2013\u0026ndash;2014. Lipids Health Dis. 2023;22(1):168.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng LL, Wang SY. Lower serum testosterone is associated with increased likelihood of arthritis. Sci Rep-Uk 2023, 13(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng HY, Zhang XY, Li YH, Cao DZ, Luo CL, Zhang Q, Zhang SZ, Jiao YZ. Age-related testosterone decline: mechanisms and intervention strategies. Reprod Biol Endocrin 2024, 22(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing EL, Song YQ, Malik VS, Liu SM. Sex differences of endogenous sex hormones and risk of type 2 diabetes - A systematic review and meta-analysis. Jama-J Am Med Assoc. 2006;295(11):1288\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShigehara K, Izumi K, Kadono Y, Mizokami A. Testosterone and Bone Health in Men: A Narrative Review. J Clin Med 2021, 10(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarakas M, Sch\u0026auml;fer S, Appelbaum S, Ojeda F, Kuulasmaa K, Br\u0026uuml;ckmann B, Berisha F, Schulte-Steinberg B, Jousilahti P, Blankenberg S et al. Testosterone Levels and Type 2 Diabetes-No Correlation with Age, Differential Predictive Value in Men and Women. Biomolecules : 2018, 8(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Zhang SH, He J, Zhang TQ, Zhu HJ, Sun RB, Yang N. Biochemical classification diagnosis of polycystic ovary syndrome based on serum steroid hormones. J Steroid Biochem 2025, 245.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalravens J, Sleumer B, Vos MJ, Snaterse G, Narinx N, Antonio L, Reyns T, Fiers T, Kema IP, Kaufman JM et al. SHBG Gene Polymorphisms and Their Influence on Serum SHBG, Total and Free Testosterone Concentrations in Men. J Clin Endocr Metab 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamachandran S, Hackett GI, Strange RC. Sex Hormone Binding Globulin: A Review of its Interactions With Testosterone and Age, and its Impact on Mortality in Men With Type 2 Diabetes. Sex Med Rev. 2019;7(4):669\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang SN, Xiao ZX, Lin FT, Liang XN, Ma XX, Wu J, Zhou XW, Zhao QH, Gao JL, Xiao QY, et al. Joint effect of testosterone and neurofilament light chain on cognitive decline in men: The Shanghai Aging Study. Alzheimers Dement. 2024;20(8):5290\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCimino N, Kang MS, Honig LS, Rutherford BR. Blood-Based Biomarkers for Alzheimer's Disease in Older Adults with Posttraumatic Stress Disorder. Journal of Alzheimer's disease reports; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang RM, Hong Y, Wu YK, Li WF, Liu WL. Simultaneous quantification of total and free testosterone in human serum by LC-MS/MS. Anal Bioanal Chem 2023, 415(27):6851\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoffat SD, Zonderman AB, Metter EJ, Kawas C, Blackman MR, Harman SM, Resnick SM. Free testosterone and risk for Alzheimer disease in older men. Neurology. 2004;62(2):188\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChandrasekaran N, Bustillos P, Starke N. Utility of Directly Measured Free Testosterone in Predicting Benefit of Testosterone Therapy in Men with Hypogonadal Symptoms and Normal Total Testosterone. J Sex Med. 2022;19(4):S17\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePyo Y, Kwon KH. Aging, testosterone and male fertility therapy: a review. J Mens Health. 2024;20(8):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalansky LB, Levy JA, Burnett AL. Testosterone and Male Sexual Function. Urol Clin N Am. 2022;49(4):627\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdelazim IA, Alanwar A, AbuFaza M, Amer OO, Bekmukhambetov Y, Zhurabekovas G, Shikanova S, Karimova B. Elevated and diagnostic androgens of polycystic ovary syndrome. Menopause Rev. 2020;19(1):1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussein RS, Dayel SB, Abahussein O. Polycystic Ovary Syndrome and Reproductive Health: A Comprehensive Review. Clin Exp Obstet Gyn 2024, 51(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSukhapure M, Eggleston K, Douglas K, Fenton A, Frampton C, Porter RJ. Free testosterone is related to aspects of cognitive function in women with and without polycystic ovary syndrome. Arch Women Ment Health. 2022;25(1):87\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhittaker J, Harris M. Low-carbohydrate diets and men's cortisol and testosterone: Systematic review and meta-analysis. Nutr Health-Uk. 2022;28(4):543\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAngele MK, Pratschke S, Hubbard WJ, Chaudry IH. Gender differences in sepsis Cardiovascular and immunological aspects. Virulence. 2014;5(1):12\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao M, Wang GZ, Xie JF. Immune dysregulation in sepsis: experiences, lessons and perspectives. Cell Death Discov 2023, 9(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Nisio A, Sabovic I, De Toni L, Santa Rocca M, Dall'Acqua S, Azzena B, Ponce MD, Foresta C. Testosterone is sequestered in dysfunctional adipose tissue, modifying androgen-responsive genes. Int J Obes. 2020;44(7):1617\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Free testosterone, Performance evaluation, Hormone markers, Chemiluminescence kit development","lastPublishedDoi":"10.21203/rs.3.rs-5969090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5969090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eFree 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe linear r was \u0026gt; 0.99, limit of the blank was 0.021 pg/mL, accuracy deviation was \u0026lt; 5%, imprecision was \u0026lt; 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe 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.\u003c/p\u003e","manuscriptTitle":"Development of Free Testosterone Chemiluminescence Detection Kit and Its Clinical Application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 09:36:43","doi":"10.21203/rs.3.rs-5969090/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7cea29e-da2f-4890-a3b6-e1e28949aa38","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-14T07:23:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-07 09:36:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5969090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5969090","identity":"rs-5969090","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}