Chemiluminescence detection kit for free testosterone and preparation method thereof

Abstract

The invention relates to a free testosterone detection kit developed by adopting a competition method in a chemiluminescence method and a preparation method, the kit is matched with a full-automatic chemiluminescence analyzer for use, and human intervention is basically not needed. Compared with the existing free testosterone detection kit, the kit has the advantages that the detection time is shortened, the detection efficiency is improved, the lowest detection limit is 0.021 ng / mL, the linear R2 is equal to 1.000, the anti-interference performance is good, the stability is 12 months, the correlation R2 with the free testosterone kit sold in the market is equal to 0.9956, and the clinical detection requirement is met. Compared with the same type of free testosterone detection kits sold in the market, the detection range is widened to 0-400 pg / mL, the accuracy and precision are both controlled within 5%, and market detection requirements are met.

Description

Technical Field

[0001] The present application belongs to the field of clinical in vitro diagnosis and medical immunology, and relates to an immunoassay reagent, and specifically to a free testosterone chemiluminescence detection kit and its preparation, which is suitable for the quantitative detection of free testosterone in the human body. Background Art

[0002] Testosterone, also known as testosterone, testosterone, has a molecular formula of C 19 H 28 O2 is a steroid hormone secreted by the male testicles or female ovaries. The adrenal glands also secrete a small amount of testosterone, which has the functions of maintaining muscle strength and quality, maintaining bone density and strength, refreshing and improving physical fitness. Testosterone is a secretory glycoprotein originally identified from the culture medium of mouse testicular support cells. It is a cytoskeleton-associated protein with a small molecular weight and can be tightly bound to the cell surface through receptor-associated proteins.

[0003] In clinical tests, the serum testosterone levels of patients with type 1 diabetes and type 2 diabetes were significantly reduced, and the serum testosterone levels were negatively correlated with blood glucose concentration and disease course. Studies have found that testosterone has a significant protective effect on pancreatic islet cells and can significantly promote insulin synthesis.

[0004] For male patients, elevated serum testosterone can be seen in the following diseases: testicular interstitial cell tumor, true precocious puberty, familial incomplete pseudohermaphroditism type II, complete or incomplete testicular feminization, male infertility syndrome, Reifenstein syndrome, and the use of androgenic drugs such as phenytoin sodium and danazol; decreased serum testosterone can be seen in the following diseases: primary male sexual dysfunction such as congenital seminiferous tubule dysgenesis, anorchia, cryptorchidism, interstitial cell dysgenesis or agenesis, hypothalamic-pituitary diseases such as hypogonadotropic hypogonadism, simple LH deficiency, and obesity-induced reproductive impotence syndrome, hyperprolactinemia, hypercortisolism, 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 aminodiopathic, amiloride, cannabis, ketoconazole, and dichlorobenzene.

[0005] For female patients, elevated serum testosterone levels can be seen in some diseases such as polycystic ovary syndrome, theca cell hyperplasia, ovarian masculinizing tumors, and adrenal hyperplasia.

[0006] Clinical detection of free testosterone levels in serum is of great significance for diagnosing related diseases. Currently, the main immunoassay method for detecting free testosterone is radioimmunoassay. This method mainly takes 0.5 mL of patient plasma, adds 0.2 mL of 0.1 mol / L sodium hydroxide and 5 mL of dichloromethane, shakes vigorously for 3 to 5 minutes, removes the plasma layer, washes the dichloromethane layer twice with distilled water, 1 mL each time, centrifuges (1500 r / min) for 3 minutes, aspirates two portions of dichloromethane, 1 mL each, blows dry in a 45 ℃ water bath, and performs radioimmunoassay. However, this method has the risk of radioactive contamination.

[0007] In contrast, chemiluminescent immunoassay is an immunoassay technology developed on the basis of enzyme-linked immunosorbent assay. It has the advantages of high sensitivity, wide detection linear range, simple operation, and high degree of automation, and has been widely used. However, there are few chemiluminescent detection kits for detecting free testosterone content at home and abroad. A free testosterone kit (CN109633180A) uses chemiluminescence, but the linear range of the kit is narrow, and the stability and accuracy of the kit are not measured. Therefore, it is necessary to develop a more sensitive, specific and safe free testosterone chemiluminescent detection kit. Summary of the invention

[0008] The present invention aims to develop a chemiluminescent detection kit for determining the free testosterone content in human body, which has the advantages of simple operation, wide linear range, fast detection speed, high sensitivity, no pollution, good specificity and low cost.

[0009] Detection principle: The free testosterone detection kit of the present invention is based on the principle of chemiluminescence competition method and uses magnetic beads as solid phase carriers. In the detection system, antibodies that specifically bind to testosterone and testosterone analogs labeled with chemiluminescent substances are included. When the sample is added to the reaction system, the free testosterone in the sample competes with the labeled testosterone analogs for the limited binding sites on the antibody. If the free testosterone content in the sample is high, more free testosterone will bind to the antibody, resulting in a reduced chance of the labeled testosterone analogs binding to the antibody; conversely, when the free testosterone content in the sample is low, the chance of the labeled testosterone analogs binding to the antibody increases. The labeled testosterone analogs bound to the antibody will generate a chemiluminescent signal. The intensity of the chemiluminescent signal is proportional to the amount of the labeled testosterone analogs bound, and the amount of the labeled testosterone analogs bound is inversely proportional to the content of free testosterone in the sample. By detecting the intensity of the chemiluminescent signal and comparing it with the intensity of the chemiluminescent signal generated by the calibrator of known concentration, the content of free testosterone in the sample can be quantitatively determined using a standard curve.

[0010] Based on the above purpose, the present invention provides a chemiluminescent detection kit for detecting the content of free testosterone in human body, comprising: Magnetic bead reagent: containing magnetic beads, the surface of which is coated with streptavidin; Calibrators; Label solution: a solution containing a testosterone analog labeled with an acridinium ester; Biotin-antibody solution: contains an antibody that specifically binds to testosterone, and the antibody is labeled with biotin; Substrate solution: used to excite the marker to produce chemiluminescent signal.

[0011] The chemiluminescent detection kit for detecting the free testosterone content in the human body as described above is characterized in that: it also includes a quality control product, a washing solution and a substrate solution. Preferably, the quality control product is prepared by diluting free testosterone with a human serum matrix to a concentration of 5 pg / mL and 150 pg / mL; preferably, the washing solution is composed of 0.05 M Tris-HCl buffer at pH 7.4 and 0.1% Tween-20 surfactant, which is used to wash the magnetic beads and remove unbound substances to reduce the impact of nonspecific binding on the detection results; further preferably, the substrate solution contains hydrogen peroxide and 0.25 mol / L sodium hydroxide at concentrations of 0.1 mol / L, respectively.

[0012] The chemiluminescent detection kit for detecting the free testosterone content in the human body as described above is characterized in that the average particle size of the magnetic beads is 2.8 μm, the concentration is 10 mg / mL, and the magnetic beads are suspended in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4) to maintain their stability and activity.

[0013] The chemiluminescent detection kit for detecting the free testosterone content in the human body as described above is characterized in that: the concentrations of the calibration substances are testosterone standard dilution solutions of 0 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, and 400 pg / mL, respectively.

[0014] The chemiluminescent detection kit for detecting the free testosterone content in the human body as described above is characterized in that the concentration of the testosterone analog in the marker solution is 1 μmol / L, and the labeling efficiency of the acridinium ester reaches more than 90%, ensuring that the marker has a high chemiluminescent activity.

[0015] The chemiluminescent detection kit for detecting the free testosterone content in the human body as described above is characterized in that: the antibody concentration in the biotin-antibody solution is 5 μg / mL, the molar ratio of biotin to antibody is 1:5, and the antibody is a free testosterone antibody, item number: FT3001, purchased from Zhenjiang Arainak Biotechnology Co., Ltd.

[0016] Another object of the present invention is to provide a method for preparing the chemiluminescent detection kit for detecting free testosterone content in human body as described above, characterized in that the steps are as follows: (1) Preparation of calibrators: Using human serum matrix, human serum was first treated to remove endogenous testosterone, and then accurate amounts of testosterone standards were added according to the predetermined concentrations to prepare calibrators with concentrations of 0 pg / mL, 5 pg / mL, 10 pg / mL, 20 npg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, and 400 pg / mL. (2) Preparation of magnetic bead reagent: Select magnetic beads with a particle size of about 2.8 μm, activate the surface of the magnetic beads with 0.1 M hydrochloric acid solution, then rinse with deionized water until neutral, mix the streptavidin solution with the magnetic beads, incubate at 37 °C for 2 h to coat the streptavidin on the surface of the magnetic beads, and finally suspend the magnetic beads in a solution containing 0.1% bovine serum albumin and 0.05 M pH 7.4 Tris-HCl buffer; Preparation of substrate solution: prepare hydrogen peroxide and sodium hydroxide stock solutions, and further prepare them into solutions with final concentrations of 0.1 mol / L and 0.25 mol / L, respectively; Preparation of marker solution: select a testosterone analog, label the testosterone analog with acridinium ester by chemical synthesis, and dilute the bound marker with diluent; Preparation of biotin-antibody solution: The antibody that specifically binds to testosterone is labeled with biotin, the molar ratio of biotin to antibody is 1:5, and the antibody concentration is adjusted to 5 μg / mL.

[0017] The preparation method of the chemiluminescent detection kit for detecting the free testosterone content in the human body as described above is characterized in that: the marker and biotin-antibody diluent are prepared as follows: 1 L of pure water is prepared, 12.05 g of Tris, 10 g of salicylic acid, and 2.0 g of NaN are weighed and stirred at room temperature for 1 h, 0.998 mL of 1 M MgCl2·6H2O, 1.027 mL of 0.1 M ZnCl2, and 1 g of BSA are added, the pH is adjusted to 7.0±0.05, filtered, and stored at 4°C.

[0018] Another object of the present invention is to provide the use of the chemiluminescent detection kit for detecting free testosterone content in human body as described above in detecting free testosterone content, characterized in that the detection of free testosterone uses plasma as a sample.

[0019] The present invention adopts the competitive method in the chemiluminescence method to develop a free testosterone detection kit, which is used in conjunction with a fully automatic chemiluminescence analyzer and basically does not require human intervention. Testosterone in the blood is divided into free testosterone and bound testosterone. Free testosterone refers to testosterone that exists in the blood in a free form. Free testosterone is in an active state. After the secreted testosterone enters the blood, only 2% exists in a free form. Compared with the existing free testosterone detection kit, the detection time is shortened, the detection efficiency is improved, the minimum detection limit is 0.021 ng / mL, and the linear R 2 =1.000, good anti-interference, stability for 12 months, and correlation with the test kits for free testosterone sold on the market is R 2 =0.9956, which meets the requirements of clinical testing. Compared with the free testosterone test kit (CN109633180A) of the same type on the market, our test range is expanded to 0-400pg / mL, and the accuracy and precision are controlled within 5%, which meets the market testing requirements. By screening testosterone monoclonal antibodies and specific analysis with antigens, highly specific antibodies were obtained as raw materials for the preparation of the kit, which greatly improved the sensitivity and specificity of the kit, providing a more accurate and more valuable commercial kit for clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the detection principle of the free testosterone detection kit of the present invention; Figure 2 Schematic diagram of the free testosterone detection kit of the present invention; Figure 3 A schematic diagram of the linear results of the free testosterone detection kit developed for the present invention; Figure 4 A schematic diagram of the results of correlation between the kit developed in the present invention and free testosterone kit samples sold on the market. DETAILED DESCRIPTION

[0021] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0022] The following are the main materials and equipment used in the implementation: Free testosterone antigen (Cat. No. FT2011), free testosterone antibody (Cat. No. FT3001), and testosterone analog (Cat. No. FT4001) were provided by Zhenjiang Arenac Biotechnology Co., Ltd. (Zhenjiang, China); magnetic bead reagents were purchased from Thermo Fisher Scientific (China), Cat. No. 88817.

[0023] Example 1: Kit preparation Preparation of calibrators: Human serum matrix was used. Human serum was first treated to remove endogenous testosterone. Then, accurate amounts of testosterone standards (purity of more than 99%) were added according to the predetermined concentrations to prepare calibrators with concentrations of 0 pg / mL, 5 pg / mL, 10 pg / mL, 20 npg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, and 400 pg / mL. During the preparation process, high-precision pipettes and balances were used to ensure the accuracy and repeatability of the calibrator concentrations.

[0024] Calibrator diluent: 1000 mL newborn calf serum, 32.0 g NaN, 43 mg tetracycline (tetracycline was dissolved in 0.1 M Tris, pH = 7.5 and then added (50 mg / 10 ml)), 0.1% ProClin 300. After stirring, the pH value was tested to be 5.5 ± 0.1, filtered with 0.22 um filter paper, and stored at 4 °C.

[0025] Preparation of magnetic bead reagent: First, select magnetic beads with a particle size of 2.8 μm, activate the surface of the magnetic beads with 0.1 M hydrochloric acid solution, and then rinse with deionized water until neutral. Next, mix the streptavidin solution (concentration of 10 μg / mL) with the magnetic beads and incubate at 37 °C for 2 h to coat the streptavidin on the surface of the magnetic beads. Finally, suspend the magnetic beads in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4).

[0026] Preparation of substrate solution: Prepare hydrogen peroxide and sodium hydroxide stock solutions, and further prepare them into solutions with final concentrations of 0.1 mol / L and 0.25 mol / L, respectively.

[0027] Selection of magnetic bead concentration: Based on the selected antibody concentration, the magnetic bead concentration was screened, and the magnetic bead concentrations were 15 mg / mL, 10 mg / mL, 7 mg / mL, and 5 mg / mL, respectively. According to the test results, when the magnetic bead concentration was 10 mg / mL, the luminescence effect was the best, so the magnetic bead concentration was determined to be 10 mg / mL. The test results are shown in Table 1.

[0028] Table 1: Comparison of luminescence values ​​of different magnetic bead concentrations in the free testosterone kit Free Testosterone Calibrator Calibrator concentration (pg / mL) Magnetic beads concentration 15 mg / mL Magnetic beads concentration 10 mg / mL Magnetic beads concentration 7 mg / mL Magnetic 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 Preparation of marker solution: select testosterone analogs, use chemical synthesis method to label acridinium ester to testosterone analogs, and dilute the bound marker with diluent. During the labeling process, optimize the reaction conditions such as temperature, time and reagent concentration to ensure that the labeling efficiency reaches more than 90%. The labeled testosterone analog solution is purified and quality tested to ensure that it meets the requirements of the kit.

[0029] Preparation of biotin-antibody solution: First, the antibody that specifically binds to testosterone is labeled with biotin. During the labeling process, the molar ratio of biotin to antibody is controlled to be 1:5. Then, the labeled antibody is dissolved in the diluent and the antibody concentration is adjusted to 5 μg / mL.

[0030] Preparation of labeling substance and biotin-antibody diluent: prepare 1 L of pure water, weigh 12.05 g of Tris, 10 g of salicylic acid, and 2.0 g of NaN3, and stir at room temperature for 1 h. Add 0.998 mL of 1 M MgCl2·6H2O, 1.027 mL of 0.1 M ZnCl2, and 1 g of BSA, adjust the pH to 7.0±0.05, filter, and store at 4°C.

[0031] Example 2: Detection steps Preparation of magnetic beads: Take out the magnetic bead reagent from the kit and gently invert to mix so that the magnetic beads are evenly suspended. Pipette 100 μL of magnetic bead reagent and add it to the reaction tube.

[0032] Sample addition: Add sample, marker solution and biotin-antibody solution to the reaction tube containing magnetic beads in sequence, with the added amount of 50 μL each.

[0033] Incubation: Place the reaction tube in an incubator and incubate at 37°C for 30 min. During the incubation, free testosterone in the sample competes with the testosterone analog in the marker solution for binding to the antibody bound to the magnetic bead surface through the biotin-streptavidin interaction.

[0034] Washing: After the incubation, use a magnetic separator to separate the magnetic beads from the reaction solution, and then wash the magnetic beads with washing solution. The amount of washing solution used is 300 μL per tube, and the number of washes is 3 times.

[0035] Add luminescent substrate: Add substrate solution to the washed magnetic bead reaction tube, the amount of substrate solution added is 100 μL. After adding the substrate, the reaction tube should be immediately placed in the chemiluminescence detector to start the chemiluminescence reaction.

[0036] Detection: In the chemiluminescence detector, the reaction tubes are detected according to the instrument's operating procedures. The instrument measures the chemiluminescence signal intensity of each reaction tube and transmits the data to the computer for analysis.

[0037] Result calculation: Draw a standard curve based on the concentration of the calibrator and the corresponding chemiluminescent signal intensity. Using the standard curve, the content of free testosterone in the sample is calculated by the chemiluminescent signal intensity of the sample, which is automatically completed by computer software.

[0038] Example 3: Performance Evaluation 1. Standard curve: The free testosterone complex described in Example 1 was diluted with a standard diluent to prepare calibration solutions S1-S8 of different concentrations, with concentrations of 0 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, and 400 pg / mL, respectively, and stored at -20 °C for use. Then, the calibrators were tested using the kit provided in Example 1 of the present invention, and the luminescence intensity values ​​corresponding to each calibrator were read respectively. The results are shown in Table 2 below. The standard curve was obtained by fitting with concentration as the horizontal axis and luminescence intensity as the vertical axis, as shown in Table 2 below. Figure 2 .

[0039] Table 2 Determination of the detection range of free testosterone test kit Calibrator concentration (pg / mL) Luminescence value (RLU) 0 7612894 5 1527381 10 763372 25 305552 50 152776 100 76842 200 38738 400 19369 2. Linear The high-concentration sample is the basic serum sample (concentration ≤ blank limit) or the calibration diluent (try to use the real high-value serum sample) with added antigen, and the low-concentration sample is the basic serum sample or the calibration diluent. The high-value sample close to the upper limit of the linear range is diluted to several concentrations according to a certain ratio (for example: linear sample preparation table), and the low-value concentration sample must be close to the lower limit of the linear range. The sample of each concentration is tested 2 to 3 times and the average value is calculated. The concentration value of the analyte (as the horizontal axis) and the average value of the test result (as the vertical axis) are fitted with a straight line using the least squares method. And calculate the correlation coefficient r within the linear range, and the acceptance standard is the correlation coefficient r ≥ 0.99.

[0040] Figure 3 This is the linear analysis of the free testosterone chemiluminescence assay kit of the present invention. The results show that the linear relationship between the theoretical concentration and the actual concentration of the sample is good, and the linear regression equation after fitting is Y=0.9990*X+0.6843, R 2 =1.000.

[0041] 3. Minimum detection limit Use the zero-concentration calibrator dilution as the sample for detection, repeat the measurement 20 times, and obtain the light signal value of the 20 measurement results. Calculate the mean (M) and standard deviation (SD) to obtain M-2SD. Perform two-point regression fitting based on the concentration and light signal value results between the zero-concentration enterprise linear reference and the adjacent concentration calibrator to obtain a linear equation. Substitute the light signal value of M-2SD into the equation to find the corresponding concentration value, which is the minimum detection limit.

[0042] The results are shown in Table 3. The concentrations of free testosterone were 0.013 pg / mL, 0.021 pg / mL, and 0.016 pg / mL, respectively, and the minimum detection limit of free testosterone was 0.021 pg / mL.

[0043] Table 3: Validation results of free testosterone detection limit Test 1 Test 2 Test 3 M 9898443.05 9230547.1 9779625.65 SD 54762.74323 65679.13733 161610.7278 M-2SD 9788917.564 9099188.825 9456404.194 LOD(pg / mL) 0.013 0.021 0.016 4. Accuracy Prepare samples with high and low concentration levels with free testosterone complex, repeat the measurement three times, calculate the relative deviation between the average value (denoted as M) and the labeled value, and calculate the relative deviation B of the measured concentration according to formula (1). Alternatively, use the reagent to be evaluated to test samples with high and low concentrations that have been determined by the reference method, repeat the measurement three times for each concentration sample, take the average value of the test results, and calculate its relative deviation from the labeled value.

[0044] B=(MT) / T×100%(1) Where: B—relative deviation; M—average value of measured concentration; T—indicative value; The above experimental results are shown in Table 4. The relative deviation of the free testosterone test kit of the present invention is within 5%, which has good accuracy and is superior to the same type of test kits in the domestic market.

[0045] Table 4: Free testosterone test kit accuracy test Concentration (pg / mL) Test 1 Test 2 Test 3 Average value (pg / mL) Relative deviation (%) 5.00 4.98 5.11 4.91 5.01 1% 200.00 202.10 199.1 201.6 200.35 -2% 5. Precision The test reagent (same batch or multiple batches) is used to test the same batch of samples multiple times to see if the results are consistent. This indicator is used as precision. Usually, the CV value within the batch is within 8%, and the CV value between batches is within 10%. According to the NCCLSEP5-A2 standard, this technology was used to measure two plasma samples of different concentrations. Free testosterone sample 1: a low-concentration sample with a concentration of 5.1 pg / mL, sample 2: a high-concentration sample with a concentration of 100 pg / mL.

[0046] (1) Intra-batch precision: The two samples were measured 20 times using three batches of reagents respectively, and the average M and standard deviation SD of the 20 measured concentrations were calculated, and the CV was calculated according to Formula 2.

[0047] CV=M / SD×100(2) Where CV is the coefficient of variation and M is the mean; SD is standard deviation.

[0048] (2) Inter-batch precision: Two samples were measured using three batches of reagents, 20 times each. The CV value was calculated according to Formula 2.

[0049] The precision of this kit is within 5%, which meets clinical requirements. See Table 5.

[0050] Table 5: Comparison of precision measurement results of free testosterone test kits 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 Fitted concentration SD 0.139379578 0.741308902 Fitting concentration CV 3% 1% 5. Thermal stability test The reagent must meet the quality standards of the product within the validity period, and the various performance indexes of the product must be stable. The stability test includes: thermal stability when stored at 2-8 ℃ for 15 months and 37 ℃ for 10 days, and the stability of each index is determined by a three-day simulation experiment. At the same time, the stability of the standard sample and the quality control material was evaluated, and the stability of the sample stored at 2-8 ℃ for 14-15 months was determined, the stability of the sample after 20 days of opening the bottle was determined, and the stability of the simulated transport sample for 3 days was evaluated. In addition, the stability of clinical samples placed under different conditions also needs to be considered. To verify the stability of the reagent, a standard experimental process needs to be carried out in a storage environment of 37 ℃, so that the experimental determination can be completed quickly. The detailed operation process requires three different batches of plasma reagents to be stored in an incubator, and the stability of the samples is determined at different time points. The sample can be tested on the 1st, 3rd, 6th and 10th days as 4 different measurement time points, and then the final test results are compared and analyzed with the reagents stored at 4 ℃, and each performance index is evaluated.

[0051] The calibrators of the configured free testosterone kit were placed at 4°C and 37°C for 1 day, 3 days, 6 days, 10 days, 20 days, 25 days, and 30 days, respectively, and the luminescence value was tested. The relative error (B%) was within ±20%. The accelerated stability of the free testosterone kit of the present invention was comprehensively analyzed to meet the test requirements. The relative error of the accelerated stability was within ±5%, which was better than similar free testosterone chemiluminescence detection kits on the market. The results are shown in Table 6 Table 6: Results of accelerated stability test of free testosterone test kit

[0052] The free testosterone test kit was taken out at the 3rd, 6th, 9th and 12th month, and the blank quality control and quality control products S2, S4 and S6 were tested respectively. The CV values ​​were within 10%, which met the specified standards. Therefore, the free testosterone test kit can be effectively stored at room temperature for 12 months. The results are shown in Table 7: Table 7: Real-time stability evaluation results of free testosterone test kit

[0053] 6. Kit specificity test The specific substances were configured to corresponding concentrations, and one batch of the free testosterone detection kit of the present invention was used to determine the specific substances and calculate the cross-reaction rate. Cross-reaction rate % = (free testosterone determination concentration / specific substance experimental concentration) × 100%. The specificity was <0.1%, which met the requirements, and the results are shown in Table 8 below.

[0054] Table 8: Free testosterone test kit specific test results Cross-reactants Experimental concentration (pg / mL) Measured concentration (pg / mL) Cross-reaction 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% Example 4: Clinical relevance assessment In order to better verify the detection effect of the kit developed in this study, a clinical performance evaluation is required. 150 samples of patient plasma (provided by the Affiliated Hospital of Jiangnan University) were collected and tested using a commercially available free testosterone detection kit, and the data were exported. The concentration in plasma was measured using a self-developed free testosterone chemiluminescence kit and compared with the measured values ​​of the commercially available kit. The values ​​measured by the homemade kit were used as the horizontal axis, and the values ​​measured by the commercially available kit were used as the vertical axis. This method was used to fit the corresponding method. The linear regression results are shown in Figure 4 The linear correlation coefficients of free testosterone were R 2 =0.9956, greater than 0.95. Its detection effect is well linearly correlated with the results of existing kits on the market, indicating that the developed kit can meet the needs of clinical testing.

[0055] In summary, after a large number of repeated experiments, the detection range of the free testosterone chemiluminescence detection kit of the present invention is 0-400 pg / mL, which is wider than the detection range of the existing free testosterone chemiluminescence detection kit (patent number: CN109633180A). The minimum detection limit is 0.021 pg / mL, and the linear test R 2=1.000, which is superior to the same type of kits on the market and the existing free testosterone chemiluminescence detection kit (patent number: CN109633180A) in terms of accuracy and stability. By comparing multiple markers, while ensuring the labeling efficiency, the lower-priced acridinium ester is selected to reduce the production cost, thereby reducing the economic pressure on patients when using the free testosterone chemiluminescence detection kit invented in this study.

Claims

1. A chemiluminescent detection kit for detecting free testosterone content in human body, characterized in that: The components include: Magnetic bead reagent: containing magnetic beads, the surface of which is coated with streptavidin; Calibrators; Label solution: a solution containing a testosterone analog labeled with an acridinium ester; Biotin-antibody solution: contains an antibody that specifically binds to testosterone, and the antibody is labeled with biotin; Substrate solution: used to excite the marker to produce chemiluminescent signal.

2. The chemiluminescent detection kit for detecting free testosterone content in human body as claimed in claim 1, characterized in that: It also includes a quality control product, a washing solution and a substrate solution. Preferably, the quality control product is prepared by diluting free testosterone with a human serum matrix to a concentration of 5 pg / mL and 150 pg / mL; preferably, the washing solution is composed of 0.05 M Tris-HCl buffer at pH 7.4 and 0.1% Tween-20 surfactant, which is used to wash the magnetic beads and remove unbound substances to reduce the impact of nonspecific binding on the test results; further preferably, the substrate solution contains hydrogen peroxide and 0.25 mol / L sodium hydroxide at concentrations of 0.1 mol / L, respectively.

3. The chemiluminescent detection kit for detecting free testosterone content in human body as claimed in claim 1 or 2, characterized in that: The magnetic beads have an average particle size of 2.8 μm and a concentration of 10 mg / mL. The magnetic beads are suspended in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer at pH 7.4 to maintain their stability and activity.

4. A chemiluminescent detection kit for detecting free testosterone content in human body as described in any one of claims 1-3, characterized in that: The calibration sample concentrations are 0 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, and 400 pg / mL of testosterone standard dilution solutions.

5. The chemiluminescent detection kit for detecting free testosterone content in human body as described in any one of claims 1-3, characterized in that: The concentration of the testosterone analog in the marker solution is 1 μmol / L, and the labeling efficiency of the acridinium ester reaches more than 90%, ensuring that the marker has a high chemiluminescent activity.

6. The chemiluminescent detection kit for detecting free testosterone content in human body as described in any one of claims 1-3, characterized in that: The antibody concentration in the biotin-antibody solution is 5 μg / mL, the molar ratio of biotin to antibody is 1:5, and the antibody is a free testosterone antibody, with a catalog number of FT3001.

7. The method for preparing a chemiluminescent detection kit for detecting free testosterone content in human body according to any one of claims 1 to 6, characterized in that: Here are the steps: (1) Preparation of calibrators: Using human serum matrix, human serum was first treated to remove endogenous testosterone, and then accurate amounts of testosterone standards were added according to the predetermined concentrations to prepare calibrators with concentrations of 0 pg / mL, 5 pg / mL, 10 pg / mL, 20 npg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, and 400 pg / mL. (2) Preparation of magnetic bead reagent: Select magnetic beads with a particle size of about 2.8 μm, activate the surface of the magnetic beads with 0.1 M hydrochloric acid solution, then rinse with deionized water until neutral, mix the streptavidin solution with the magnetic beads, incubate at 37 °C for 2 h to coat the surface of the magnetic beads with streptavidin, and finally suspend the magnetic beads in a solution containing 0.1% bovine serum albumin and 0.05 M pH 7.4 Tris-HCl buffer; (3) Preparation of substrate solution: prepare hydrogen peroxide and sodium hydroxide stock solutions, and further prepare them into solutions with final concentrations of 0.1 mol / L and 0.25 mol / L, respectively; (4) Preparation of marker solution: Select a testosterone analog, label the testosterone analog with acridinium ester by chemical synthesis, and dilute the bound marker with diluent; (5) Preparation of biotin-antibody solution: The antibody that specifically binds to testosterone is labeled with biotin, the molar ratio of biotin to antibody is 1:5, and the antibody concentration is adjusted to 5 μg / mL.

8. The preparation method of the chemiluminescent detection kit for detecting free testosterone content in human body as claimed in claim 7, characterized in that: Preparation of labeling substance and biotin-antibody diluent: prepare 1 L of pure water, weigh 12.05 g of Tris, 10 g of salicylic acid, and 2.0 g of NaN3, stir at room temperature for 1 h, add 0.998 mL of 1 M MgCl2·6H2O, 1.027 mL of 0.1 M ZnCl2, and 1 g of BSA, adjust the pH to 7.0±0.05, filter, and store at 4 ℃.

9. The use of a chemiluminescent detection kit for detecting free testosterone content in human body as claimed in any one of claims 1 to 6 in detecting free testosterone content, characterized in that: The free testosterone is detected using plasma as a sample.

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