A urine nuclear matrix protein 22 quantum dot fluorescent immunochromatographic quantitative detection method and kit

Abstract

This invention discloses a quantum dot fluorescent immunochromatographic quantitative detection method and kit for urinary NMP22, belonging to the field of in vitro diagnostic technology in biomedicine. This invention uses quantum dot fluorescent microspheres as markers and prepares NMP22 monoclonal antibody conjugates using the EDC / NHS activation method. An immunochromatographic test strip composed of a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad is constructed. Based on the principle of a double-antibody sandwich immunoreaction, a quantitative standard curve is established using the T / C fluorescence ratio method to achieve accurate quantitative detection of NMP22 in urine. This invention optimizes the chromatographic system parameters for the characteristics of the urinary matrix, resulting in excellent detection sensitivity and specificity. The operation is simple, with results obtained in 15 minutes. No large-scale testing equipment is required, enabling real-time single-sample testing. It is suitable for early auxiliary diagnosis and postoperative recurrence monitoring of bladder cancer, with low testing cost, facilitating its widespread application in primary healthcare institutions.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a quantitative detection method and kit for urinary nuclear matrix protein 22 by quantum dot fluorescence immunochromatography. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system and the fifth most common malignant tumor worldwide. According to GLOBOCAN's 2020 global cancer statistics, there were approximately 573,278 new cases of bladder cancer and approximately 212,546 deaths throughout the year. The incidence rate in men is about four times that in women. Clinical data shows that about 75% of bladder cancer patients are initially diagnosed with non-muscle-invasive bladder cancer. Even after standard treatment, about 40% of patients experience recurrence, and 10% progress to muscle-invasive bladder cancer. The high recurrence rate necessitates long-term, regular follow-up monitoring for patients. Early diagnosis and early detection of recurrence of bladder cancer are crucial for improving patient prognosis and reducing mortality.

[0003] Currently, the mainstream methods for bladder cancer diagnosis and follow-up include cystoscopy and urine cytology. Cystoscopy is considered the gold standard for diagnosis, but it is an invasive procedure with low patient tolerance, high cost, and frequent follow-ups can increase the risk of urinary tract infections, resulting in a significant financial burden. Urine cytology is a non-invasive test with high specificity, but its sensitivity is relatively low, especially for low-grade urothelial carcinoma. Furthermore, result interpretation heavily relies on the pathologist's experience, lacking standardized quantitative evaluation criteria, thus limiting its clinical application. While multicolor fluorescence in situ hybridization (FISH) can improve detection sensitivity to some extent, its cumbersome procedures, long testing cycle, and high cost make it difficult to apply to large-scale screening and point-of-care testing scenarios.

[0004] Due to its advantages of being non-invasive, convenient to sample, and repeatable, urinary tumor marker detection has become an important research direction for the non-invasive diagnosis of bladder cancer. Nuclear matrix protein 22 (NMP22) is an important component of the nuclear matrix and participates in key physiological processes such as DNA synthesis, RNA transcription, and cell mitosis. After bladder cancer cells undergo apoptosis, they release large amounts of NMP22 into the urine, resulting in significantly higher NMP22 levels in the urine of bladder cancer patients than in normal individuals. It is currently the urinary protein biomarker approved by the US FDA for the adjunctive diagnosis and postoperative monitoring of bladder cancer.

[0005] Currently available NMP22 testing products have significant limitations: imported colloidal gold test reagents can only achieve qualitative detection, and clinical data show that their sensitivity for detecting newly diagnosed bladder cancer is about 54%, with even lower sensitivity for recurrent cases, making it difficult to meet the accuracy requirements for early screening and recurrence monitoring. Moreover, the procurement cost of imported reagents is high. Although domestically available magnetic particle chemiluminescence NMP22 test reagents can achieve quantitative detection, they require a large-scale, fully automated chemiluminescence analyzer, resulting in high equipment purchase and maintenance costs and expensive reagents. This makes them unsuitable for batch, real-time testing and difficult to promote in primary healthcare institutions and outpatient rapid testing scenarios.

[0006] Fluorescent lateral flow immunochromatography combines the high sensitivity of fluorescence detection with the convenience of immunochromatography, enabling quantitative point-of-care testing (POCT) and representing a significant development direction in the field of point-of-care testing (POCT). Quantum dots, as a new generation of fluorescent nanolabeling materials, possess characteristics such as high fluorescence quantum yield, broad excitation spectrum, narrow and symmetrical emission spectrum, and excellent resistance to photobleaching. Compared to traditional fluorescent labels, they can significantly improve the sensitivity and stability of immunochromatographic detection. Currently, quantum dot immunochromatography has been applied in various fields such as virus detection and serum protein detection, but there is still no quantitative detection kit for urinary NMP22. Furthermore, the complex composition of urine samples makes general-purpose immunochromatographic systems prone to non-specific binding, failing to directly adapt to the detection needs of urine samples. Therefore, there is an urgent need to develop targeted and optimized systems. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a quantitative detection method and kit for urinary nuclear matrix protein 22 using quantum dot fluorescence immunochromatography, which solves the problems mentioned in the background.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a quantum dot fluorescence immunochromatographic quantitative detection method for urinary nuclear matrix protein 22, comprising the following steps: (1) Preparation of quantum dot labeled antibody: Quantum dot fluorescent microspheres were activated by EDC / NHS activation method, and the activated quantum dot fluorescent microspheres were coupled with NMP22 monoclonal labeled antibody. After blocking, washing and purification, quantum dot labeled NMP22 monoclonal antibody was obtained. (2) Preparation of immunochromatographic test strips: The quantum dot fluorescently labeled NMP22 monoclonal antibody was pre-sprayed and cured on the conjugate pad; the detection line and control line were pre-sprayed and dried on the nitrocellulose membrane; a PVC backing plate was taken and the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were sequentially overlapped and pasted along the chromatography direction; the strips were cut and assembled into the cartridge; the quantum dot labeled NMP22 monoclonal antibody was pre-sprayed and cured on the conjugate pad; the detection line and control line were etched and dried on the nitrocellulose membrane, the detection line was coated with NMP22 capture monoclonal antibody that recognizes specific antigenic epitopes of NMP22, and the control line was coated with goat anti-mouse IgG secondary antibody; the strips were dried and ready for use. (3) Establishing a quantitative standard curve: Prepare a series of NMP22 recombinant antigen standard solutions with different concentration gradients, add them to the sample application end of the test strip for chromatographic reaction, and detect the fluorescence intensity value T of the detection line and the fluorescence intensity value C of the control line using a fluorescence immunoassay analyzer. Fit the NMP22 antigen concentration as the abscissa and the T / C ratio as the ordinate to obtain the quantitative standard curve. (4) Quantitative detection of urine sample: Take the urine sample to be tested and add it to the sample application end of the test strip. After the chromatographic reaction, detect the T / C ratio and substitute it into the quantitative standard curve to calculate the concentration of nuclear matrix protein 22 in the urine sample.

[0009] Preferably, step (1) specifically includes: Take 2.5 mg / mL quantum dot fluorescent microspheres, add 100 mM MES buffer (pH 6.0) and mix well; add EDC working solution and NHS working solution sequentially, vortex quickly, and react at room temperature for 30 min to complete activation; centrifuge at 20,000 rpm for 10 min at 4 °C, discard the supernatant, resuspend and wash in 20 mM BS buffer (pH 7.4–8.0), repeat centrifugation and washing, and sonicate to resuspend the microspheres; add 10–20 μg NMP22 monoclonal antibody, mix well, and react at 120 min to complete coupling; add BSA to a final concentration of 1%, and block at room temperature for 60 min; centrifuge, discard the supernatant, wash repeatedly with BS buffer, and finally resuspend and store at 4 °C for later use; Both the EDC working solution and the NHS working solution were prepared with MES buffer at pH 6.0 and a concentration of 10 mg / mL, and were prepared and used immediately. The quantum dot fluorescent microspheres had a particle size of 85 nm or 125 nm.

[0010] Preferably, in step (2), both the sample pad and the conjugate pad are made of glass fiber and are soaked in a pretreatment solution for 30 minutes before use and then dried overnight at 37°C. The pretreatment solution is based on Tris-HCl buffer solution with pH 7.2~8.0 and contains blocking protein, surfactant, stabilizer, polyethylene oxide (PEO) and preservative. In the pretreatment solution, the blocking protein is BSA or casein with a mass fraction of less than 0.5%; the surfactant is Tween-20 or S9, wherein the mass fraction of Tween-20 is 0.5%~1.5% and the mass fraction of S9 is 0.5%~1%; the stabilizer is trehalose or sucrose with a mass fraction of less than 5%; and the preservative is PC-300 or Junqing preservative.

[0011] Preferably, in step (2), the amount of quantum dot labeled antibody sprayed on the conjugate pad is 2~5 μg / cm; the coating solution of the detection line is a 20mM phosphate buffer with pH 7.2~7.4 containing 1%~3% sucrose, and the concentration of the coated NMP22 capture monoclonal antibody is not higher than 1.5mg / mL; after spraying, it is dried at 37℃ overnight; The nitrocellulose membrane in step (2) has a creeping speed of 90~150s; before scribing and spraying, the nitrocellulose membrane is placed in an environment with a relative humidity of 45%~65% to equilibrate.

[0012] Preferably, in steps (3) and (4), the chromatography reaction time is 15 min; the excitation wavelength of the fluorescence immunoassay analyzer is 350 nm to 400 nm, and the emission detection wavelength is 615 nm.

[0013] Preferably, the NMP22 monoclonal labeled antibody and the NMP22 capture monoclonal antibody are prepared using hybridoma technology. Specifically, Balb / c mice are immunized with the NMP22 recombinant antigen, and spleen cells from the immunized mice are fused with SP2 / 0 myeloma cells using PEG1450. The fused cells are screened using HAT medium, and a stable antibody-secreting hybridoma cell line is obtained by subcloning using the limiting dilution method. The hybridoma cell line is inoculated into the peritoneal cavity of Balb / c mice to prepare ascites fluid, and the ascites fluid is purified by protein A chromatography column to obtain a high-affinity IgG type monoclonal antibody. The labeled antibody and the capture antibody recognize specific antigenic epitopes of NMP22.

[0014] This invention also proposes a quantitative detection kit for urinary nuclear matrix protein 22 using quantum dot fluorescence immunochromatography, comprising quantum dot fluorescence immunochromatographic test strips and NMP22 recombinant antigen standard; The quantum dot fluorescent immunochromatographic test strip includes a PVC backing plate, and a sample pad, a binding pad carrying quantum dot-labeled antibodies, a nitrocellulose membrane coated with detection lines and control lines, and an absorbent pad that are sequentially overlapped and pasted on the PVC backing plate along the chromatography direction. The binding pad is immobilized with NMP22 monoclonal antibody labeled with quantum dot fluorescent microspheres; The nitrocellulose membrane is provided with a detection line and a control line in sequence along the chromatography direction. The detection line is coated with an NMP22 capture monoclonal antibody that recognizes the specific antigenic epitope of NMP22 with the labeled antibody. The control line is coated with goat anti-mouse IgG secondary antibody.

[0015] Preferably, the quantum dot fluorescent microspheres have a particle size of 85 nm or 125 nm; the quantum dot fluorescent microspheres are activated by EDC / NHS, conjugated with NMP22 monoclonal labeled antibody, and blocked by BSA.

[0016] Preferably, the sample pad and the conjugate pad are made of glass fiber and are soaked in a pretreatment solution and dried. The pretreatment solution is based on Tris-HCl buffer solution with pH 7.2 to 8.0 and contains less than 0.5% by mass of blocking protein, 0.5% to 1.5% of surfactant, less than 5% of sucrose or trehalose, polyethylene oxide (PEO), and preservatives. The detection line is coated with an antibody concentration of 0.5-1.5 mg / mL, and the coating solution is a 20 mM phosphate buffer solution with pH 7.2-7.4 containing 1%-3% sucrose; the nitrocellulose membrane has a creep rate of 90-150 s. The NMP22 recombinant antigen standard is an NMP22 recombinant protein solution with at least 5 concentration gradients, used to construct a standard curve for quantitative detection.

[0017] Preferably, the detection conditions of the kit are: chromatography reaction time 15 min, excitation wavelength 350 nm~400 nm, emission wavelength 615 nm, and quantification is achieved by detecting the T / C fluorescence ratio.

[0018] This invention addresses the problems of insufficient sensitivity, inaccurate quantification, high detection cost, and difficulty in achieving real-time detection in existing urine NMP22 detection technologies. It develops a quantum dot fluorescence immunochromatographic quantitative detection method and kit for urine nuclear matrix protein 22, with the following specific benefits: 1. This invention uses quantum dot fluorescent microspheres as antibody markers, leveraging the high-intensity fluorescence signal of quantum dots to enhance the detection signal-to-noise ratio. Simultaneously, it screens high-affinity monoclonal antibodies that recognize specific NMP22 antigenic epitopes to construct a double-antibody sandwich detection system, ensuring the specificity and stability of antigen-antibody binding. A quantitative standard curve is established using the T / C fluorescence ratio method, which can offset systematic errors caused by the chromatography process and sample matrix. Compared to traditional colloidal gold qualitative detection, this significantly improves detection sensitivity and result accuracy, providing reliable quantitative data support for the early auxiliary diagnosis and postoperative recurrence monitoring of bladder cancer.

[0019] 2. The detection method of this invention does not require complex sample pretreatment. Urine samples can be directly added for detection, and quantitative results can be obtained in 15 minutes. The detection only requires a small portable fluorescence immunoassay analyzer, without the need for large-scale fully automated detection equipment, which can realize point-of-care testing. It is suitable for rapid screening in urology clinics and can also meet the testing needs of primary medical institutions and health check-up centers. The individual test strip format can avoid the waste of batch reagents, which is especially suitable for application scenarios with small sample volumes and effectively controls the detection cost.

[0020] 3. This invention optimizes the entire immunochromatographic system based on the matrix characteristics of urine samples: by matching the quantum dot particle size with the nitrocellulose membrane pore size, the chromatography flow rate is controlled to ensure sufficient antigen-antibody reaction; the formulation of the sample pad and conjugate pad pretreatment solution is optimized, and by screening the types and concentrations of blocking proteins, surfactants and stabilizers, the non-specific binding of impurities in urine is effectively reduced, and false positive interference is reduced, ensuring both detection sensitivity and detection specificity.

[0021] 4. The reagent kit of this invention is independently developed from antibody preparation to chromatography system construction, without relying on imported reagents and equipment. Compared with imported colloidal gold reagents and chemiluminescence detection schemes, the reagent cost and supporting equipment cost of this kit are significantly reduced, which can not only reduce the burden of testing costs for patients, but also help reduce the pressure on medical insurance payments. At the same time, it can improve my country's independent research and development capabilities for urine biomarker detection reagents for bladder cancer and reduce dependence on imported products. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of an immunochromatographic test strip; Figure 2 The principle and flowchart of quantitative detection; Figure 3 Example graph of standard curve for quantitative detection; Figure 4 This is a schematic diagram of the process flow for preparing NMP22 monoclonal antibody; Figure 5 This is a schematic diagram of the antibody-coupled reaction process of quantum dot fluorescent microspheres. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and parameter optimization experiments. The scope of protection of the present invention is not limited to the following embodiments. All technical solutions implemented based on the core content of the present invention fall within the scope of protection of the present invention. Experimental operations in the following embodiments that do not specify specific conditions were performed in accordance with conventional experimental conditions in the art and the manufacturer's instructions.

[0024] Example 1, Preparation of NMP22 monoclonal antibody: This embodiment uses hybridoma technology to screen for high-affinity monoclonal antibodies that recognize specific antigenic epitopes of NMP22, which are used as labeling antibodies and capture antibodies, respectively. The preparation process is as follows: Figure 4 As shown, the specific steps are as follows: Experimental materials: 3 male Balb / c mice aged 6-8 weeks; 100 μg of recombinant NMP22 antigen expressed in Escherichia coli (molecular weight 48.7 kDa); water-soluble adjuvant; SP2 / 0 myeloma cell line; PEG1450; 50×HAT medium and 50×HT medium; hybridoma cell supplementation factor; fetal bovine serum; 100× penicillin antibody; blank 1640 medium; erythrocyte lysis buffer; protein A affinity chromatography column; BCA protein concentration assay kit.

[0025] Animal immunization: ① Dissolve 100 μg of NMP22 recombinant antigen in 250 μL of 10 mM, pH 7.4 phosphate buffer, aliquot into 5 tubes, 50 μL per tube, and store at -20°C. Do not repeatedly freeze and thaw.

[0026] ② Take one tube of antigen solution, add 50 μL of water-soluble adjuvant, and gently mix by pipetting (no emulsification required); use a 1 mL sterile syringe to inject the mixture subcutaneously into Balb / c mice at two points.

[0027] ③ A second booster immunization should be administered 2 weeks later, with the same immunization method and dosage as in step ②.

[0028] ④ Blood was collected from the tail tip of mice in the third week after immunization, and the OD value at 450 nm was measured by indirect ELISA to evaluate serum antibody titer.

[0029] ⑤ Three days before cell fusion, mice that met the titer target were given a tail vein pulse immunization, injecting an equal amount of NMP22 recombinant antigen.

[0030] SP2 / 0 myeloma cell culture: ① Place 25cm 2 Sterilize the culture flask with UV for 30 min, add 5 mL of 1640 complete culture medium preheated at 37℃; after rapidly thawing the SP2 / 0 cells, transfer them to the culture flask, shake well, and place them in a 37℃, 5% CO2 incubator for aeration. Passage the cells when the cell confluence reaches 70% or more.

[0031] ② Take 75cm 2 Sterilize the culture flasks with UV light for 30 min; remove the old culture medium, add 2 mL of 0.25% trypsin, and digest at 37°C for 5-10 min; add 5.5 mL of complete culture medium to stop digestion, disperse the cells by pipetting, and then passage at a ratio of 1:5 to 1:10 to 75 cm⁻¹ cells. 2 Continue culturing in the culture flask.

[0032] Cell fusion: ① Take immunized mice, collect blood from their eyeballs, euthanize them by cervical dislocation, disinfect them by soaking in 75% ethanol for 5 minutes, and transfer them to a biosafety cabinet; sterilize the surgical instruments and 200-mesh cell sieve by autoclaving and irradiate with ultraviolet light for 30 minutes in advance; dilute 10× red blood cell lysis buffer to 1× working solution for later use.

[0033] ② Place a 200-mesh cell sieve at the mouth of a 50 mL centrifuge tube and moisten it with blank 1640 medium.

[0034] ③ Remove the mouse spleen under aseptic conditions, grind it thoroughly on a sieve, rinse the sieve with 5 mL of blank 1640 culture medium, and collect the spleen cell suspension.

[0035] ④ Centrifuge at 1000 rpm for 10 min and discard the supernatant; gently tap the bottom of the tube to break up the precipitate, add 6 mL of red blood cell lysis working solution, and lyse at room temperature for 5 min; centrifuge again and discard the supernatant, resuspend and wash with blank culture medium to obtain purified spleen B cells.

[0036] ⑤ Count the number of spleen cells and logarithmic growth phase SP2 / 0 cells separately, mix them in a ratio of 5:1 to 10:1, centrifuge at 1000 rpm for 10 min, discard the supernatant, and gently tap the bottom of the tube to break up the cells.

[0037] ⑥ Add 1 mL of preheated PEG1450 at 37℃ dropwise over 1 min, while slowly rotating the centrifuge tube during the process; continue stirring slowly for 2 min after the addition is complete.

[0038] ⑦ Slowly add preheated blank 1640 medium to terminate the fusion: first add 1 mL (1 min), then add 3 mL (3 min), stirring while adding; finally add 10 mL of medium, mix well, and incubate in a water bath at 37°C for 5 min.

[0039] ⑧ Centrifuge and discard the supernatant. Resuspend the cells in 200 mL of HAT selective medium and dispense 200 μL into 10 96-well culture plates. Incubate the plates in an incubator and change half of the medium every 2-3 days.

[0040] ⑨ On the 8th day of culture, replace with HT medium: aspirate 100 μL of the old medium and add 120 μL of HT medium to each well to continue culture.

[0041] Single-clonal cell line screening: ① After 10 days of culture, monoclonal colonies were labeled under a microscope, and antibody secretion in the cell supernatant was detected by indirect ELISA on days 12-15.

[0042] ② Perform limiting dilution on antibody-positive wells, seed 0.5 cells per well into 96-well plates, and continue culturing.

[0043] ③ After culturing for about 5 days, select wells containing only a single colony to retest the antibody activity in the supernatant; if the positive rate of single colony wells reaches 100%, it is determined to be a stable monoclonal cell line; if it does not meet the standard, select the well with the highest positive value to repeat the subcloning until a 100% positive monoclonal cell line is obtained.

[0044] ④ Screen multiple positive cell lines that recognize different epitopes of NMP22, expand their culture, and then freeze them in liquid nitrogen for preservation.

[0045] Ascites preparation and antibody purification: ① Eight-week-old female Balb / c mice were selected and each mouse was sensitized for 15 days by intraperitoneal injection of 0.5 mL of ascites-specific adjuvant.

[0046] ② Wash hybridoma cells three times with 1×PBS, resuspend in physiological saline and adjust the concentration to 2×10⁻⁶. 6 0.5 mL was injected intraperitoneally into each mouse.

[0047] ③ About 7 days after inoculation, when the mice’s abdomens are significantly swollen and their movement is restricted, they are euthanized by cervical dislocation, and ascites is aseptically extracted.

[0048] ④ Centrifuge the ascites fluid at 3000 rpm for 10 min, take the clear intermediate layer, and purify it by protein A affinity chromatography to obtain monoclonal antibodies; determine the protein concentration by BCA method, verify the purity ≥95% by SDS-PAGE, and screen high affinity IgG type antibodies as labeling antibodies and capture antibodies, respectively.

[0049] Example 2, Preparation of quantum dot-labeled NMP22 monoclonal antibody: This embodiment uses the EDC / NHS activation method to prepare quantum dot-antibody conjugates. The coupling reaction process is as follows: Figure 5 As shown, the specific steps are as follows: Reagent preparation: 2.5 mg / mL quantum dot fluorescent microspheres (85 nm or 125 nm particle size); 100 mM MES buffer (pH 6.0); EDC and NHS prepared into 10 mg / mL working solutions using pH 6.0 MES buffer, freshly prepared and used immediately; 20 mMMBS buffer (pH 7.4~8.0); 10% BSA solution; purified NMP22 monoclonal labeled antibody.

[0050] Microsphere activation: Take 50 μL of quantum dot microspheres, add 50 μL of 100 mL MME S buffer (pH 6.0) and mix well; add 4 μL of LEDC working solution and 4 μL of NHS working solution in sequence, vortex quickly to mix well, and react at room temperature for 30 min to complete the activation of carboxyl groups on the surface of microspheres.

[0051] Antibody conjugation and blocking: ① After activation, centrifuge at 4℃ and 20000rpm for 10min, discard the supernatant; add 100μL of 20mMBS buffer to resuspend, repeat centrifugation and washing once to remove residual activator.

[0052] ② Add 100 μL BS buffer and sonicate to resuspend the microspheres; add 10~20 μg NMP22 monoclonal labeled antibody, mix well and shake at room temperature for 120 min to achieve covalent coupling of antibody and microspheres.

[0053] ③ Add 10 μL of 10% BSA solution to make the final BSA concentration 1%, and block at room temperature with shaking for 60 min to block the remaining active sites and reduce non-specific binding.

[0054] Purification and preservation: After blocking, centrifuge and discard the supernatant, wash twice with 20 mMBS buffer; finally, resuspend in 50 μL BS buffer to obtain a quantum dot labeled antibody working solution with a concentration of about 2.5 mg / mL, and store at 4°C in the dark for later use.

[0055] Note: The blocking process can be combined with small molecule blocking agents such as ethanolamine and glycine, or PEG and PVP can be added to optimize the microsphere dispersibility; the coupling reaction can be optimized in the pH range of 6.0~8.0 to obtain the best coupling efficiency.

[0056] Example 3, Preparation of quantum dot fluorescent immunochromatographic test strips: Sample pad and conjugate pad pretreatment: ① Preparation of pretreatment solution: Using Tris-HCl buffer solution with pH 7.2~8.0 as the base, add blocking protein, surfactant, stabilizer, polyethylene oxide (PEO) and preservative; the basic formula is: pH 7.6 Tris-HCl, 0.3% BSA, 1.0% Tween-20, 2% sucrose, appropriate amount of PEO, 0.05% PC-300 preservative.

[0057] ② Take a glass fiber sample pad and a bonding pad, immerse them completely in the pretreatment solution for 30 minutes; remove and drain, then dry overnight at 37°C, and store in a sealed, dry container.

[0058] Conjugate pad coating: Take quantum dot labeled antibody working solution, add bromocresol purple indicator, and use a gold sprayer to uniformly spray onto the pretreated conjugate pad at a spray volume of 2~5μg / cm; dry at 37℃ overnight, and store in a sealed, dry place.

[0059] NC membrane coating: ① Select a nitrocellulose membrane (NC membrane) with a crawling speed of 90~150s, and place it in an environment with a relative humidity of 45%~65% for 30 minutes before scribing.

[0060] ② Dilute the NMP22 capture antibody to 0.5-1.5 mg / mL using 20 mM phosphate buffer (pH 7.2-7.4) containing 1%-3% sucrose as the coating solution for the detection line; at the same time, prepare the coating solution for the quality control line (goat anti-mouse IgG secondary antibody).

[0061] ③ Use an automatic film coating machine to spray the test lines and quality control lines on the NC film at intervals, dry at 37°C overnight, and store in a sealed, dry place.

[0062] Test strip assembly: The overall structure of the immunochromatographic test strip is as follows Figure 1 As shown, the sample pad, the binding pad carrying quantum dot labeled antibody, the nitrocellulose membrane coated with detection lines and control lines, and the absorbent pad are sequentially overlapped and pasted on the PVC backing plate along the chromatography direction. Each layer partially overlaps to ensure smooth liquid flow. After assembly, the sample pad is cut into 3-4 mm wide test strips, which are then placed in a cartridge, sealed, and dried for storage.

[0063] Key system parameter gradient optimization: To balance detection sensitivity and specificity and reduce non-specific interference from the urine matrix, this invention optimizes and screens multiple sets of gradients for key parameters such as sample pad / conjugate pad treatment solution formulation, antibody coating concentration, NC membrane climbing rate, and quantum dot particle size. The specific parameter gradients are shown in the table below: Table 1. Optimization gradient of key parameters for the immunochromatographic system

[0064] Example 4: Establishment of a quantitative standard curve: 1. Dilute the NMP22 recombinant antigen with a negative urine matrix to create standard solutions with 6-8 concentration gradients, covering the linear range of clinical testing.

[0065] 2. Add 80-100 μL of each concentration standard solution to the sample well of the test strip and react at room temperature for 15 min; use a fluorescence immunoassay analyzer to detect the fluorescence, with an excitation wavelength of 350-400 nm and an emission wavelength of 615 nm. Read the fluorescence intensity T of the detection line and the fluorescence intensity C of the control line, and calculate the T / C ratio.

[0066] 3. A standard curve was established using a four-parameter fitting method, with NMP22 antigen concentration as the x-axis and the T / C ratio as the y-axis. The regression equation and correlation coefficient were then obtained for subsequent quantitative calculations. A typical quantitative standard curve is shown below. Figure 3 As shown, the regression equation and correlation coefficient are obtained, which are used for subsequent quantitative calculations of the sample.

[0067] Example 5, Quantitative detection of urine samples: This embodiment utilizes the principle of double-antibody sandwich immunochromatography to achieve quantitative detection of NMP22 in urine samples. The overall detection principle and process are as follows: Figure 2 As shown, the specific operation steps are as follows: 1. Collect fresh urine samples from the subjects for direct testing; if the sample is turbid, centrifuge at 3000 rpm for 5 minutes and collect the supernatant.

[0068] 2. Remove the test strip, add 80~100μL of the sample to be tested, and start the instrument to start the automatic timing; insert the instrument into the fluorescence immunoassay analyzer to read the T / C ratio and obtain the concentration of NMP22 in the urine.

[0069] Example 6, Reagent kit performance verification: Limit of detection determination: Repeat the testing with at least 20 NMP22-negative urine samples, and calculate the mean and standard deviation of the blank values; calculate the blank limit (LOB) using the following formula:

[0070] In the formula, The mean of the blank sample test results is given, and the standard deviation of the blank sample test results is given by SDK. The actual detection limit of the method is determined by combining the test results of low concentration samples.

[0071] Precision verification: Three quality control samples with high, medium and low concentrations were selected, and the samples were repeatedly tested 10 times within the batch and continuously tested for 3 days between batches. The coefficient of variation was calculated. The results showed that the CV of both the intra-batch and inter-batch samples was less than 10%, indicating good detection precision.

[0072] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A quantum dot fluorescence immunochromatographic quantitative detection method for urinary nuclear matrix protein 22, characterized in that, Includes the following steps: (1) Preparation of quantum dot labeled antibody: Quantum dot fluorescent microspheres were activated by EDC / NHS activation method, and the activated quantum dot fluorescent microspheres were coupled with NMP22 monoclonal labeled antibody. After blocking, washing and purification, quantum dot labeled NMP22 monoclonal antibody was obtained. (2) Preparation of immunochromatographic test strips: The quantum dot fluorescently labeled NMP22 monoclonal antibody was pre-sprayed and cured on the conjugate pad; the detection line and control line were pre-sprayed and dried on the nitrocellulose membrane; a PVC backing plate was taken and the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were sequentially overlapped and pasted along the chromatography direction; the strips were cut and assembled into the cartridge; the quantum dot labeled NMP22 monoclonal antibody was pre-sprayed and cured on the conjugate pad; the detection line and control line were etched and dried on the nitrocellulose membrane, the detection line was coated with NMP22 capture monoclonal antibody that recognizes specific antigenic epitopes of NMP22, and the control line was coated with goat anti-mouse IgG secondary antibody; the strips were dried and ready for use. (3) Establishing a quantitative standard curve: Prepare a series of NMP22 recombinant antigen standard solutions with different concentration gradients, add them to the sample application end of the test strip for chromatographic reaction, and detect the fluorescence intensity value T of the detection line and the fluorescence intensity value C of the control line using a fluorescence immunoassay analyzer. Fit the NMP22 antigen concentration as the abscissa and the T / C ratio as the ordinate to obtain the quantitative standard curve. (4) Quantitative detection of urine sample: Take the urine sample to be tested and add it to the sample application end of the test strip. After the chromatographic reaction, detect the T / C ratio and substitute it into the quantitative standard curve to calculate the concentration of nuclear matrix protein 22 in the urine sample.

2. The quantum dot fluorescence immunochromatographic quantitative detection method for urinary nuclear matrix protein 22 according to claim 1, characterized in that, Step (1) specifically includes: Take 2.5 mg / mL quantum dot fluorescent microspheres, add 100 mM MES buffer (pH 6.0) and mix well; add EDC working solution and NHS working solution sequentially, vortex quickly, and react at room temperature for 30 min to complete activation; centrifuge at 20,000 rpm for 10 min at 4 °C, discard the supernatant, resuspend and wash in 20 mM BS buffer (pH 7.4–8.0), repeat centrifugation and washing, and sonicate to resuspend the microspheres; add 10–20 μg NMP22 monoclonal antibody, mix well, and react at 120 min to complete coupling; add BSA to a final concentration of 1%, and block at room temperature for 60 min; centrifuge, discard the supernatant, wash repeatedly with BS buffer, and finally resuspend and store at 4 °C for later use; Both the EDC working solution and the NHS working solution were prepared with MES buffer at pH 6.0 and a concentration of 10 mg / mL, and were prepared and used immediately. The quantum dot fluorescent microspheres had a particle size of 85 nm or 125 nm.

3. The quantum dot fluorescence immunochromatographic quantitative detection method for urinary nuclear matrix protein 22 according to claim 1, characterized in that, In step (2), both the sample pad and the conjugate pad are made of glass fiber. Before use, they are soaked in the pretreatment solution for 30 minutes and then dried overnight at 37°C. The pretreatment solution is based on Tris-HCl buffer solution with pH 7.2~8.0 and contains blocking protein, surfactant, stabilizer, polyethylene oxide (PEO) and preservative. In the pretreatment solution, the blocking protein is BSA or casein with a mass fraction of less than 0.5%; the surfactant is Tween-20 or S9, wherein the mass fraction of Tween-20 is 0.5%~1.5% and the mass fraction of S9 is 0.5%~1%; the stabilizer is trehalose or sucrose with a mass fraction of less than 5%; and the preservative is PC-300 or Junqing preservative.

4. The quantum dot fluorescence immunochromatographic quantitative detection method for urinary nuclear matrix protein 22 according to claim 1, characterized in that, In step (2), the amount of quantum dot labeled antibody sprayed on the conjugate pad is 2~5 μg / cm; the coating solution of the detection line is 20mM phosphate buffer with pH 7.2~7.4 containing 1%~3% sucrose, and the concentration of the coated NMP22 capture monoclonal antibody is not higher than 1.5mg / mL; after spraying, it is dried at 37℃ overnight; The nitrocellulose membrane in step (2) has a creeping speed of 90~150s; before scribing and spraying, the nitrocellulose membrane is placed in an environment with a relative humidity of 45%~65% to equilibrate.

5. The quantum dot fluorescence immunochromatographic quantitative detection method for urinary nuclear matrix protein 22 according to claim 1, characterized in that, In steps (3) and (4), the chromatography reaction time is 15 min; the excitation wavelength of the fluorescence immunoassay analyzer is 350 nm to 400 nm, and the emission detection wavelength is 615 nm.

6. The quantum dot fluorescence immunochromatographic quantitative detection method for urinary nuclear matrix protein 22 according to claim 1, characterized in that, The NMP22 monoclonal labeled antibody and NMP22 capture monoclonal antibody were prepared using hybridoma technology. Specifically, Balb / c mice were immunized with the NMP22 recombinant antigen. Spleen cells from the immunized mice were fused with SP2 / 0 myeloma cells using PEG1450. The fused cells were screened using HAT medium and subcloned using the limiting dilution method to obtain a stable antibody-secreting hybridoma cell line. The hybridoma cell line was inoculated into the peritoneal cavity of Balb / c mice to prepare ascites fluid. The ascites fluid was purified by protein A chromatography to obtain a high-affinity IgG type monoclonal antibody. The labeled antibody and the capture antibody recognize specific antigenic epitopes of NMP22.

7. A quantitative detection kit for urinary nuclear matrix protein 22 using quantum dot fluorescence immunochromatography, characterized in that, Including quantum dot fluorescent immunochromatographic test strips and NMP22 recombinant antigen standards; The quantum dot fluorescent immunochromatographic test strip includes a PVC backing plate, and a sample pad, a binding pad carrying quantum dot-labeled antibodies, a nitrocellulose membrane coated with detection lines and control lines, and an absorbent pad that are sequentially overlapped and pasted on the PVC backing plate along the chromatography direction. The binding pad is immobilized with NMP22 monoclonal antibody labeled with quantum dot fluorescent microspheres; The nitrocellulose membrane is provided with a detection line and a control line in sequence along the chromatography direction. The detection line is coated with an NMP22 capture monoclonal antibody that recognizes the specific antigenic epitope of NMP22 with the labeled antibody. The control line is coated with goat anti-mouse IgG secondary antibody.

8. The detection kit according to claim 7, characterized in that, The quantum dot fluorescent microspheres have a particle size of 85 nm or 125 nm; after activation by EDC / NHS, the quantum dot fluorescent microspheres are conjugated with NMP22 monoclonal labeled antibody and blocked by BSA.

9. The detection kit according to claim 7, characterized in that, The sample pad and conjugate pad are made of glass fiber and are soaked and dried in a pretreatment solution. The pretreatment solution is based on Tris-HCl buffer solution with pH 7.2~8.0 and contains less than 0.5% by mass of blocking protein, 0.5%~1.5% of surfactant, less than 5% of sucrose or trehalose, polyethylene oxide (PEO) and preservatives. The detection line is coated with an antibody concentration of 0.5-1.5 mg / mL, and the coating solution is a 20 mM phosphate buffer solution with pH 7.2-7.4 containing 1%-3% sucrose; the nitrocellulose membrane has a creep rate of 90-150 s. The NMP22 recombinant antigen standard is an NMP22 recombinant protein solution with at least 5 concentration gradients, used to construct a standard curve for quantitative detection.

10. The detection kit according to claim 7, characterized in that, The detection conditions of the kit are: chromatography reaction time 15 min, excitation wavelength 350 nm~400 nm, emission wavelength 615 nm, and quantification is achieved by detecting the T / C fluorescence ratio.

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