Chemiluminiscence detection kit for urine and bladder cancer antigen and preparation method of chemiluminiscence detection kit

Abstract

The invention provides a UBC-CK8 / 18 detection kit based on chemiluminescence, and the kit is characterized in that an antibody is immobilized through a streptavidin-biotin system to form a magnetic bead-biotinylated antibody-antigen-acridinium ester labeled antibody compound; after washing, the substrate solution excites the acridinium ester to generate a photon signal, and a relative luminescence unit (RLUs) is captured by a full-automatic chemiluminescence immunoassay instrument, so that rapid and low-cost clinical detection of bladder cancer is realized, and the detection performance is good.

Description

Technical Field

[0001] The present application relates to a chemiluminescent immunoassay kit, and specifically to a chemiluminescent detection kit for urinary bladder cancer antigen cytokeratins 8 and 18 (UBC-CK8 / 18) and its preparation. Background Art

[0002] 1. Clinical needs for cancer diagnosis: - Epidemiological burden: The incidence of bladder cancer in China is 5.8 / 100,000 (NCCR 2023), of which 45% have progressed to the muscle invasive stage (MIBC) at the time of diagnosis. The 5-year survival rate of patients in advanced stages is less than 30%.

[0003] - Imbalance in diagnostic and treatment resources: Primary hospitals rely on low-sensitivity ultrasound (sensitivity 60%), while the gold standard cystoscopy has a penetration rate of less than 20% at the primary level and is an invasive procedure.

[0004] 2. Limitations of Existing Biomarker Detection Technologies Existing technology Core flaws Clinical impact Urine cytology Low sensitivity (35-45%), dependent on pathologist experience High missed diagnosis rate, especially for low-grade tumors (stage Ta) NMP22 ELISA Insufficient detection limit (5 ng / mL) and susceptible to hemolysis / infection interference (false positive rate > 25%) Misdiagnosis leading to unnecessary cystoscopy FISH testing The operation is complicated (fluorescence microscope is required), takes more than 3 days, and costs more than 800 yuan per time Limited to tertiary hospitals, difficult to popularize Traditional UBC-CK8 / 18 ELISA Low sensitivity, detection limit of only 0.73 ng / mL, narrow linear range, and low degree of automation Unable to meet the needs of early detection of minimal lesions The urinary bladder cancer antigen (UBC) test detects bladder cancer by measuring fragments of cytokeratins 8 and 18 (UBC-CK8 / 18) in urine. Existing research suggests that high levels of cytokeratins 8 and 18 in urine can rapidly detect urothelial bladder cancer and are important markers for the clinical screening and detection of bladder cancer (Research Progress of Bladder Cancer Biomarkers in Urine, Dong Taotao et al., 2021). CN101368961B discloses a UBC detection kit using monoclonal antibodies. However, this kit can only detect UBC-CK8 / 18 levels in serum, not urine. Furthermore, it has low sensitivity and a narrow detection range, failing to meet clinical testing requirements. Therefore, there is an urgent need for a UBC-CK8 / 18 detection kit with high sensitivity, wide detection range, simple and accurate operation, which can realize rapid, convenient and low-cost detection of clinical urine samples and can be applied to primary medical care (initial screening of hematuria in community hospitals / clinics) and specialist monitoring (postoperative follow-up in urology outpatient clinics, which can replace 20% of cystoscopy examinations). Summary of the Invention

[0005] In order to address the deficiencies in the above-mentioned prior art, the present application provides a UBC-CK8 / 18 detection kit based on chemiluminescence, which immobilizes antibodies through a streptavidin-biotin system to form a "magnetic bead-biotinylated antibody-antigen-acridinium ester-labeled antibody" complex; after washing, the substrate solution excites the acridinium ester to produce a photon signal, and the relative luminescence units (RLUs) are captured by a fully automatic chemiluminescence immunoassay, thereby realizing rapid and low-cost clinical detection of bladder cancer with good detection performance.

[0006] This application provides a UBC-CK8 / 18 chemiluminescent detection kit, comprising the following components: Magnetic bead reagent: contains magnetic beads, the surface of which is coated with streptavidin; UBC-CK8 / 18 calibrator; Labeling solution: Contains UBC-CK8 / 18 detection antibody labeled with acridinium ester; Biotin-antibody solution: contains capture antibodies that specifically bind to UBC-CK8 / 18 and are labeled with biotin; Substrate solution: used to excite the marker to produce chemiluminescent signal.

[0007] In some embodiments, the UBC-CK8 / 18 chemiluminescence detection kit further includes quality control products and washing solution.

[0008] In some embodiments, the UBC-CK8 / 18 chemiluminescent detection kit, wherein 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 MTris-HCl buffer to maintain their stability and activity.

[0009] In some embodiments, the UBC-CK8 / 18 chemiluminescent detection kit, wherein the concentrations of the calibrators are 0 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 250 ng / mL, respectively.

[0010] In some embodiments, in the UBC-CK8 / 18 chemiluminescent detection kit, the concentration of the UBC-CK8 / 18 detection antibody in the marker solution is 0.5 μg / mL.

[0011] In some embodiments, in the UBC-CK8 / 18 chemiluminescent detection kit, the concentration of the UBC-CK8 / 18 capture antibody in the label solution in the biotin-antibody solution is 3 μg / mL.

[0012] In some embodiments, the UBC-CK8 / 18 chemiluminescent detection kit is used to detect bladder cancer.

[0013] The present application also provides a method for preparing the UBC-CK8 / 18 chemiluminescent detection kit as described above, comprising the following steps: (1) Preparation of calibrators: Using phosphate buffer as the calibrator matrix, the UBC-CK8 / 18 complex was prepared into calibrators with concentrations of ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 250 ng / mL; (2) Preparation of magnetic bead reagent: 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, then rinse with deionized water until neutral, mix the streptavidin solution with the magnetic beads, incubate at 37 °C for 2 h, and suspend the coated magnetic beads in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer; (3) Preparation of substrate solution: The substrate solution consists of hydrogen peroxide and sodium hydroxide; (4) Preparation of marker solution: Conjugate the detection antibody with acridinium ester, dissolve the conjugated product in diluent, and adjust the final antibody concentration; (5) Preparation of biotin-antibody solution: Label the capture antibody that specifically binds to UBC-CK8 / 18 with biotin and adjust the final antibody concentration.

[0014] In some embodiments, the final concentration of the magnetic beads in step (2) is adjusted to 10 mg / mL.

[0015] In some implementations, the antibody concentration in the label solution is 0.5 μg / mL, and the antibody concentration in the biotin-antibody solution is 3 μg / mL. Beneficial effects

[0016] This invention overcomes the technical bottlenecks of UBC-CK8 / 18 detection in terms of sensitivity, anti-interference and accessibility through chemiluminescent signal amplification and fully automated platform integration, providing a solution for early diagnosis of bladder cancer that meets the needs of primary healthcare in China. Its advantages are: (1) Improved detection sensitivity: The detection limit of the traditional ELISA method (0.73 ng / mL) was broken, and the detection limit reached 0.07 ng / mL, with a linear range of 0-250 ng / mL (R=0.9999).

[0017] (2) Enhance anti-interference ability: solve the false positive problem caused by hematuria and inflammation samples, and improve specificity.

[0018] (3) Lowering the operational threshold: Developing a fully automatic, rapid (less than 45 minutes), room-temperature stable detection system to achieve integrated “sample in, result out” detection, suitable for scenarios with limited resources and grassroots scenarios.

[0019] (4) Compared with traditional testing, which requires diluting urine 10 times, urine does not need to be diluted and can be directly tested on the machine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the detection principle of the urine bladder cancer antigen (UBC-CK8 / 18) detection kit of the present invention; Figure 2 Schematic diagram of the urine bladder cancer antigen (UBC-CK8 / 18) detection kit of the present invention; Figure 3 Schematic diagram of the linear results of the urine bladder cancer antigen (UBC-CK8 / 18) detection kit developed for the present invention; Figure 4 Schematic diagram of the correlation between the test results of the kit developed for the present invention and the urine bladder cancer antigen (UBC-CK8 / 18) kit available 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 raw materials used in the present study: urinary bladder cancer antigen (catalog number: MA2341), urinary bladder cancer antigen monoclonal antibody (catalog number: capture antibody (6E10-F7), detection antibody (2B3-D11), provided by Wuxi Mosinus Technology Co., Ltd. (Wuxi, China); magnetic bead reagent was purchased from Thermo Fisher Scientific (China), catalog number 88817.

[0023] Example 1: Preparation method of the kit 1. Preparation of magnetic bead reagent Magnetic beads with an average particle size of 2.8 μm were activated with 0.1 M hydrochloric acid solution and rinsed with deionized water until neutral. The activated magnetic beads were mixed with 10 μg / mL streptavidin solution and incubated at 37°C for 2 hours to complete coating. The coated magnetic beads were suspended in 0.05 M Tris-HCl buffer (pH 7.4) containing 0.1% bovine serum albumin and the concentration was adjusted to 10 mg / mL.

[0024] 2. Construction of the Calibrator System Calibrators: Using phosphate buffer as the matrix, add urinary bladder cancer antigen (UBC-CK8 / 18) complex standard to prepare standard solutions with a concentration gradient of 0 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 250 ng / mL; Preparation method of calibrator dilution: Add 12.11 g Tris, 17.53 g NaCl, 2 g NaN3, 0.02 g tetracycline, and 0.02 g neomycin sulfate in sequence, adjust the pH to 7.5±0.05, add 4 mL Tween-20, 100 g BSA, and 10 g casein, stir until completely dissolved, add purified water to make up to 1000 mL, sterilize through a 0.22 μm filter membrane, and store at 4°C.

[0025] 3. Preparation of marker solution The detection antibody was conjugated to an acridinium ester, and the labeling efficiency was ≥90% by optimizing the temperature, time, and concentration parameters. The conjugated product was dissolved in diluent and the final antibody concentration was adjusted to 0.5 μg / mL.

[0026] 4. Preparation of Biotin-Antibody Solution Take 250 μL of capture antibody (1 mg / mL) and add 4 μL of Sulfo-NHS-LC-Biotin solution (1 mg / mL). After incubation with vortexing at 37°C for 1 hour, transfer the solution to a 14,000 Da dialysis bag and dialyze against 500 mL of PBS buffer for 4 hours and overnight (16-18 hours). Determine the concentration by UV spectrophotometry (OD280nm / 1.4). Add 0.1% BSA and 0.1% Proclin 300 to adjust the final concentration to 3 μg / mL.

[0027] 5. Preparation of other solutions Dilution: Add 12.05 g Tris, 5.82 g NaCl, and 2.0 g NaN3 to 1 L of pure water. Supplement with 0.998 mL of 1 M MgCl2·6H2O, 1.027 mL of 0.1 M ZnCl2, and 2 mL of goat serum; Adjust the pH to 8.0 ± 0.05, filter sterilize and store at 4°C.

[0028] Substrate solution: composed of 0.1 mol / L hydrogen peroxide and 0.25 mol / L sodium hydroxide, used to stimulate chemiluminescent signals.

[0029] Washing buffer: Contains 0.05 M Tris-HCl buffer (pH 7.4) and 0.1% Tween-20 surfactant to eliminate nonspecific binding.

[0030] Example 2: Urinary bladder cancer antigen (UBC-CK8 / 18) detection method Step 1: Magnetic bead suspension treatment Take the magnetic bead reagent in the kit and vortex mix to evenly disperse the magnetic particles in the suspension.

[0031] Step 2: Construction of immune response system Add the following into the reaction tube in order: 30 μL of sample to be tested 30 μL marker solution Biotin-antibody solution 30 μL Step 3: Antigen-antibody complex formation The reaction tube was placed in an incubator at 37°C for 20 minutes to allow the UBC-CK8 / 18 antigen to specifically bind to the biotinylated antibody and the acridinium ester-labeled antibody to form a "double antibody sandwich" complex.

[0032] Step 4: Solid phase separation and washing Add 100 μL of magnetic bead reagent to the reaction system and incubate for 3 minutes to complete the immobilization of the complex; use a magnetic separator to adsorb the magnetic beads and discard the liquid phase; wash the magnetic beads with 300 μL of washing solution each time, repeat 3 times to eliminate nonspecific binding.

[0033] Step 5: Chemiluminescent signal excitation Add 100 μL of substrate solution to the washed magnetic beads and immediately transfer to the reaction chamber of the chemiluminescence detector.

[0034] Step 6: Signal acquisition and quantitative analysis The detector captures the chemiluminescence intensity in real time and converts it into relative luminescence units (RLUs). Through the built-in analysis system, a standard curve is established with the calibrator concentration (0-250 ng / mL) as the horizontal axis and the corresponding RLUs as the vertical axis to automatically calculate the UBC-CK8 / 18 content of the sample to be tested.

[0035] Example 3: Performance Evaluation 1. Determination of Antibody Working Concentration and Standard Curve To determine the optimal detection conditions, a titering method was used. Specifically, the biotin-labeled antibody solution was diluted to a concentration gradient of 1 μg / mL, 3 μg / mL, and 5 μg / mL. Simultaneously, the acridinium ester-labeled antibody solution was diluted to a concentration gradient of 0.3 μg / mL, 0.5 μg / mL, and 0.7 μg / mL. Calibrator solutions were prepared at three concentration levels: 0 ng / mL (blank control, point A), 25 ng / mL (point C), and 100 ng / mL (point E).

[0036] Through systematic experimental testing and data analysis, we ultimately determined the optimal antibody working concentration combination: 3 μg / mL for biotinylated antibody and 0.5 μg / mL for acridinium ester-labeled antibody. This concentration combination achieved optimal assay performance, as shown in the table below (represented by 1).

[0037] Table 1 Urinary bladder cancer antigen (UBC-CK8 / 18) checkerboard assay data

[0038] Preparation of calibrators and establishment of standard curves The urinary bladder cancer antigen (UBC-CK8 / 18) complex prepared in Example 1 was serially diluted using standard diluent to prepare a series of calibrator solutions (S1-S6) with concentrations of 0 ng / mL (S1), 5 ng / mL (S2), 20 ng / mL (S3), 50 ng / mL (S4), 100 ng / mL (S5), and 250 ng / mL (S6). The prepared calibrator solutions were stored at 4°C until use.

[0039] Subsequently, the series of calibrator solutions (S1-S6) were tested using the kit provided in Example 1 of the present invention. The chemiluminescence intensity values ​​corresponding to each concentration of the calibrator were recorded (see Table 2 for detailed test results).

[0040] Based on the test data in Table 2, the concentration of urinary bladder cancer antigen (UBC-CK8 / 18) complex was used as the horizontal axis (X axis) and the measured chemiluminescence intensity value was used as the vertical axis (Y axis). Four-parameter fitting was performed to establish a standard curve. The fitting results are shown in Figure 2. Figure 2 shown.

[0041] Table 2 Detection range of urine bladder cancer antigen (UBC-CK8 / 18) test kit Calibrator concentration (ng / mL) Luminescence value (RLU) 0 2143 5 10021 20 41329 50 102131 100 210329 250 540211 2. Linear To verify the linear range of the test kit of the present invention, the following experimental method was used: Sample preparation: Use calibrators spiked with target antigen as high-concentration samples (near the upper limit of the expected linear range) and standard dilutions as low-concentration samples (near the lower limit of the expected linear range).

[0042] Gradient dilution: The high-concentration sample is diluted in a gradient manner according to a preset ratio to obtain a series of concentration gradient samples covering the expected linear range (from near the lower limit to near the upper limit).

[0043] Testing and data collection: Each concentration gradient sample prepared above was tested three times. The results of each test were recorded, and the arithmetic mean of the test results of each concentration sample was calculated.

[0044] Data analysis and fitting: The theoretical concentration value of the target analyte in the sample is used as the independent variable (abscissa, X-axis), and the average value of the test results of the corresponding sample is used as the dependent variable (ordinate, Y-axis). The least squares method is used for linear regression fitting.

[0045] Judgment criteria: Calculate the correlation coefficient (r) within the linear range. Set the acceptance criterion for linear relationship as: correlation coefficient r ≥ 0.99.

[0046] Verification results: The linear analysis was performed using the chemiluminescence assay kit for urine bladder cancer antigen (UBC-CK8 / 18) provided in Example 1 of the present invention. The results were as follows: Figure 3 The data demonstrates a good linear relationship between the theoretical and measured sample concentrations, with a calculated correlation coefficient r > 0.99, meeting the pre-defined acceptance criteria. The linear regression equation obtained using the least squares method is: Y = 0.9860 * X - 0.2106.

[0047] 3. Limit of Blank (LOB) Determination The limit of detection of blank (LOB) was determined according to the following steps: Blank sample test: Use zero concentration calibrator solution as the blank sample.

[0048] Repeated measurement: Repeat the measurement of the blank sample 20 times, and record the chemiluminescence signal value obtained in each measurement.

[0049] Data calculation: Calculate the arithmetic mean (M) and standard deviation (SD) of the chemiluminescence signal values ​​obtained from 20 blank sample measurements.

[0050] Critical value determination: Based on the calculated M and SD, determine the critical value M + 2SD of the blank sample signal distribution.

[0051] Standard curve establishment (two-point method): Using the zero-concentration calibrator (company linear reference) and the known concentration values ​​of its adjacent low-concentration calibrator and its corresponding average chemiluminescence signal values, a linear equation (Y = aX + b) between concentration (X) and chemiluminescence signal value (Y) is established through two-point regression fitting.

[0052] LOB Calculation: Substitute the critical signal value (M + 2SD) determined in Step 4 into the linear equation (Y = aX + b) established in Step 5 and solve for the corresponding concentration (X). The concentration value obtained from this calculation is the blank limit of detection (LOB) for the analyte.

[0053] Measurement results: As shown in Table 3, three independent LOB assays for urinary bladder cancer antigen (UBC-CK8 / 18) performed using the above method yielded concentrations of 0.07 ng / mL, 0.06 ng / mL, and 0.04 ng / mL, respectively. Based on these three experimental results, the limit of blank (LOB) for the present kit for detecting urinary bladder cancer antigen (UBC-CK8 / 18) was determined to be 0.07 ng / mL.

[0054] Table 3 Verification results of blank detection limit of urine bladder cancer antigen (UBC-CK8 / 18) Test 1 Test 2 Test 3 M 2106.75 2105.45 2089.45 SD 65.57 61.88 53.38 M+2SD 2237.89 2229.22 2196.20 LOB (ng / mL) 0.07 0.06 0.04 4. Accuracy Sample preparation: Use the urinary bladder cancer antigen (UBC-CK8 / 18) complex to prepare two different concentrations of test samples: high concentration level and low concentration level.

[0055] Repeated testing and calculation: Repeat the test for each concentration level for 3 times. Calculate the arithmetic mean of the concentrations of the 3 test results (denoted as M).

[0056] Deviation calculation: Calculate the relative deviation (B) of the average measured concentration (M) relative to the labeled concentration (T) of the sample according to formula (1): B = (M - T) / T × 100% (1) Where: B represents relative deviation (%); M represents the mean value of the measured concentrations; T represents the labeled concentration value of the sample.

[0057] Table 4 Accuracy test of urine bladder cancer antigen (UBC-CK8 / 18) kit Concentration (ng / mL) Test 1 Test 2 Test 3 Average value (ng / mL) Relative deviation (%) 20 21.10 20.32 19.23 20.22 1.08% 100 97.22 98.32 98.99 98.18 -1.82% 5. Precision The reagent is tested multiple times on the same batch of samples to determine if the results are consistent, which is considered imprecision. Typically, the intra-batch CV value is within 8%, and the inter-batch CV value is within 10%. According to the NCCLS EP5-A2 standard, two UBC-CK8 / 18 samples (quality control products) with different concentrations were measured using this technique. Urinary bladder cancer antigen (UBC-CK8 / 18) sample 1 (quality control product 1) was a low-concentration sample with a concentration of 20 ng / mL; sample 2 (quality control product 2) was a high-concentration sample with a concentration of 100 ng / mL.

[0058] (1) Intra-batch precision: The two samples were measured 20 times using three batches of reagents, and the mean M and standard deviation SD of the 20 measured concentrations were calculated, and the CV was calculated according to Formula 2.

[0059] CV=M / SD×100%---------(2) Where CV is the coefficient of variation and M is the mean; SD is standard deviation.

[0060] (2) Inter-batch precision: Two samples were measured using three batches of reagents, 20 times each batch, and the CV value was calculated according to Formula 2.

[0061] The precision of this kit is within 5%, which meets clinical requirements. See Table 5.

[0062] Table 5 Imprecision measurement results of the urine bladder cancer antigen (UBC-CK8 / 18) detection kit Low value quality control High-value quality control Intra-batch CV 2.68% 2.59% Inter-batch CV 3.12% 3.06% 6. Thermal stability test Reagents must meet product quality standards and maintain stable performance throughout their shelf life. Stability testing includes storage at 2-8°C for 12 months and storage at 37°C for 10 days to assess stability. Validation requires a standard 37°C protocol to ensure rapid testing. Stability is assessed at four time points: Days 1, 3, 6, and 10. Final results are compared to reagents stored at 4°C, and all performance indicators are evaluated.

[0063] A comprehensive analysis of the accelerated stability of the present UBC-CK8 / 18 kit confirmed that the assay met the test requirements and outperformed similar commercially available chemiluminescent detection kits for UBC-CK8 / 18. The results are shown in Table 6.

[0064] Table 6 Accelerated stability test results of the urine bladder cancer antigen (UBC-CK8 / 18) kit

[0065] The Urine Bladder Cancer Antigen (UBC-CK8 / 18) test kit was taken out at 3, 6, 9, and 12 months, and the quality control samples were tested. The CV values ​​were within 10%, meeting the specified standards. Therefore, the Urine Bladder Cancer Antigen (UBC-CK8 / 18) 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 the urine bladder cancer antigen (UBC-CK8 / 18) kit

[0066] 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. Urine samples from 164 patients (provided by the Affiliated Hospital of Jiangnan University) were collected and tested using a commercially available urine bladder cancer antigen (UBC-CK8 / 18) detection kit, and the data were exported. The concentration in urine was determined using a self-developed urine bladder cancer antigen (UBC-CK8 / 18) chemiluminescence kit, and compared with the measured values ​​of the commercially available Elisa kit. The values ​​measured by the self-made kit were used as the horizontal axis, and the values ​​measured by the commercially available kit were used as the vertical axis. The corresponding method was fitted using this method. The obtained linear regression results are shown in Figure 4 The linear correlation coefficient for urinary bladder cancer antigen (UBC-CK8 / 18) was r = 0.9859. This test showed a good linear correlation with the results of existing kits on the market, indicating that the developed kit can meet clinical testing needs.

[0067] Table 8 Technical advantages and expected results (compared with existing UBC-CK8 / 18 detection kits) parameter CN101368961B (ELISA) This invention (CLIA) Detection sensitivity 0.73 ng / mL 0.07 ng / mL Linear range 0.73—17.42 µg / L 0—250 µg / L Detection time 4 hours 30 minutes Cost per test 120 yuan 3 yuan

Claims

1. A UBC-CK8 / 18 chemiluminescence detection kit, characterized in that: Contains the following components: Magnetic bead reagent: contains magnetic beads, the surface of which is coated with streptavidin; UBC-CK8 / 18 calibrator; Labeling solution: Contains UBC-CK8 / 18 detection antibody labeled with acridinium ester; Biotin-antibody solution: contains capture antibodies that specifically bind to UBC-CK8 / 18 and are labeled with biotin; Substrate solution: used to excite the marker to produce chemiluminescent signal.

2. The UBC-CK8 / 18 chemiluminescent detection kit according to claim 1, wherein: Also includes quality control products and washing fluids.

3. The UBC-CK8 / 18 chemiluminescent detection kit according to claim 1, wherein: The average particle size of the magnetic beads is 2.8 μm, and the concentration is 10 mg / mL. The magnetic beads are suspended in a solution containing 0.1% bovine serum albumin and 0.05 MTris-HCl buffer to maintain their stability and activity.

4. The UBC-CK8 / 18 chemiluminescent detection kit according to any one of claims 1 to 3, wherein: The concentrations of the calibrators were 0 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 250 ng / mL, respectively.

5. The UBC-CK8 / 18 chemiluminescent detection kit according to any one of claims 1 to 3, wherein: The concentration of the UBC-CK8 / 18 detection antibody in the marker solution was 0.5 μg / mL, and the detection antibody was 2B3-D11.

6. The UBC-CK8 / 18 chemiluminescent detection kit according to any one of claims 1 to 3, wherein: The concentration of the UBC-CK8 / 18 capture antibody in the label solution in the biotin-antibody solution was 3 μg / mL, and the capture antibody was 6E10-F7.

7. The UBC-CK8 / 18 chemiluminescent detection kit according to any one of claims 1 to 3, wherein: The kit is used for detecting bladder cancer.

8. The method for preparing the UBC-CK8 / 18 chemiluminescent detection kit according to any one of claims 1 to 7, wherein: Here are the steps: (1) Preparation of calibrators: Using phosphate buffer as the calibrator matrix, the UBC-CK8 / 18 complex was prepared into calibrators with concentrations of ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 250 ng / mL; (2) Preparation of magnetic bead reagent: 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, then rinse with deionized water until neutral, mix the streptavidin solution with the magnetic beads, incubate at 37 °C for 2 h, and suspend the coated magnetic beads in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer; (3) Preparation of substrate solution: The substrate solution consists of hydrogen peroxide and sodium hydroxide; (4) Preparation of marker solution: Conjugate the detection antibody with acridinium ester, dissolve the conjugated product in diluent, and adjust the final antibody concentration; (5) Preparation of biotin-antibody solution: Label the capture antibody that specifically binds to UBC-CK8 / 18 with biotin and adjust the final antibody concentration.

9. The method for preparing the UBC-CK8 / 18 chemiluminescent detection kit according to claim 8, wherein: The final concentration of the magnetic beads in step (2) was adjusted to 10 mg / mL.

10. The method for preparing the UBC-CK8 / 18 chemiluminescent detection kit according to claim 8, wherein: The antibody concentration in the label solution was 0.5 μg / mL, and the antibody concentration in the biotin-antibody solution was 3 μg / mL.

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