Immunochromatographic test strip and kit for rapid detection of human urine nuclear matrix protein 22

Abstract

The invention discloses an immunochromatographic test strip and a kit for rapid detection of human urine nuclear matrix protein 22, the test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad connected in sequence, a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane, the detection line is coated with a blocker-treated NMP22 antibody, the quality control line is coated with a blocker-treated DNP antigen, the blocker includes 1% anti-heterophilic antibody agent, 0.2mg / mL anti-rheumatoid factor agent, 1% anti-HAMA agent, 0.02mol / L PBS, 5% sucrose and 0.1% PC300, and the above percentages are mass percentages. The immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 of the present invention has a fast detection speed, and the test result is obtained in 1-15min, without the need for an instrument, simple operation, low price, and is suitable for primary medical institutions, and is suitable for popularization and self-testing.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to an immunochromatographic test strip and a kit for rapidly detecting human urine nuclear matrix protein 22. Background Art

[0002] NuMA is a protein that makes up a large proportion of nuclear matrix proteins (NMPs) and is released from cells to detectable levels as a result of cell death (e.g., apoptosis). NuMA released from bladder tumor cells in urine is detected using a monoclonal antibody that recognizes a specific region of NuMA. The antigenic portion of NuMA detected is called NMP22 (nuclear matrix protein 22).

[0003] Chinese patent application CN202310325363.1 discloses a method for detecting the nuclear matrix protein Nmp22, a bladder cancer marker. The method uses immunocytochemical staining, uses NMP22 rabbit anti-human monoclonal antibody to bind to bladder exfoliated cell antigens in urine, and then adds the corresponding primary antibody labeled with horseradish peroxidase (HRP) sheep anti-mouse to bind, and then adds a color developing substrate based on HRP-mediated catalysis. Abnormal cells will be stained brown-yellow, and then counterstained with hematoxylin staining solution. The nuclei of normal cells appear blue, and the cytoplasm is not stained. Aqueous mounting medium or neutral resin mounting medium is used, and the cell staining results are evaluated under an optical microscope.

[0004] Chinese patent application CN202110746113.6 discloses markers and a detection kit for bladder cancer detection by jointly detecting nuclear matrix protein 22 (NMP22), carcinoembryonic antigen (CEA), bladder tumor antigen (BTA) and cytokeratin 19 fragment (CYFRA21-1).

[0005] Chinese patent application CN202010319759.1 discloses an immunochromatographic kit for detecting MCM5 and NMP22 in urine for the diagnosis of bladder cancer.

[0006] Chinese patent application CN201410733177.2 discloses a preparation method and application of a bladder cancer marker - NMP22 immunosensor constructed based on carbon nanotubes / PdPt nanocages, and the detection limit of the bladder cancer marker - NMP22 can reach 0.21pg / mL.

[0007] Chinese patent application CN201510793361.0 discloses a preparation method and application of a three-antibody dual-channel immunosensor for simultaneous detection of human nuclear matrix protein and carcinoembryonic antigen. The detection limit of this method for NMP22 is 12 pg / ml.

[0008] Chinese patent application CN201410574169.8 discloses an immunochromatographic test paper for rapid detection of bladder cancer using anti-nuclear matrix protein 22 antibodies and a preparation method thereof. This method can only detect samples with a content greater than 10U / ml. For samples with a content less than 10U / ml, there will be a certain amount of missed detection and it cannot meet the needs of all patients.

[0009] The NMP22 detection methods disclosed in the above patent documents do not provide corresponding clinical sensitivity and specificity data, and their clinical applications are uncertain; moreover, the operation is cumbersome, requires instrument assistance, and is costly.

[0010] Chinese patent application CN201610126380.2 discloses molecular markers for non-invasive diagnosis of bladder cancer, and specifically discloses that the detection sensitivity in this scheme is 77% and the specificity is 76%.

[0011] Currently, there is Abbott's colloidal gold detection method for urine nuclear matrix protein 22 on the market, which can only detect samples greater than 5U / ml. It is also unable to detect positive results for patients with samples below 5U / ml. The overall sensitivity of Abbott's product is 69.6%, and the specificity is 84.9%, so there is a certain amount of missed detections and false detections. Summary of the invention

[0012] In view of the above-mentioned defects of the prior art, the present invention provides an immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22, which improves the specificity of detection by coating the NMP22 antibody treated with a blocking agent on the detection line, and the clinical specificity is as high as 95%. To this end, the present invention also provides an immunochromatographic kit for rapid detection of human urine nuclear matrix protein 22.

[0013] The first aspect of the present invention provides an immunochromatographic test strip for rapid detection of human urinary nuclear matrix protein 22, the test strip comprising a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad connected in sequence, the nitrocellulose membrane being provided with a detection line and a quality control line in sequence, the detection line being coated with a NMP22 antibody treated with a blocker, the quality control line being coated with a DNP antigen treated with a blocker, the blocker comprising 0.1%-2% of a heterophilic antibody blocker, 0.05mg / mL-1mg / mL of an anti-rheumatoid factor, 0.1%-2% of an anti-HAMA agent, 0.01mol / L-0.2mol / L of a buffer, 1%-5% of a protective agent and 0.05%-0.1% of a biological preservative, the above percentages being percentages by mass.

[0014] As a preferred embodiment, the conjugate pad is coated with latex microsphere-labeled NMP22 antibody and latex microsphere-labeled anti-DNP antibody, and the molar ratio of latex microsphere-labeled NMP22 antibody to latex microsphere-labeled anti-DNP antibody is 1:(0.1-1). Preferably, it is 1:0.2.

[0015] As a preferred embodiment, the method of labeling antibodies with latex microspheres is as follows:

[0016] S1. Add the antibody to the coupling solution to pretreat the antibody;

[0017] S2, adding the latex microspheres into an activator to activate the latex microspheres;

[0018] S3. Add the pretreated antibody to S2 and mix well to obtain the latex microsphere-labeled antibody.

[0019] The coupling solution is MES, PBS or boric acid solution, with a concentration of 0.05 mol / L and a pH of 6.5, 7.0 or 7.4. In order to optimize the coupling effect, DTT is also added in step S1.

[0020] As a preferred embodiment, the activators are EDC and NHS, and the microspheres are activated by EDC and NHS, and the molar ratio of EDC, NHS to microspheres is 1-100, preferably 20-60. In order to optimize the activation effect, when using EDC and NHS to activate the microspheres, MES solution is also added to enhance the activation effect.

[0021] As a more preferred embodiment, the method of labeling antibodies with latex microspheres is as follows:

[0022] S1. Take 0.2 mg of antibody, dilute it to 1 mg / mL with PBS coupling solution of pH=7.4, take 2 μL of DTT and add it to the antibody, react at room temperature for 2 hours; ultrafilter to collect the antibody; use the above coupling solution to dilute the antibody to 0.06 mg / mL, add 10 mg / ml NHS, and react at room temperature for 30 minutes;

[0023] S2, take 12.5 μL of latex microspheres with a solid content of 4%, add 1 mL of MES solution with a pH of 6 and mix well;

[0024] S3, prepare 10 mg / mL EDC, add 20 μL, mix well, centrifuge and discard the supernatant;

[0025] S4. Add 60 μg of the antibody treated in step S1 and mix well.

[0026] As a preferred embodiment, the NMP22 antibody is a NMP22 monoclonal antibody or a NMP22 polyclonal antibody, preferably a NMP22 monoclonal antibody.

[0027] As a preferred embodiment, a bottom plate is provided at the bottom end of the test strip.

[0028] The sample pad, the binding pad, the nitrocellulose membrane and the water absorption pad are arranged in sequence on the bottom plate.

[0029] As a preferred embodiment, the bottom plate is a PVC board or a PVP board.

[0030] As a preferred embodiment, the sample pad is a glass cellulose membrane or a polyester cellulose membrane.

[0031] As a more preferred embodiment, the sample pad is a glass cellulose membrane.

[0032] As a preferred embodiment, the sample pad is treated by immersion or ultrasound, preferably ultrasound.

[0033] As a preferred embodiment, the treatment solution used to treat the sample pad is selected from phosphate buffer, borate buffer or tris buffer.

[0034] As a more preferred embodiment, the concentration of the treatment solution used to treat the sample pad is 0.01-0.1 mol / L, and more preferably, 0.05-0.1 mol / L.

[0035] As a preferred embodiment, a marker line is provided on one side of the nitrocellulose membrane close to the conjugate pad.

[0036] As a preferred embodiment, the distance between the marker line and the binding pad is 1.5 mm.

[0037] As a preferred embodiment, the marker line is coated with a mixture of NMP22 antibody labeled with latex microspheres and anti-DNP antibody labeled with latex microspheres.

[0038] As a more preferred embodiment, the molar ratio of the latex microsphere-labeled NMP22 antibody to the latex microsphere-labeled anti-DNP antibody is 1:(0.1-1).

[0039] As a more preferred embodiment, the molar ratio of the latex microsphere-labeled NMP22 antibody to the latex microsphere-labeled anti-DNP antibody is 1:0.2.

[0040] As a preferred embodiment, the heterophilic antibody blocker is selected from HBR1, HBR2, HBR3, HBR5, HBR6, HBR9, HBR10, HBR11, 3BX484, 3KC535, 3KC701, 3KC542, 3KC564 or 3KC565 produced by Shenzhen Yawei Century Technology Co., Ltd., the anti-rheumatoid factor is MK33 produced by Roche Diagnostics, the anti-HAMA agent is selected from C8001, C8005, C8003 or C8004 produced by Nanjing Jingda Biotechnology Co., Ltd., the buffer is selected from any one of PBS buffer, tris buffer, and borate buffer; the protective agent is selected from sucrose or trehalose; the biological preservative is selected from any one of proclin300, proclin950, proclin150, and proclin200.

[0041] The second aspect of the present invention provides an immunochromatographic kit for rapid detection of human urinary nuclear matrix protein 22, comprising the above-mentioned test strip.

[0042] As a preferred embodiment, the kit further comprises a card case for holding the test strip. The card case comprises a bottom groove, an upper cover and an observation window, the test strip is located in the bottom groove, the upper cover is located above the bottom groove, the upper cover is provided with a sample addition hole for adding a sample to the sample pad, and the observation window is provided on the upper cover for observing the strips on the detection line and the quality control line.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) The immunochromatographic kit for rapid detection of human urine nuclear matrix protein 22 of the present invention has a fast detection speed, and the detection result can be obtained in 1-15 minutes. It does not require any instrument, is simple to operate, and is low in price. It is suitable for primary medical institutions and is suitable for popularization and self-testing.

[0045] (2) The immunochromatographic kit for rapid detection of human urinary nuclear matrix protein 22 of the present invention can improve the patient's professional requirements for instrument operation, personnel, environment, etc., as well as the defect of bladder cancer patients' dependence on cystoscopy, and can accelerate the screening of suspected cases, quickly confirm the diagnosis and treatment, and effectively reduce the patient's pain and medical costs.

[0046] (3) The immunochromatographic kit for rapid detection of human urine nuclear matrix protein 22 of the present invention has a detection sensitivity of 90% and a specificity of 95%.

[0047] (4) The immunochromatographic kit for rapid detection of human urinary nuclear matrix protein 22 of the present invention has a marker line disposed on the side of the nitrocellulose membrane close to the binding pad, thereby extending the shelf life of the kit to 5 years.

[0048] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the structure of the immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 of the present invention.

[0050] Wherein: sample pad 1, conjugate pad 2, detection line 3, quality control line 4, absorbent pad 5, bottom plate 6, nitrocellulose membrane 7, marker line 8. DETAILED DESCRIPTION

[0051] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific illustrations. However, the invention is not limited to the following implementation cases.

[0052] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0053] The reagent names are listed below:

[0054] Anti-HAMA Agents: Human Anti-Mouse Antibodies

[0055] MES: MES

[0056] EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0057] NHS: N-hydroxysuccinimide

[0058] NMP22: Human urinary nuclear matrix protein 22

[0059] DNP antigen: nucleoprotein antigen

[0060] Anti-DNP antibody: Anti-nucleoprotein antibody

[0061] DTT: dithiothreitol

[0062] PVC: Polyvinyl chloride

[0063] PVP: Polyvinylpyrrolidone

[0064] BSA: bovine serum albumin

[0065] PB or PBS: Phosphate buffered saline

[0066] tris: Tris-HCl buffer

[0067] The PC300 mentioned below refers to proclin300.

[0068] The percentages referred to below are by mass.

[0069] Reagents:

[0070] 1. Activation solution: MES aqueous solution, concentration is 0.05 mol / L, pH values ​​are 6.0, 6.5, 7.0 respectively.

[0071] 2. Coupling solution

[0072] Table 1

[0073]

[0074] 3. Preservative solution

[0075] Table 2

[0076]

[0077] 4. Coating fluid

[0078] Table 3

[0079]

[0080] 5. Combined pad treatment solution

[0081] Table 4

[0082]

[0083] 6. Sample pad treatment solution

[0084] Table 5

[0085]

[0086] 7. Blocking solution: includes 0.05 mol / L boric acid and 1% BSA, pH 7.4.

[0087] 8. Blockers

[0088] The blocking agent involved in the present application is an improvement on the basis of conventional coating solution, which is composed of 0.02 mol / L PBS, 5% sucrose or trehalose and 0.1% proclin300, and the above percentages are all mass percentages.

[0089] The blocker of the present application includes 0.1%-2% heterophilic antibody blocker, 0.05mg / mL-1mg / mL anti-rheumatoid factor, 0.1%-2% anti-HAMA agent, 0.01mol / L-0.2mol / L buffer, 1%-5% protective agent and 0.05%-0.1% biological preservative, and the above percentages are mass percentages.

[0090] Heterophilic antibody blocker: used to eliminate heterophilic interference in human samples. Heterophilic antibodies in human samples can cause false positive or false negative interference in immunoassays. The interference of heterophilic antibodies is eliminated by adding heterophilic antibody blockers. Commonly used heterophilic antibody blockers can be selected from the models HBR1, HBR2, HBR3, HBR5, HBR6, HBR9, HBR10, HBR11, 3BX484, 3KC535, 3KC701, 3KC542, 3KC564, and 3KC565 produced by Shenzhen Yawei Century Technology Co., Ltd. The above optional heterophilic antibody blockers have been proven to have the same effect in this application and are not limited by model when used.

[0091] The anti-rheumatoid factor used was MK33 produced by Roche Diagnostics, with the product number: 11939661103.

[0092] The anti-HAMA agent can be mouse serum, rabbit serum or sheep serum, and can be selected from the product numbers C8001, C8005, C8003 or C8004 produced by Nanjing Jingda Biotechnology Co., Ltd.

[0093] Buffer: Use any one of PBS, Tris-HCl buffer, or borate buffer.

[0094] Protective agent: Sucrose or trehalose can be used.

[0095] Biological preservative: Choose any one of proclin300, proclin950, proclin150, proclin200.

[0096] The blockers obtained by any combination of the above components can be used in this application. For example, the various types of heterophilic antibody blockers can be replaced without affecting the final performance. For the convenience of description, the blockers in Table 6 are used as examples for subsequent experiments.

[0097] Table 6

[0098]

[0099] Example 1

[0100] like Figure 1The invention discloses an immunochromatographic test strip for rapid detection of human urinary nuclear matrix protein 22, comprising a sample pad 1, a conjugation pad 2, a nitrocellulose membrane 7 and a water absorbent pad 5 which are connected in sequence, and a detection line 3 and a quality control line 4 are arranged on the nitrocellulose membrane 7 in sequence, wherein the detection line 3 is located on a side close to the conjugation pad 2, and the quality control line 4 is located on a side close to the water absorbent pad 5.

[0101] A bottom plate 6 is arranged at the bottom of the test strip. The bottom plate 6 is a PVC plate or a PVP plate. The sample pad 1, the binding pad 2, the nitrocellulose membrane 7 and the water absorbent pad 5 are arranged on the bottom plate 6 in sequence.

[0102] The sample pad 1 is a glass cellulose membrane or a polyester cellulose membrane, preferably a glass cellulose membrane. The sample pad 1 is treated by immersion or ultrasound, preferably ultrasound. The treatment solution for treating the sample pad 1 is selected from phosphate buffer, borate buffer or Tris-HCl buffer, and its concentration is 0.01-0.1 mol / L, preferably 0.05-0.1 mol / L. The sample pad 1 is soaked in the treatment solution for 6 hours, and then dried at 35°C for 12 hours. Use a strip cutter to cut into 10 mm sample pads 1 for later use.

[0103] The conjugate pad 2 is a glass cellulose membrane or a polyester cellulose membrane, preferably a polyester cellulose membrane. The conjugate pad 2 is treated by immersion or ultrasound, preferably ultrasound. The treatment solution for treating the conjugate pad 2 is selected from phosphate buffer, borate buffer or Tris-HCl buffer, and its concentration is 0.01-0.1 mol / L, preferably 0.05-0.1 mol / L. Soak the conjugate pad 2 in the treatment solution for 6 hours, and then dry it at 35°C for 12 hours. Use a strip cutter to cut the conjugate pad 2 into 10 mm pieces for later use.

[0104] The conjugate pad 2 is coated with latex microsphere-labeled NMP22 antibody and latex microsphere-labeled anti-DNP antibody, and the molar ratio of the latex microsphere-labeled NMP22 antibody and the latex microsphere-labeled anti-DNP antibody is 1:(0.1-1).

[0105] The latex microspheres are colored latex microspheres. Preferably, the surface groups of the colored latex microspheres are selected from any one of -NH2, -OH, -COOH, -FITC, -NHS and -Tosyl. The particle size of the colored latex microspheres is 80nm-400nm, preferably 100-300nm.

[0106] When labeling antibodies with latex microspheres, it is usually necessary to add an activator and a coupling agent to the system of latex microspheres and antibodies. Common activators are EDC and NHS. The molar ratio of EDC, NHS to microspheres is 1-100, preferably 20-60.

[0107] Commonly used coupling agents are MES, PBS or boric acid solution. There is a strict order of adding the activator and the coupling agent. The activator is added first to activate the latex microspheres, and then the coupling agent is added to couple the latex microspheres with the antibody. In order to enhance the activation effect, when the activator activates the latex microspheres, an activation buffer, such as MES buffer, needs to be added.

[0108] The inventor of the present application discovered in an accidental experiment that changing the order of adding the activator and the coupling agent, that is, first adding the coupling agent to pretreat the antibody and then adding the pretreated antibody to the latex microspheres activated by the activator, can improve the detection sensitivity.

[0109] A test line 3 (T line) and a quality control line 4 (C line) are provided on the nitrocellulose membrane 7. The test line 3 is coated with NMP22 antibody, and the quality control line 4 is coated with DNP antigen. The NMP22 antibody coated on the test line 3 is usually the NMP22 antibody treated with a coating solution, that is, the NMP22 antibody is diluted to a preset concentration using a coating solution. Commonly used coating solutions include PBS solution or citric acid solution, and the optional pH values ​​are 6.5, 7.0, and 7.4, respectively. Similarly, the quality control line 4 is also treated in the same way using a coating solution. The NMP22 antibody and DNP antigen treated with the coating solution are stenciled on the nitrocellulose membrane 7 using a film-stenciling gold spraying instrument, and then dried for 48 hours at 37°C and a humidity of <30%, to obtain a test line 3 and a quality control line 4 that meet the experimental requirements.

[0110] The present applicant discovered in experimental exploration that the detection line 3 (T line) and the quality control line 4 (C line) treated with conventional coating solution had poor specificity. The inventor of the present application creatively introduced a blocker and coated the NMP22 antibody and DNP antigen with the blocker, thereby significantly improving the specificity.

[0111] The NMP22 antibody is a NMP22 monoclonal antibody or a NMP22 polyclonal antibody, preferably a NMP22 monoclonal antibody.

[0112] An immunochromatographic kit for rapid detection of human urine nuclear matrix protein 22 includes, in addition to the above-mentioned test strip, a card case for holding the test strip. The card case includes a bottom groove, an upper cover and an observation window, the test strip is located in the bottom groove, the upper cover is located above the bottom groove, a sample addition hole for adding a sample to a sample pad 1 is provided on the upper cover, and an observation window is provided above the upper cover for observing the strips on the detection line 3 and the quality control line 4.

[0113] A method for preparing an immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 comprises the following steps:

[0114] 1) Preparation of nitrocellulose membrane 7: NMP22 antibody and DNP antigen are coated on nitrocellulose membrane respectively and dried for later use;

[0115] 2) Preparation of conjugate pad 2: mixing the NMP22 monoclonal antibody labeled with latex microspheres and the anti-DNP antibody labeled with latex microspheres, spraying the mixture on the glass cellulose membrane, and drying the mixture for later use;

[0116] 3) Assembling the test strip: bonding the nitrocellulose membrane 7 to the bottom plate 6, overlapping the absorbent pad 5 at one end of the quality control line 4 close to the nitrocellulose membrane 7, overlapping the binding pad 2 at one end of the detection line 3 close to the nitrocellulose membrane 7, and connecting the sample pad 1 to the other side of the binding pad 2; then cutting it into test strips of the required width.

[0117] The method for using the immunochromatographic kit for rapid detection of human urine nuclear matrix protein 22 comprises the following steps:

[0118] S1. Collect the urine sample to be tested and restore it to room temperature;

[0119] S2. Take 30-100 μL of the sample to be tested, drop the sample to be tested onto the sample pad 1, and let it stand at room temperature for 1-15 minutes. The antigen in the sample to be tested reacts with the latex group and chromatographs along the nitrocellulose membrane 7, reacting with the detection line 3 and the quality control line 4 respectively, and observing the bands at the detection line 3 and the quality control line 4.

[0120] If bands appear on both the test line 3 and the quality control line 4, it is judged as positive;

[0121] If there is no band at the test line 3 and a band appears at the quality control line 4, it is judged as negative;

[0122] If there is no band on quality control line 4, it is considered that there is a problem with the operation or the detection reagent, and retesting is required. Example 2

[0123] A method for preparing an immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 comprises the following steps:

[0124] 1. Preparation of nitrocellulose membrane

[0125] (1) Preparation of blocking agent: Prepare the blocking agent according to Table 6.

[0126] (2) Dilute the NMP22 monoclonal antibody and DNP antigen with blocking agents to a concentration of 1.5 mg / mL or 2 mg / mL for the NMP22 monoclonal antibody and 1.5 mg / mL for the DNP antigen. Scratch the nitrocellulose membrane using a gold streaking instrument. The line containing the NMP22 monoclonal antibody is used as the test line (T line), and the line containing the DNP antigen is used as the quality control line (C line). Then dry the membrane at 37°C and a humidity of <30% for 48 h for later use.

[0127] 2. Preparation of conjugate pad

[0128] The NMP22 monoclonal antibody labeled with latex microspheres and the anti-DNP antibody labeled with latex microspheres were mixed, sprayed on a glass cellulose membrane, and dried for later use.

[0129] Method for labeling antibodies with latex microspheres:

[0130] S1. Take 0.2 mg of antibody and dilute it to 1 mg / mL with PBS (0.02 mol / L) with pH=7.4 coupling solution. Take 2 μL of DTT and add it to the antibody. Let it react at room temperature for 2 hours. Ultrafilter and collect the antibody. Use the above coupling solution to dilute the antibody to 0.06 mg / mL, add 10 mg / ml NHS and let it react at room temperature for 30 minutes.

[0131] S2, take 12.5 μL of latex microspheres with a solid content of 4%, add 1 mL of MES solution (0.05 mol / L) with a pH of 6 and mix well;

[0132] S3, prepare 10 mg / mL EDC, add 20 μL, mix well, centrifuge and discard the supernatant;

[0133] S4, add 60 μg of the antibody treated in step S1, rotate and mix at room temperature for 150 min, add blocking solution, and rotate and mix at room temperature for 150 min;

[0134] S5. Centrifuge and discard the supernatant, add 1 mL of preservation solution (containing boric acid), mix well and disperse, and store at 2-8°C.

[0135] The NMP22 monoclonal antibody labeled with latex microspheres and the anti-DNP antibody labeled with latex microspheres were obtained by the above method, mixed, sprayed on a glass cellulose membrane, and dried for later use.

[0136] 3. Assemble the test strip: glue the nitrocellulose membrane 7 on the bottom plate, overlap the absorbent pad 5 at one end of the quality control line 4 close to the nitrocellulose membrane 7, overlap the binding pad 2 at one end of the detection line 3 close to the nitrocellulose membrane 7, and connect the sample pad 1 to the other side of the binding pad 2; then cut it into test strips of required width.

[0137] The preparation method of the test strip of this embodiment is different from the conventional method in two aspects: first, the NMP22 antibody coated on the detection line 3 and the DNP antigen coated on the quality control line 4 are treated with a blocker, so that the detection line 3 is coated with the NMP22 antibody treated with the blocker, and the quality control line 4 is coated with the DNP antigen treated with the blocker; second, when the latex microspheres are labeled with antibodies, the order of adding the activator and the coupling agent is different, and the coupling agent is first added to pretreat the antibody, and then the pretreated antibody is added to the latex microsphere system activated by the activator.

[0138] 30 μL of each of the samples with NMP22 concentrations of 0 U / mL, 0.5 U / mL, 2 U / mL, and 10 U / mL and 30 urine samples (S1-S30) randomly selected from healthy people were taken and tested using the test strips prepared in this example. The test results are shown in Table 7.

[0139] Table 7

[0140]

[0141] It can be seen from the table that when the concentration of NMP22 antibody coated on test line 3 is 1.5 mg / mL, the sensitivity is 2 U / mL; and when the concentration of NMP22 antibody coated on test line 3 is 2 mg / mL, the sensitivity is 0.5 U / mL.

[0142] Urine samples from 360 patients with different stages of bladder cancer and 180 patients without bladder cancer or bladder tumors were tested. The test results are shown in Table 8.

[0143] Table 8

[0144]

[0145] As can be seen from Table 8, the overall specificity was 95% and the overall sensitivity was 90.28% for the urine samples of 360 patients with different stages of bladder cancer and 180 patients without bladder cancer or bladder tumors.

[0146] When the inventors of this application explored the preparation method of the test strip, they tried to prepare it in a conventional way, that is, first, the NMP22 antibody treated with a coating solution (0.02M phosphate buffer, pH=7.4) was coated on the test line, and the DNP antigen treated with a coating solution (0.02M phosphate buffer, pH=7.4) was coated on the quality control line; second, when the latex microspheres were labeled with antibodies, an activator was added first, and then a coupling agent was added. Specifically:

[0147] Regarding the coated antibodies on the test line: NMP22 monoclonal antibody and DNP antigen were diluted with 0.02M phosphate buffer (pH=7.4) respectively to a concentration of 2mg / mL for NMP22 monoclonal antibody. The membrane was streaked on the nitrocellulose membrane using a gold streaking instrument. The membrane containing NMP22 monoclonal antibody was used as the test line (T line), and the membrane containing DNP antigen was used as the quality control line (C line). The membrane was then dried at 37°C and humidity <30% for 48 hours for use.

[0148] About latex microsphere labeled antibodies:

[0149] 1. Take 12.5 μL of latex microspheres with a solid content of 4%, add 1 mL of activation buffer (MES, concentration of 0.05 mol / L) and mix well.

[0150] 2. Prepare EDC 10 mg / mL and NHS 10 mg / mL, add equal volumes, mix well, rotate to mix for 30 minutes, centrifuge and discard the supernatant.

[0151] 3. Add 1 mL of coupling solution (MES, concentration is 0.05 mol / L) and mix well. Add 60 ug of antibody and rotate and mix at room temperature for 150 min. Add blocking solution and rotate and mix at room temperature for 150 min.

[0152] 4. Centrifuge and discard the supernatant, add 1 ml of preservation solution (including 1% BSA, 0.50% Tween 20, 5% sucrose, 0.10% PC300, boric acid), mix well and store at 2-8 degrees.

[0153] The other processing steps were exactly the same. The test strips prepared by this method were used to test urine samples of 360 patients with different stages of bladder cancer and 180 patients without bladder cancer or bladder tumors. The overall specificity (the proportion of negative samples measured) was 69.44%, and the overall sensitivity (the proportion of positive samples measured) was 55.56%. The statistical results are as follows:

[0154] Table 9

[0155]

[0156] By comparing Table 8 and Table 9, it can be seen that the detection sensitivity and specificity are significantly improved by improving the antibody coating method on the detection line and the latex microsphere labeling antibody method on the conjugate pad, so that the overall specificity is increased from 69.44% to 95%, and the overall sensitivity is increased from 55.56% to 90.28%.

[0157] Example 3

[0158] Different from Example 2, the preparation method of the nitrocellulose membrane is different. The detection line and quality control line on the nitrocellulose membrane are not the NMP22 monoclonal antibody and DNP antigen treated with a blocker, but the NMP22 monoclonal antibody and DNP antigen treated with 0.02 M phosphate buffer (pH = 7.4) to a concentration of 2 mg / mL of the NMP22 monoclonal antibody.

[0159] The test samples with NMP22 concentrations of 0 U / mL, 0.5 U / mL, and 2 U / mL and 30 urine samples from healthy people randomly selected were taken and tested using the test strips prepared in this example. The test results are shown in Table 10.

[0160] Table 10

[0161]

[0162] As can be seen from Table 10, when the NMP22 antibody is treated with phosphate buffer, the sensitivity can still reach 0.5 U / mL when the concentration of the NMP22 antibody coated on the test line is 2 mg / mL.

[0163] Urine samples from 360 patients with different stages of bladder cancer and 180 patients without bladder cancer or bladder tumors were tested. The test results are shown in Table 11.

[0164] Table 11

[0165]

[0166] As can be seen from Table 11, the overall specificity was 44.44% and the overall sensitivity was 91.66% for the urine samples of 360 patients with different stages of bladder cancer and 180 patients without bladder cancer and bladder tumors.

[0167] It can be seen that when the NMP22 antibody on the test line is not treated with a blocker, its detection sensitivity is not affected, but the specificity changes greatly, increasing the risk of false positives.

[0168] Example 4

[0169] Different from Example 2, a marker line 8 is provided on the nitrocellulose membrane 7 near the conjugate pad 2 and 1.5 mm away from the conjugate pad 2. A mixture of NMP22 antibody labeled with latex microspheres and anti-DNP antibody labeled with latex microspheres is coated on the marker line 8. The molar ratio of NMP22 antibody labeled with latex microspheres and anti-DNP antibody labeled with latex microspheres is 1:(0.1-1), and the molar ratio of 1:0.2 is selected in this embodiment. After mixing, 1 μL / cm is drawn on the nitrocellulose membrane 1.5 mm near the conjugate pad, and then dried for 48 hours at 37°C and humidity <30%.

[0170] The method of labeling antibodies with latex microspheres is a conventional method, and can be carried out in the present application in the following manner:

[0171] 1. Take 12.5 μL of latex microspheres with a solid content of 4%, add 1 ml of activation solution (MES, concentration of 0.05 mol / L) and mix well.

[0172] 2. Prepare EDC 10mg / ml and NHS 10mg / ml, add equal volumes, mix well, rotate to mix for 30min, centrifuge and discard the supernatant.

[0173] 3. Add 1 mL of coupling solution (MES, concentration is 0.05 mol / L) and mix well. Add 60 ug of antibody and rotate and mix at room temperature for 150 min. Add blocking solution and rotate and mix at room temperature for 150 min.

[0174] 4. Centrifuge and discard the supernatant, add 1 ml of preservation solution (including 1% BSA, 0.50% Tween 20, 5% sucrose, 0.10% PC300, boric acid), mix well and store at 2-8 degrees.

[0175] The samples with NMP22 concentrations of 0 U / mL, 0.5 U / mL, and 2 U / mL and 30 urine samples from healthy people randomly selected were taken and tested using the test strips prepared in this example (the concentration of NMP22 antibody coated on the test line was 2 mg / mL). The test results are shown in Table 12.

[0176] Table 12

[0177]

[0178] It can be seen from Table 12 that when the concentration of the NMP22 antibody coated on the detection line is 2 mg / mL, the sensitivity can still reach 0.5 U / mL, and the added marker line 8 has no effect on the detection sensitivity.

[0179] Urine samples from 360 patients with different stages of bladder cancer and 180 patients without bladder cancer or bladder tumors were tested. The test results are shown in Table 13.

[0180] Table 13

[0181]

[0182] Urine samples from 360 patients with different stages of bladder cancer and 180 patients without bladder cancer or bladder tumors were tested. The overall specificity was 95.56% and the overall sensitivity was 90.00%. There was no significant effect on sensitivity and specificity.

[0183] Comparative Example 1 Investigating the effect of the presence of the marker line on the shelf life of the test strip

[0184] The test strips prepared in Example 2 and Example 4 were used to test urine samples from 360 patients with different stages of bladder cancer and 180 patients without bladder cancer or bladder tumors, and the changes in sensitivity and specificity within five years were compared. The comparison results are shown in Table 14.

[0185] Table 14

[0186]

[0187] It can be seen from the test results in Table 14 that the test strip prepared using Example 4 had no difference within five years, reaching 0.5 U / ml, and the performance of the test strip prepared using Example 2 decreased significantly after the second year.

[0188] 360 urine samples of patients with different stages of bladder cancer and 180 urine samples without bladder cancer and bladder tumors were tested. The overall specificity of the test strip prepared in Example 4 within five years was 95.00%, 95.56%, 95%, 96.11%, 95.56%, 95%, and the overall sensitivity was 90.00%, 90%, 90.28%, 90.28%, 90.00%, 90.56%, respectively. As time goes by, the sensitivity and specificity of the test strip detection are not significantly affected; the test strip prepared in Example 2 has an overall specificity of 95.00%, 95.56%, 100%, 100%, 100%, 100%, and an overall sensitivity of 90%, 83.33%, 41.67%, 26.00%, 13.89% respectively within five years. %, it can be seen that the sensitivity of the test strip prepared in Example 2 is significantly reduced after the second year.

[0189] It can be seen that the test strip prepared in Example 4 can extend the service life of the test strip by providing the marker line 8 on the nitrocellulose membrane 7.

[0190] Comparative Example 2 Investigating the effects of different marking processes on test sensitivity

[0191] The experimental group was the same as Example 2. The difference between the control group and the experimental group was that the labeling process was different. The control group used a conventional method to prepare the conjugate pad. The method of labeling the antibody with latex microspheres was as follows:

[0192] 1. Take 12.5 μL of latex microspheres with a solid content of 4%, add 1 ml of activation buffer (MES, concentration of 0.05 mol / L) and mix well.

[0193] 2. Prepare EDC 10mg / ml and NHS 10mg / ml, add equal volumes, mix well, rotate to mix for 30min, centrifuge and discard the supernatant.

[0194] 3. Add 1 mL of coupling solution (MES, concentration is 0.05 mol / L) and mix well. Add 60 ug of antibody and rotate and mix at room temperature for 150 min. Add blocking solution and rotate and mix at room temperature for 150 min.

[0195] 4. Centrifuge and discard the supernatant, add 1 ml of preservation solution (including 1% BSA, 0.50% Tween 20, 5% sucrose, 0.10% PC300, boric acid), mix well and store at 2-8 degrees.

[0196] The samples with NMP22 concentrations of 0 U / mL, 0.5 U / mL, and 2 U / mL and 30 urine samples randomly selected from healthy people were tested using the test strips prepared for the experimental group and the control group, respectively. The test results are shown in Table 15.

[0197] Table 15

[0198]

[0199] As can be seen from Table 15, the detection sensitivity of the conjugate pad prepared by the conventional labeling method (control group) is 10U / mL, while the detection sensitivity of the conjugate pad prepared by the optimized labeling method (experimental group) is 0.5U / mL, and the sensitivity is greatly improved.

[0200] In order to exclude the possibility that the reagents used in conventional labeling methods may affect the detection sensitivity, the corresponding changes in detection sensitivity under different conditions were investigated.

[0201] 1. Adjust the type and pH of the coupling solution. When labeling antibodies with latex microspheres, different types and pH coupling solutions were used to test their sensitivity. The results are shown in Table 16.

[0202] Table 16

[0203]

[0204] As can be seen from Table 16, the coupling solution PBS pH6.5 (0.02 mol / L) and MES pH6.5 (0.05 mol / L) have a corresponding detection sensitivity of up to 10 U / mL, but the risk of false positives is high.

[0205] This study explores the effects of changing the material of the conjugate pad and the way it is treated in conventional labeling methods on the detection sensitivity.

[0206] 2. Adjust the material of the bonding pad

[0207] The sensitivity of the conjugate pads made of different materials was tested respectively, and the results are shown in Table 17.

[0208] The glass cellulose membrane and polyester cellulose membrane were immersed in a borate buffer (pH = 7.4) containing 0.5% Tween 20, 0.9% sodium chloride, 0.5% BSA, 0.5% casein, 2.5% PVP10, 0.1% PC300 0.05M for 6 hours, and then dried at 35°C for 12 hours. The results are shown in Table 17.

[0209] Table 17

[0210]

[0211] It can be seen from the test results in Table 17 that the polyester cellulose membrane is relatively superior, but its sensitivity is only 10U / mL.

[0212] 3. Adjust the processing method of the conjugate pad and sample pad

[0213] The conjugate pad and sample pad were treated in different ways and their sensitivities were tested respectively. The results are shown in Table 18.

[0214] Soaking method: Soak the polyester cellulose membrane in a borate buffer (pH=7.4) containing 0.5% Tween 20, 0.5% BSA, 0.9% sodium chloride, 0.5% casein, 2.5% PVP10, 0.1% PC300 0.05M for 6 hours, and then dry it at 35°C for 12 hours.

[0215] Ultrasonic method: The polyester cellulose membrane was immersed and ultrasonicated for 0.5 h in a borate buffer (pH = 7.4) containing 0.5% Tween 20, 0.5% BSA, 0.9% sodium chloride, 0.5% casein, 2.5% PVP10, 0.1% PC300 0.05M, and then dried at 35 °C for 12 h.

[0216] Table 18

[0217]

[0218] It can be seen from the test results in Table 18 that the detection sensitivities corresponding to the immersion and ultrasonic methods are comparable, both at 10 U / mL.

[0219] 4. Adjust the processing liquid of the sample pad

[0220] The glass cellulose membrane was incubated in PBS buffer (pH=7.4) containing 0.1% Tween 20, 0.9% NaCl, 1% casein, 1% PVP10, 1% PEG20K, 0.1% PC300 0.05M. 0.1M PBS buffer (pH=7.4), 0.1% Tween 20, 0.9% sodium chloride, 1% casein, 1% PVP10, 1% PEG20K, 0.1% PC300 0.05M Tris-HCl buffer (pH=7.4), 0.1% Tween 20, 0.9% sodium chloride, 1% casein, 1% PVP10, 1% PEG20K, 0.1% PC300 Soak in 0.1M Tris-HCl buffer (pH=7.4) for 6 hours, and then dry at 35°C for 12 hours. The results are shown in Table 19.

[0221] Table 19

[0222]

[0223] It can be seen from the test results in Table 19 that no matter whether PBS, Tris-HCl or boric acid is used as the sample pad treatment solution, the sensitivity is only 10 U / mL.

[0224] Therefore, when preparing the conjugate pad, the sensitivity can only reach 10U / mL by using the conventional labeling method, and changing other conditions will not improve the sensitivity.

[0225] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. An immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22, the test strip comprising a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorbent pad connected in sequence, characterized in that: The nitrocellulose membrane is provided with a detection line and a quality control line in sequence, the detection line is coated with a NMP22 antibody treated with a blocker, and the quality control line is coated with a DNP antigen treated with a blocker, wherein the blocker comprises 1% heterophilic antibody blocker, 0.2 mg / mL anti-rheumatoid factor, 1% anti-HAMA agent, 0.02 mol / L buffer, 5% protective agent and 0.1% biological preservative, and the above percentages are mass percentages; The conjugate pad is coated with latex microsphere-labeled NMP22 antibody and latex microsphere-labeled anti-DNP antibody, and the molar ratio of the latex microsphere-labeled NMP22 antibody and the latex microsphere-labeled anti-DNP antibody is 1:(0.1-1); Method for labeling antibodies with latex microspheres: S1. Add the antibody to the coupling solution to pretreat the antibody; S2, adding the latex microspheres into an activator to activate the latex microspheres; S3. Add the pretreated antibody to S2 and mix well to obtain the latex microsphere-labeled antibody.

2. The immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 according to claim 1, characterized in that: The coupling solution is MES, PBS or boric acid solution, and the activating agent is EDC and NHS.

3. The immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 according to claim 1, characterized in that: A marker line is arranged on one side of the nitrocellulose membrane close to the conjugate pad.

4. The immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 as claimed in claim 3, characterized in that: The distance between the marker line and the conjugate pad is 1.5 mm.

5. The immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 according to claim 3, characterized in that: The marker line is coated with a mixture of latex microsphere-labeled NMP22 antibody and latex microsphere-labeled anti-DNP antibody.

6. The immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 according to claim 5, characterized in that: The molar ratio of the latex microsphere-labeled NMP22 antibody to the latex microsphere-labeled anti-DNP antibody is 1:(0.1-1).

7. The immunochromatographic test strip for rapid detection of human urine nuclear matrix protein 22 according to claim 1, characterized in that: The heterophilic antibody blocker is selected from HBR1, HBR2, HBR3, HBR5, HBR6, HBR9, HBR10, HBR11, 3BX484, 3KC535, 3KC701, 3KC542, 3KC564 or 3KC565 produced by Shenzhen Yawei Century Technology Co., Ltd., the anti-rheumatoid factor is MK33 produced by Roche Diagnostics, the anti-HAMA agent is selected from C8001, C8005, C8003 or C8004 produced by Nanjing Jingda Biotechnology Co., Ltd., the buffer is selected from any one of PBS, Tris-HCl buffer, and borate buffer; the protective agent is selected from sucrose or trehalose; the biological preservative is selected from any one of proclin300, proclin950, proclin150, and proclin200.

8. An immunochromatographic kit for rapid detection of human urine nuclear matrix protein 22, characterized in that: The invention comprises the test strip according to any one of claims 1 to 7.

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