CN103033625A - Human bladder cancer cellular chemiluminescent detection kit and preparation method thereof - Google Patents

Abstract

The invention relates to the field of immunoanalysis medicine, and specifically provides a kit for detecting human bladder cancer cells by a chemiluminescence analysis method based on immunomagnetic particles and a preparation method thereof. According to the present invention, the kit of the present invention mainly includes: 1) Bladder cancer EJ cell standard; 2) Magnetic particles coated with monoclonal antibodies that specifically recognize bladder cancer cell surface antigens; 3) Enzyme-labeled cells that specifically recognize bladder cancer Monoclonal antibody to cell surface antigen; 4) Chemiluminescence substrate acted on by the enzyme in 3); 5) Reaction tube or microwell plate; 6) Magnetic separator matched with the reaction tube or microwell plate in 5) .

Description

A kind of human bladder cancer cell chemiluminescent detection kit and its preparation method

technical field

The present invention relates to the fields of immune analysis and clinical diagnostic medicine, specifically, the present invention provides a chemiluminescent detection kit for human bladder cancer cells and a preparation method thereof.

Background technique

Bladder cancer is the most common tumor in the urinary system. Hematuria is one of the early clinical symptoms of bladder cancer patients, including gross hematuria and microscopic hematuria. However, hematuria also occurs in urinary tract inflammation and stone symptoms, and is not a specific symptom of bladder cancer. It is precisely because the early symptoms of bladder cancer are not obvious that hematuria is found, and bladder tumors in patients generally develop into larger tumor nodules. Finding a specific diagnostic method for bladder cancer has important clinical application value for the diagnosis of early bladder cancer.

There are several methods for early diagnosis of bladder cancer:

1) Urinalysis: Urinalysis is the first step after hematuria occurs, in order to rule out whether it is caused by inflammation of the urinary tract, and it is not possible to determine whether the patient has bladder cancer;

2) Analysis of urine exfoliated cells: observation of urine exfoliated cells in urine sediment under a microscope can identify and distinguish deteriorated tumor cells from normal cells. However, the sensitivity is not high, and early bladder cancer is easily missed;

3) Ultrasound, CT or MRI imaging observation: the size and degree of deterioration of the tumor can be directly observed by image, and the accuracy is greatly improved compared with the previous method. Despite the continuous improvement of imaging technology in recent years, the sensitivity has been continuously improved, but the observation of small tumors is still not enough, and it is easy to miss detection;

4) Cystoscope observation: Cystoscope enters the bladder through the urinary tract for visual observation in the bladder to detect early tumors. It can detect most of the patients missed in 2) and 3).

At present, the most specific method for the early diagnosis of bladder cancer cells is observation through cystoscopy, and the application of fluorescent cystoscopy improves the detection sensitivity. However, patients generally need clinical follow-up observation, so that tumor occurrence or recurrence can be detected early. However, cystoscopy can cause physical discomfort to the patient. In addition, the specificity of fluorescent dyes required for fluorescent cystoscopy detection is not high at present, and has certain negative effects on the human body. Therefore, in the early diagnosis of bladder cancer, people have been exploring more specific and sensitive detection methods, using specific tumor markers as much as possible to achieve convenient and rapid detection.

Realizing the detection of bladder cancer markers or cells in urine samples, real-time detection has important application value in clinical diagnosis. Currently commercially available bladder cancer markers include: human complement factor H-related protein (bladder tumor antigen, BTA kit), high molecular weight carcinoembryonic antigen, and two bladder cancer cell-related adhesion molecules (Scimedx Corp, USA). company's Immunocyt kit), nuclear matrix protein 22 (NMP22), aneuploidy of chromosomes 3, 7, and 17, and deletion of the P16 tumor suppressor 9p21 locus (UroVysion kit). BTA refers to human supplement factor H-related protein. BTA detection requires professional operators in a standard laboratory, with a sensitivity of 57-83% and a specificity of 60-92%. The specificity of patients with hematuria can only reach 46%. In addition, benign prostatic hyperplasia, kidney stones, and urinary tract infections all affect the specificity of BTA detection; the Immunocyt kit uses immunofluorescence to detect urine exfoliated cells. Although the sensitivity and specificity are high, in order to achieve higher accuracy, it needs to be tested A large number of urine exfoliated cells are detected, so it has high application value in the treatment and prognosis of bladder cancer patients, but the application is not satisfactory in early diagnosis; among them, NMP22 is currently the most widely used bladder cancer marker, and the ELISA kit Marketization has realized the convenient and fast detection of early diagnosis of bladder cancer, and the detection sensitivity and specificity are also high, but in the case of viral infection, kidney/bladder stones, etc., false positives may still occur; the UroVysion kit uses multiple The fluorescence in situ hybridization (FISH) technology of the target, although the sensitivity and specificity have been greatly improved, and the US FDA has approved it, but similar to the Immunocyt kit, it needs to detect a large number of urine exfoliated cells.

Combined with the current application of bladder cancer markers and the diagnosis status of bladder cancer, it is concluded that: on the one hand, new specific bladder cancer markers are still the focus of research, and the preparation of early diagnosis kits to achieve rapid detection is important in the diagnosis of bladder cancer. On the one hand, it has important clinical value; on the other hand, the cell detection in urine samples can avoid the influence of non-specific symptoms such as inflammation and hematuria, and the accuracy is the highest.

Contents of the invention

(1) Solved technical problems

This invention focuses on the early diagnosis of bladder cancer and the follow-up observation of bladder cancer patients, and intends to solve the current difficult problem in the diagnosis of bladder cancer, that is, to use two specific monoclonal antibodies to identify bladder cancer cells to realize double antibody recognition of bladder cancer cells And capture, combined with highly sensitive chemiluminescence immunoassay method, can quantitatively detect bladder cancer cells exfoliated from urine, does not require expensive detection equipment, is convenient for clinical promotion, has good market application value, and will make up for the shortage of existing kits in the market.

The purpose of the present invention is to provide a specific method for capturing and identifying bladder cancer cells, and a highly sensitive detection kit.

Another object of the present invention is to provide a kit for early diagnosis and postoperative monitoring of bladder cancer.

Another object of the present invention is to provide a method for preparing the above kit.

(2) Technical solution

The present invention specifically provides the following kits:

[1] A kit for detecting human bladder cancer cells, the kit comprising two monoclonal antibodies that specifically recognize surface antigens of bladder cancer cells.

[2] The kit according to [1], comprising:

1) Bladder cancer EJ cell standard;

2) Magnetic particles coated with monoclonal antibodies that specifically recognize surface antigens of bladder cancer cells;

3) Enzyme-labeled monoclonal antibodies that specifically recognize bladder cancer cell surface antigens;

4) a chemiluminescent substrate acted by the enzyme in 3);

5) Reaction tube or microporous plate, the reaction tube or microporous plate is preferably sealed with a protein-free blocking solution, the protein-free blocking solution is 0.02M phosphate containing 1.0% fish hydrolyzed gelatin, pH7.2 buffer;

6) A magnetic separator matched with the reaction tube or microplate in 5).

[3] The kit according to [2], wherein the bladder cancer EJ cell standard is a serial dilution of bladder cancer EJ cells.

[4] The kit according to [2], wherein the monoclonal antibody specifically recognizing the surface antigen of bladder cancer cells described in 2) is a monoclonal antibody prepared by a hybridoma cell line with a deposit number of CGMCC No.6906 Antibody BCMab2, which is covalently bonded to the magnetic particles.

[5] The kit according to [2], wherein the magnetic particles described in 2) are silicon oxide hydroxyl magnetic particles having a particle diameter of 100 nm to 1 μm.

[6] The kit according to [2], wherein the enzyme-labeled monoclonal antibody specifically recognizing bladder cancer cell surface antigen described in 3) is an enzyme-labeled BCMab1 monoclonal antibody, and the BCMab1 monoclonal antibody It is prepared from the hybridoma cell line with the preservation number CGMCC No. 3845 (for the preparation of the BCMab1 monoclonal antibody, please refer to the Chinese patent application number: 201010251384.6).

[7] The kit according to [2], wherein the enzyme described in 3) is horseradish peroxidase, alkaline phosphatase or luciferase.

[8] The kit according to [2], wherein the reaction tube described in 5) is a polystyrene tube, polyethylene tube, polypropylene tube or glass tube with optical transparency; the microwell plate is White microplates suitable for detection of chemiluminescent reactions.

The present invention also provides a method for preparing the kit, the method comprising the following steps:

1) Preparation of bladder cancer EJ cell standard product: progressively diluting the bladder cancer EJ cell solution to prepare a stepwise dilution of bladder cancer EJ cell;

2) preparing streptavidin-modified magnetic particles;

3) Two monoclonal hybridoma cell lines with preservation numbers of CGMCC No.3845 and CGMCC No.6906 were used to prepare monoclonal antibodies BCMab1 and BCMab2 that can specifically recognize surface antigens of bladder cancer cells;

4) Preparation of biotinylated monoclonal antibody BCMab2: coupling biotin-N-hydroxysuccinimide with free lysine of the antibody under slightly alkaline conditions;

5) Using the streptavidin-modified magnetic particles prepared in 2) and the biotinylated monoclonal antibody BCMab2 prepared in 4), through the specific binding reaction of streptavidin and biotin, a biological Magnetic particles coated with primed monoclonal antibody BCMab2;

6) Preparation of enzyme-labeled monoclonal antibody BCMab1: using carbodiimide (EDC) coupling method to realize the combination of the amino group on the antibody and the carboxyl group on the enzyme molecule;

7) preparing the chemiluminescence substrate solution acted by the enzyme in 6);

8) Assemble into a finished product.

Preferably, the method further comprises the steps of:

a) after the preparation of the magnetic particles coated with the biotinylated monoclonal antibody BCMab2, block the prepared magnetic particles coated with the biotinylated monoclonal antibody BCMab2 with a blocking solution to reduce non-specific adsorption, The blocking solution comprises 0.2%-1.0% bovine serum albumin, 0.5%-1.0% casein, 0.5%-1.0% fish hydrolyzed gelatin, and 0.02M phosphate buffer at pH 7.2; and

b) Block the reaction tube and the microwell plate with a protein-free blocking solution, which is 0.02M phosphate buffer containing 1.0% fish hydrolyzed gelatin and pH 7.2.

The kit of the present invention can sensitively and quickly detect the bladder cancer cells exfoliated in urine, and can detect early exfoliated malignant tumor cells, and the sensitivity is higher than that of urine component assay and urine cytology assay currently on the market. The detection sensitivity of the patent of the present invention reaches the detection of one malignant bladder cancer cell for each analysis.

The kit of the present invention is also applicable to the detection of tumor cells of bladder cancer tissue digested cells, and the detection of bladder cancer cells circulating in the blood.

The kit of the present invention adopts a double-antibody sandwich method in which immunomagnetic particles are specifically captured and enzyme-labeled monoclonal antibodies are specifically recognized, thereby realizing the specific detection of bladder cancer cells. This kit can exclude interference from normal, inflammatory epithelial cells, and other blood cells, and only specifically recognize bladder cancer cells.

The patent of the invention adopts the immunomagnetic particle method to capture the bladder cancer cells shed in urine, which is easy to operate and short in detection time, which is conducive to large-scale clinical application.

Two kinds of monoclonal antibodies that specifically recognize the surface antigen of bladder cancer cells are used in the present invention:

1) BCMab1: prepared from a hybridoma cell line with the preservation number CGMCC No.3845, which was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on May 21, 2010 (CGMCC, Beijing, China) , the antibody and its preparation are described in Chinese patent application number: 201010251384.6; and

2) BCMab2: prepared from a hybridoma cell line with the preservation number CGMCC No.6906, which was deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms on November 27, 2012 (CGMCC, Beijing, China) .

Description of drawings

Figure 1. The linear standard curve obtained by the prepared kit using EJ cells as a model.

Figure 2. Comparison of the luminescence values of EJ, Lovo, HCV29 cell lines and PBS detected by this method. EJ: bladder cancer cell line; Lovo: rectal cancer cell line; HCV29: normal epithelial cell line.

Figure 3. Kit detection sensitivity. Bladder cancer cell lines: EJ, T24, 5637, BIU-87; BCU-Ta: urine samples from patients with bladder cancer at stage Ta; BCU-T1: urine samples from patients with bladder cancer at stage T1.

Figure 4. The kit of the present invention is applied to the detection of bladder cancer and normal samples.

Fig. 5. ROC curve of the clinical assay results of the kit of the present invention.

Detailed ways

Example 1 Preparation of a new bladder cancer cell chemiluminescence detection kit of the present invention

1. Preparation of standardized bladder cancer cells

Bladder cancer EJ cell line (CRL-2888 ^TM ) was purchased from American Type Culture Collection (ATCC) and cultured in DMEM medium (Gibco, 12100-046) containing 10% fetal bovine serum. Add 1-2 mL of trypsin (Invitrogen, R-001-100) to the cultured cells with good adherence, digest at 37°C for 2 minutes, add an appropriate amount of medium, centrifuge the digested cells, discard the supernatant, and resuspend in In the cryopreservation solution prepared by adding fetal bovine serum and 10% dimethyl sulfoxide (DMSO), the cells were frozen at -80°C at a concentration of 10 ⁶ cells/mL, and each tube was frozen in 500 μL. During the transportation of the kit, this component needs to be stored and transported separately in dry ice.

2. Preparation of monoclonal antibody BCMab2 hybridoma cells

Frozen EJ cells were taken and cultured in DMEM medium. After 3 weeks of culture in good condition, 1× ¹⁰⁷ EJ cells in the logarithmic phase of growth were taken and immunized with 6-week-old BALB/c mice (commercially purchased from Beijing Weiwei Co., Ltd. Tonglihua Experimental Animal Technology Co., Ltd.), immunized 4 times a week, and 1 month after immunization, the spleen cells of the mice were collected. And prepare mouse myeloma cell Sp2/0 (CRL-1772, ATCC) and mouse spleen cell fusion, according to the reference (Kirk, AD, et al.Nat Med 1999, 5, 686-693) described hybridoma Screening of monoclonal strains of EJ cells.

Then, the selected positive hybridoma cells were cloned and cultured (limited dilution method, using mouse peritoneal macrophages as feeder cells). After 2-3 rounds of cloning culture, stable hybridoma cell clones capable of producing high-titer monoclonal antibodies were obtained. The hybridoma cell clones were expanded and cultured, and kept frozen. The obtained hybridoma cell line can secrete monoclonal antibody BCMab2, which can specifically recognize EJ cells and other bladder cancer cells.

3. Preparation of monoclonal antibodies BCMab1 and BCMab2

There are two monoclonal hybridoma cell lines: the hybridoma cell line with the preservation number CGMCC No.3845 can obtain BCMab1 antibody (for details, see Chinese patent application number: 201010251384.6); the hybridoma cell line with the preservation number CGMCC No.6906 strains can obtain BCMab2 antibodies. 5×10 ⁶ hybridoma cells in the logarithmic growth phase were taken and injected into the peritoneal cavity of BALB/c mice at a cell concentration of 1×10 ⁷ , and the ascites was collected 10 days later. Purify monoclonal antibodies from ascitic fluid by the following steps:

1. Centrifuge the collected ascites at 2500r/min, take the supernatant, and double-dilute the ascites supernatant with 0.01M pH7.4 PBS.

2. Add an equal volume of saturated ammonium sulfate to the above ascites, and stir at room temperature for 1 hour;

3. Centrifuge at 11000r/min, 4°C for 20 minutes, and discard the supernatant. Resuspend in an appropriate amount of 0.01M pH7.4PBS, add half the volume of ascites saturated ammonium sulfate, and stir overnight at 4°C.

4. The above ascites was centrifuged at 11000r/min, 4°C for 20 minutes, and the supernatant was discarded. Dissolve the precipitate in 0.01M pH 7.4 PBS, and then dialyze with 0.01M pH 7.4 PBS to obtain thick antibody spheres.

5. Install the Protein-G gel column to the AKTA protein purification instrument;

6. Centrifuge the antibody pellet at 11000r/min at 4°C for 20 minutes, and take the supernatant. The supernatant passed through the Protein-G gel column pre-equilibrated with 0.02M pH7.0 PBS containing 0.15M NaCl at a flow rate of 0.5mL/min, and incubated for 1 hour after loading;

7. 0.02M pH7.0 PBS containing 0.15M NaCl to elute impurities;

8. 0.2M pH2.8 glycine buffer elutes the antibody peak;

9. The eluted antibody solution is concentrated by ultrafiltration, and the antibody concentration is measured to complete the purification of the antibody.

4. Preparation of immunomagnetic particles

1. Biotinylation of Monoclonal Antibody BCMab2

Take 10 mg of the purified monoclonal antibody BCMab2 (2 mg/mL), and dialyze it with 0.1 M carbonate buffer, pH 9.5, to obtain an antibody solution in the carbonate buffer system. Biotin-N-hydroxysuccinimide (NHS-Biotin, Cat. No. H1759) was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL. Then 200 uL of biotin solution was added to the antibody solution, stirred at 4°C for 6 hours, and 20 uL of 1M NH ₄ Cl solution was added to terminate the reaction. The reactant was dialyzed in 0.05M, pH7.4 phosphate buffer solution to obtain the biotinylated antibody BCMab2.

2. Streptavidin Modification of Magnetic Particles

Take 20 mg of silicon oxide hydroxyl magnetic particles (Shanghai Aorun Micro-Nano Co., Ltd., SM1-015A, SM1-035, SM1-050, SM1-100), remove the storage buffer under the action of an external magnetic field, add 2 mL of 1.25% glutaraldehyde to activate , after stirring at room temperature for 2 hours, under an external magnetic field, let stand for 5 minutes, discard the supernatant, wash with 0.01M pH7.4 phosphate buffer three times, and resuspend in 0.05M, pH9.5 carbonic acid buffer solution. Afterwards, 1 mg streptavidin (purchased from Sigma-Aldrich, S4762) was added, stirred at room temperature for 2 hours, washed three times with 0.01 M phosphate buffer solution of pH 7.4, and resuspended in phosphate buffer solution at a constant concentration of 4 mg/ mL to obtain streptavidin-modified magnetic particles.

3. Preparation of Immunomagnetic Particles

20 mg of streptavidin-modified magnetic particles (4 mg/mL) were placed in a magnetic field. After the magnetic particles had completely settled, the supernatant was removed, and 10 mg of biotinylated antibody BCMab2 (2 mg/mL) was added, stirred at 4°C for 4 Hour. Then wash four times with 0.01M pH7.4 phosphate buffer, and finally resuspend in antibody buffer solution (0.01M pH7.4 phosphate buffer, 2% bovine serum albumin, 0.05% Tween-20, 0.05% proclin -300), to obtain immunomagnetic particles. Preferably, the prepared magnetic particles coated with biotinylated monoclonal antibody BCMab2 are blocked with a blocking solution containing 0.2% to 1.0% bovine serum albumin, 0.5% to 1.0% Casein, 0.5% to 1.0% fish hydrolyzed gelatin, 0.02M phosphate buffer at pH 7.2. The prepared immunomagnetic particles were stored at 4°C at a concentration of 4 mg/mL.

5. Selection of magnetic particle size

The smaller the particle size of the magnetic particles, the more uniform the dispersion in the solution, but when it is less than 100nm, the sedimentation speed will slow down under the action of an external magnetic field, which will increase the washing time during the application of the kit. Therefore, the magnetic particles larger than 100nm are selected for this kit. particles. Magnetic particles are dispersed in the reaction system during the immune reaction, which can speed up the reaction to a certain extent. In the luminol-H ₂ O ₂ luminescence system, it has a certain catalytic effect. When the particle size is 100nm to 1μm, the luminescence kinetic curve of the luminol-H ₂ O ₂ system can last for 30 minutes to 2 hours. between. When the thickness increases to more than 1 μm, the catalytic ability decreases, and the duration of the luminescence plateau of the luminescence kinetic curve of the luminol-H ₂ O ₂ system decreases (below 15min).

6. Preparation of enzyme-labeled monoclonal antibody BCMab1

Taking horseradish peroxidase (HR, purchased from Sigma, P6782) labeled antibody as an example, the carbodiimide (EDC) coupling method was used, and the specific process was as follows: 10 mg HRP was dissolved in 0.1 M citrate at pH 6.0 In the buffer solution (78.84mg citric acid and 476.44mg sodium citrate were added to 1L deionized water), carbodiimide (EDC, dissolved in marinyl ethanesulfonic acid) was added to react with the carboxyl group on the enzyme molecule; after 10min, N -Hydroxysulfosuccinimide (NHS), will replace the EDC molecule, and an activated carboxyl group will be formed between the enzyme molecule and the NHS molecule; 1 mg of monoclonal antibody BCMab1 will react with the activated enzyme molecule to realize the amino group on the antibody and the enzyme molecule Activation of the carboxyl group reaction at room temperature for 2 hours to obtain the HRP-labeled monoclonal antibody BCMab1; add saturated ammonium sulfate solution dropwise to the labeled antibody solution, and stir while adding until the concentration of saturated ammonium sulfate drops to 1/3; stand at 4°C Set aside for 1h, centrifuge at 8000rpm for 10min, transfer the supernatant to a new tube, resuspend the pellet with an equal volume of PBS; repeat the above operation 3 times, collect the supernatant to obtain the purified horseradish peroxidase-labeled antibody BCMab1, add an equal volume Glycerol, stored at -20°C for later use.

The coupling of alkaline phosphatase or luciferase to BCMab1 is similar to the above steps, and the specific amount of reagents can be adjusted and optimized appropriately.

7. Preparation of sample diluent

Prepare sample diluent, the specific preparation is: in 1L deionized water, add 3.628gNa ₂ HPO ₄ 12H ₂ O, 0.272g KH ₂ PO ₄ , 0.2g KCl, 8g NaCl, 18g urea, 0.05g uric acid, 1mL fetal bovine Blood, 1mL proclin-300 shake and mix well, adjust the pH to 7.4, store the solution at 4°C, and use it as a dilution buffer solution for urine sediment samples and frozen cells.

Eight, chemiluminescent substrate solution

The preparation method of the chemiluminescent substrate liquid of horseradish peroxidase (HR) used in the present invention:

Based on 1000mL of the chemiluminescence substrate A solution, including 1.7716g luminol, 0.05g 4-hydroxybiphenyl, 0.012g 4-iodophenylboronic acid, 11.4g boric acid, 4.9g borax, the pH value is 8.0-10.0;

Based on 1000mL of the chemiluminescent substrate B solution, including 0.329g carbamide peroxide, 1ml Tween-20, 51.58g Na ₂ HPO4·12H ₂ O, 8.74g NaH ₂ PO ₄ ·2H ₂ O, the pH value is 7.0-7.6 .

How to use: A and B liquid two-component reagents, mix equal volumes according to the usage amount before use.

9. Sealing treatment of reaction tubes and microplates

Preferably, the reaction tube and the microwell plate are blocked with a protein-free blocking solution, which is 0.02M phosphate buffer containing 1.0% fish hydrolyzed gelatin and pH 7.2.

Example 2 Method of using the bladder cancer urine exfoliated cytochemical method photoimmunoassay assay kit of the present invention

1. Sample pretreatment

Take the first morning urine or the second morning urine sample. If it is tested immediately, the sample does not need to be processed and can be tested directly. If it is not possible to test on the same day, the sample needs to be frozen: take 10mL of urine, centrifuge at 3000rpm for 5min, suspend the urine sediment in 500uL of cell freezing solution (fetal bovine serum containing 10% DMSO), put it at 4°C for 2 hours, and then Store at -80°C for later use.

2. Detection method

Before using this kit for experiments, first adjust the incubator or water bath to 37°C; then take out the magnetic particle solution, HRP-labeled antibody and each buffer solution prepared in Example 1, and place them at room temperature to equilibrate to room temperature; then take out the frozen samples and bladder cancer EJ cell standard samples and place them in a 37°C water bath for rapid rethawing, centrifuge to remove the supernatant, and resuspend in 1mL of cell diluent. The initial concentration of EJ cells was 5×10 ⁵ cells/mL, and then serially diluted with cell diluent to obtain a series of cell standards, namely 5×10 ⁵ cells/mL, 1×10 ⁵ cells/mL, 5×10 ^{4 cells} pcs/mL, 1×10 ⁴ pcs/mL, 5×10 ³ pcs/mL, 5×10 ² pcs/mL, 20 pcs/mL. Then, prepare 200-1000 μL, 20-200 μL, 1-10 μL range microsamplers and corresponding tips and check whether the chemiluminescence instrument is working normally.

The specific operation steps of using the kit of the present invention to detect are as follows:

(1) Microwell plate chemiluminescence detection kit

Take a white 96 microwell plate and add 50 μL of urine sample or serial calibration cell solution (5×10 ⁵ cells/mL, 1×10 ⁵ cells/mL, 5×10 ⁴ cells/mL, 1×10 ⁴ cells/ mL, 5×10 ³ cells/mL, 5×10 ² cells/mL, 20 cells/mL). Then, 5 μL each of horseradish peroxidase-labeled antibody solution and immunomagnetic particle solution were added successively, and the reaction was shaken at 37° C. for 60 min. Add 150 μL of washing solution to each well, oscillate on a microplate shaker (MH-1, Kylin-Bell Instruments) for 10 seconds, and then place it on a magnetic separator (purchased from NEB, S1511S) for a 96-well plate , remove the supernatant. Repeat the above washing steps for 4 washes. Add 100 μL of chemiluminescence substrate solution to each well, mix thoroughly, and then measure the luminescence intensity (RLU) sequentially on a plate chemiluminescence instrument (Beijing Hamamatsu Photonics Co., Ltd., BHP9504). Theoretically, the number of standard cells in each well is 25000/analysis, 5000/analysis, 2500/analysis, 500/analysis, 250/analysis, 25/analysis, 1/analysis. See Figure 1 for the linear relationship between the number of cells and the luminescence value, where the ordinate RLU is the relative luminous intensity, and the abscissa is the number of detected cells (unit: cell/analysis). In the range of 1-25000 cells/analysis, the correlation coefficient R ² =0.9984, Y=12511+33.651X; in the range of 1-500 cells/analysis, the correlation coefficient R ² =0.9968, Y=1037+92.21X.

According to the size of the RLU, select an appropriate range of the standard curve, and substitute the RLU of each sample to be tested into the equation of the standard curve to obtain the number of tumor cells in the urine sample to be tested.

(2) Tubular Chemiluminescence Detection Kit

After numbering the reaction tubes, add 200 μL of urine samples or a series of calibration cell solutions, mix with 20 μL each of the magnetic particle solution and HRP-labeled BCMab1 antibody solution, and shake at 37°C for 60 minutes. Then place it on a multi-functional magnetic separator (Shanghai Aorun Micro-Nano New Material Technology Co., Ltd., MS-12), discard the supernatant after 3 minutes, add 300 μL of washing solution to each tube, mix well, and place it on a magnetic separator Let it stand for 3 minutes, discard the supernatant, repeat 4 times, discard the washing solution at last, add 200 μL of chemiluminescence substrate solution to each tube, mix well, and then test in a tube chemiluminescence measuring instrument (Germany Berthold Technologies GmbH & Co. KG) to measure the luminous intensity (RLU) of each tube sequentially. The number of tumor cells in the urine sample to be tested was determined in a manner similar to that in (1) above.

The methodological index of embodiment 3 kits of the present invention

The test kit prepared in Example 1 is tested according to the conventional manufacturing and testing procedures in the art, and the results are as follows:

1. Determination of kit precision

(1) Precision experiment of EJ cell standard

Extract 10 kits from the kit prepared in Example 1, and measure 1×10 ³ cells/mL EJ cell standard 5 times according to the operation described in Example 2. The coefficient of variation of the measurement results was calculated, and the measurement results are shown in Table 1. The results showed that the coefficient of variation was between 3.5% and 10%.

Table 1 EJ Cell Standard Reproducibility Experiment

(2) Sample precision experiment

10 kits were extracted from the kits prepared in Example 1, and the urine exfoliated cells of two bladder cancer patients were measured according to the operation described in Example 2. Calculate the coefficient of variation for the measured concentrations. The measurement results of the three batches of kits in Example 1 are shown in Table 2 and Table 3, and the results show that the coefficient of variation is less than 9.0%.

Table 2. Precision determination of urine exfoliated cell samples from Patient 1

Table 3. Precision determination of urine exfoliated cell samples from Patient 2

2. Determination of kit accuracy

Urine exfoliated cell samples were taken from two bladder cancer patients, and the tumor cell concentrations of the two samples were determined to be 4×10 ³ and 2×10 ³ cells/mL, respectively, by the kit prepared in Example 1. Add an equal volume of EJ cell standard solution 4×10 ³ cells/mL and 2×10 ³ cells/mL, operate as described in Example 2, measure each concentration three times, and calculate the recovery rate. The results are shown in Table 4, indicating that the recovery rate was between 93.0% and 102.0%.

Table 4. Accuracy Measurements

3. Kit stability test

For the kit of Example 1, other reagents except the frozen EJ cell standard were subjected to accelerated experiments at 37°C for 7 days, and the results of determining the maximum and minimum luminous intensity of the kit, the recovery rate of the analyte, etc. showed that The kit indexes of Example 1 were all within the normal range. The main components of the kit in Example 1 were followed up for 8 months at 2-8°C, and the results showed that all indicators fully met the requirements. Considering the occurrence of freezing of the kit, the kit of Example 1 was frozen at -20°C for 7 days, and the measurement results also showed that all the indicators of the kit were completely normal. Bladder cancer EJ cells cryopreserved at -80°C were revived and cultured after 8 months, and stained with Trypan Blue (Beyond, C0011) for living cell technology, and more than 95% of the cell viability remained good. It can be seen from the above results that the kit can be stored at 2-8°C for at least 6 months.

4. Kit specificity test

Add normal urothelial cell line HCV29 or other tumor cell lines such as Hela (breast cancer, CCL-2 ^TM ), Jurkat (leukemia, CRL-1990 ^TM ), lovo (rectal cancer, CCL-219 ^{TM )} in healthy human urine ), K562 (chronic myeloid leukemia, CTL-243 ^TM ), HepG2 (liver cancer, CRL-10741 ^TM ), the concentration of added cells reached 10 ⁵ cells/mL, and the specificity test was carried out. The above cell lines were purchased from the American Type Culture Collection (ATCC). By detecting the luminescence value, the effect of the addition of non-bladder cancer cells on the reaction system was compared. The results are shown in FIG. 2 , adding normal urothelial cells or other tumor cells to the urine of healthy people resulted in lower background values. In addition, the PBS buffer simulates the blank buffer, and compared with the luminescence value of bladder cancer EJ cells and T24 cell solutions (10 ³ cells/mL), it also only produces low background values, which shows that the specificity and sensitivity of this method are high.

Add 10 μL of human peripheral blood (taken from the venous blood of a healthy person) to 1 mL of healthy quality control urine sample to obtain a macroscopic hematuria sample; in addition, add 50 human peripheral blood red blood cells in one field of view under a high-power microscope to obtain microscopic hematuria samples to investigate the influence of hematuria on this kit. The measurement results showed that the magnetic particles did not capture red blood cells non-specifically, nor did the BCMab1-HRP antibody bind non-specifically, and the final result was not affected by red blood cells.

5. Kit sensitivity

The sensitivity of the kit is based on the minimum number of tumor cells that can be detected. Four bladder cancer cell lines EJ, T24 (HTB-4 ^TM , ATCC), 5637 (HTB-9 ^TM , ATCC), BIU-87 (gifted by the Shanghai Institute of Biochemical Cells, Chinese Academy of Mann, Z1) for accurate counting followed by limiting dilution. Take 50 μL of reaction from each sample, and get 1, 2, 5 cells/analysis by limiting dilution, and then use this research method kit for determination. The measured luminescence values are shown in Figure 4. In addition, urine exfoliated cell samples of bladder cancer with Tis, Ta, T1b, T2a, and T2b stages were first stained with the Swiss-Giemsa staining kit (Nanjing Jiancheng Technology Co., Ltd., D010) to determine the concentration of urinary exfoliated tumor cells, and then performed Limiting dilution. Take 50 μL of reaction for each sample, obtain 1, 2, 5 cells/analysis (cell/assay) by limiting dilution, and then use the kit of this research method to measure. The measured luminescence values are shown in Figure 3. It can be seen from the figure that the luminescence value of a single cell is significantly higher than the blank value (PBS is used as a blank control), that is, a single bladder cancer cell can be detected.

Prove through a large amount of experiments, the kit methodology index of the present invention is as follows:

Detection range: the detection concentration of bladder cancer cells is 20 cells/mL～5× ¹⁰⁶ cells/mL, that is, 1 cell/analysis～25000 cells/analysis;

Sensitivity: The minimum detection limit is 1 cell/analysis;

Precision: less than 9% (n=5)

The method of the invention has the advantages of high sensitivity, strong specificity, wide detection range, simple operation, no radioactive pollution, low kit cost, strong clinical applicability, and is more suitable for clinical detection and screening laboratories in my country. Therefore, the present invention provides a sensitive, accurate, fast and specific method for the clinical detection of urinary shedding tumor cells of bladder cancer, early diagnosis of bladder cancer and dynamic monitoring.

Example 4 Application of the kit of the present invention in the determination of clinical samples of bladder cancer patients

Collect 205 examples of urine samples from patients with bladder cancer, and 389 examples of urine samples from normal people (including healthy people, patients with benign urinary system diseases such as inflammation and calculi), and use the kit of the present invention to detect. The comparison of luminescence value (RLU) is as follows: Figure 4 shows. It can be seen that there is a significant difference in the measured luminescence value between bladder cancer and normal people. Therefore, through the detection of urine exfoliated cells, patients with bladder cancer can be well distinguished from normal people. According to the ROC curve analysis (as shown in Figure 5), three different critical values are respectively taken: (1) 3126 (RLU), the sensitivity (sensitivity) is the highest, and the specificity (specificity) is acceptable, and the sensitivity and specificity are respectively 100% and 72%; (2) 20699 (RLU), the sensitivity and specificity were optimal at the same time, the sensitivity and specificity were 93% and 89% respectively. 56875 (RLU) has the highest specificity and acceptable sensitivity, the sensitivity and specificity are 72% and 99% respectively.

Table 5. Sensitivity and specificity of the kit of the present invention at different cut-off values.

Using EJ cells as a standard, quantitative detection was carried out to obtain a standard measurement curve (as shown in Figure 1). The kit of the invention is used to detect the urine samples of bladder cancer patients, and then the number of tumor cells in the actual samples can be calculated according to the sample detection results and the standard curve. At the same time, the exfoliated cell smears of the urine samples of bladder cancer patients were prepared, and the urine exfoliated cells were stained with the rapid Riggy's staining kit (Nanjing Jiancheng Technology Co., Ltd., D010), and the number of tumor cells was counted. The cell smear method is compared with the test results of the kit of the present invention, and the results are shown in Table 6. It can be seen that the sensitivity of this method is significantly higher than that of the traditional cell smear method.

Table 6. Cell smear and urine shedding tumor cell number detection result of kit of the present invention

Claims (10)

1. A test kit for detecting human bladder cancer cells, said test kit comprising two kinds of monoclonal antibodies specifically recognizing surface antigens of bladder cancer cells. 2. test kit according to claim 1, said test kit comprises: 1) Bladder cancer EJ cell standard; 2) Magnetic particles coated with monoclonal antibodies that specifically recognize surface antigens of bladder cancer cells; 3) Enzyme-labeled monoclonal antibodies that specifically recognize bladder cancer cell surface antigens; 4) a chemiluminescent substrate acted by the enzyme in 3); 5) Reaction tube or microporous plate, the reaction tube or microporous plate is preferably sealed with a protein-free blocking solution, the protein-free blocking solution is 0.02M phosphate containing 1.0% fish hydrolyzed gelatin, pH7.2 buffer; 6) A magnetic separator matched with the reaction tube or microplate in 5). 3. The kit according to claim 2, wherein the bladder cancer EJ cell standard is a serial dilution of bladder cancer EJ cells. 4. test kit according to claim 2, wherein, the monoclonal antibody of the specific recognition bladder cancer cell surface antigen described in 2) is the monoclonal antibody prepared by the hybridoma cell line of CGMCC No.6906 by preservation number BCMab2, which is covalently bound to magnetic particles. 5. The kit according to claim 2, wherein the magnetic particles described in 2) are silicon oxide hydroxyl magnetic particles with a particle diameter of 100 nm to 1 μm. 6. The test kit according to claim 2, wherein, the monoclonal antibody of the enzyme-labeled specific recognition bladder cancer cell surface antigen described in 3) is an enzyme-labeled BCMab1 monoclonal antibody, and the BCMab1 monoclonal antibody is composed of Prepared from a hybridoma cell line with preservation number CGMCC No.3845. 7. The kit according to claim 2, wherein the enzyme described in 3) is horseradish peroxidase, alkaline phosphatase or luciferase. 8. kit according to claim 2, wherein, the reaction tube described in 5) is polystyrene tube, polyethylene tube, polypropylene tube or glass tube with optical transparency; Described microwell plate is applicable White microplate for the detection of chemiluminescent reactions. 9. prepare the method for kit according to claim 1 or 2, described method comprises the following steps: 1) Preparation of bladder cancer EJ cell standard product: progressively diluting the bladder cancer EJ cell solution to prepare a stepwise dilution of bladder cancer EJ cell; 2) preparing streptavidin-modified magnetic particles; 3) Two monoclonal hybridoma cell lines with preservation numbers of CGMCC No.3845 and CGMCC No.6906 were used to prepare monoclonal antibodies BCMab1 and BCMab2 that can specifically recognize surface antigens of bladder cancer cells; 4) Preparation of biotinylated monoclonal antibody BCMab2: coupling biotin-N-hydroxysuccinimide with free lysine of the antibody under slightly alkaline conditions; 5) Using the streptavidin-modified magnetic particles prepared in 2) and the biotinylated monoclonal antibody BCMab2 prepared in 4), through the specific binding reaction of streptavidin and biotin, a biological Magnetic particles coated with primed monoclonal antibody BCMab2; 6) Preparation of enzyme-labeled monoclonal antibody BCMab1: using carbodiimide (EDC) coupling method to realize the combination of the amino group on the antibody and the carboxyl group on the enzyme molecule; 7) preparing the chemiluminescence substrate solution acted by the enzyme in 6); 8) Assemble into a finished product. 10. The method of claim 9, further comprising the steps of: a) after the preparation of the magnetic particles coated with the biotinylated monoclonal antibody BCMab2, block the prepared magnetic particles coated with the biotinylated monoclonal antibody BCMab2 with a blocking solution to reduce non-specific adsorption, The blocking solution comprises 0.2%-1.0% bovine serum albumin, 0.5%-1.0% casein, 0.5%-1.0% fish hydrolyzed gelatin, and 0.02M phosphate buffer at pH 7.2; and b) Block the reaction tube and the microwell plate with a protein-free blocking solution, which is 0.02M phosphate buffer containing 1.0% fish hydrolyzed gelatin and pH 7.2.

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