Immuno-chromatographic test strip for detecting nmp22, its application and kit

Abstract

The application provides an immunochromatography test paper for detecting NMP22, application and kit thereof, and relates to the technical field of biological detection.The immunochromatography test paper provided by the application comprises a bottom plate, a sample pad, a combination pad, a detection pad and a water absorption paper which are sequentially laminated on the bottom plate; the sample pad is a glass fiber membrane or a polyester film which is impregnated by a sample pad treatment solution; the sample pad treatment solution comprises 3-9 g / L of tris-hydroxymethyl aminomethane, 3-7 g / L of sodium caseinate, 5-10 g / L of polyvinylpyrrolidone-K10, 5-15 g / L of bovine serum albumin, 0.1-0.3 g / L of Proclin 300, 5-15 g / L of Standapoles-1, 1-4 g / L of ethylenediaminetetraacetic acid disodium salt dihydrate, 2-4 g / L of Tetronic 1307, and the rest is water.The immunochromatography test paper has the advantages of high sensitivity, good specificity, high detection efficiency and the like.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to an immunochromatographic test strip for detecting NMP22, its application, and a reagent kit. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system. Currently, the traditional main methods for bladder cancer diagnosis and postoperative follow-up are cystoscopy and urine cytology. While cystoscopy is accurate and reliable, it is an invasive procedure, causing some trauma to patients and having a high acceptance threshold; it is also prone to missing cases of carcinoma in situ and squamous cell carcinoma. Urine cytology, as a non-invasive detection method, is recommended as an adjunct to cystoscopy and is widely used for bladder cancer screening; however, this method has low sensitivity for well-differentiated or low-grade tumors. Therefore, finding a new, non-invasive, simple method that can assist in the diagnosis of early-stage bladder cancer is of great significance in overcoming the limitations of existing bladder cancer detection methods.

[0003] Nuclear matrix protein 22 (NMP22) is a subunit of the nuclear mitotic apparatus protein, whose main function is to control normal cell mitosis. When cells become malignant, the distribution of nuclear genetic material at the end of mitosis is extremely abnormal, leading to a surge in NMP22 synthesis, which is released into the urine with shed cells. Studies by Mbeutcha et al. have found that when bladder cancer cells rupture after apoptosis, NMP22 is released into the urine, and the concentration of NMP22 in the urine of bladder cancer patients is significantly higher than in normal individuals. With increasing clinical research in recent years, NMP22 has been recognized as an important biomarker for bladder cancer.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide an immunochromatographic test strip for detecting NMP22, enabling rapid and accurate detection of NMP22.

[0006] A second objective of this invention is to provide the application of the above-mentioned immunochromatographic test strip in the preparation of bladder cancer detection products.

[0007] A third objective of this invention is to provide a reagent kit.

[0008] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides an immunochromatographic test strip for detecting NMP22, the immunochromatographic test strip comprising a base plate and a sample pad, a conjugation pad, a detection pad and absorbent paper sequentially stacked on the base plate; The binding pad contains a marker-labeled antibody against nuclear matrix protein 22; The detection pad is provided with a detection line and a control line; the detection line is coated with a nuclear matrix protein 22-coated antibody. The nuclear matrix protein 22 labeled antibody and the nuclear matrix protein 22 coated antibody recognize different epitopes of nuclear matrix protein 22; The sample pad is a glass fiber membrane or polyester membrane impregnated with a sample pad treatment solution; the sample pad treatment solution includes 3~9 g / L tris(hydroxymethyl)aminomethane, 3~7 g / L sodium caseinate, 5~10 g / L polyvinylpyrrolidone-K10, 5~15 g / L bovine serum albumin, 0.1~0.3 g / L Proclin 300, 5~15 g / L Standapoles-1, 1~4 g / L disodium ethylenediaminetetraacetate dihydrate, and 2~4 g / L Tetronic 1307.

[0009] As a further technical solution, the conjugate pad also contains a marker-labeled chicken IgY antibody; As a further technical solution, the quality control line is coated with goat anti-chicken IgY antibody.

[0010] As a further technical solution, the markers include latex microspheres, colloidal gold, colloidal selenium, fluorescent particles, or quantum dots.

[0011] As a further technical solution, the detection pad is a nitrocellulose membrane.

[0012] As a further technical solution, the bonding pad is a hydrophilic microporous structure material, which is a glass fiber membrane or a polyester membrane.

[0013] As a further technical solution, the sample pad treatment solution includes 5~8 g / L tris(hydroxymethyl)aminomethane, 3~7 g / L sodium caseinate, 7~10 g / L polyvinylpyrrolidone-K10, 3~8 g / L bovine serum albumin, 0.1~0.3 g / L Proclin 300, 7~10 g / L Standapoles-1, 2~3 g / L disodium ethylenediaminetetraacetate dihydrate, and 2~3 g / L Tetronic 1307.

[0014] As a further technical solution, the sample pad treatment solution comprises 7 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 7.5 g / L polyvinylpyrrolidone-K10, 5 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L tandapoles-1, 2.5 g / L disodium ethylenediaminetetraacetate dihydrate, and 3 g / L Tetronic 1307.

[0015] Secondly, the present invention provides the application of the above-mentioned immunochromatographic test strip in the preparation of bladder cancer detection products.

[0016] Thirdly, the present invention provides a kit comprising the above-described immunochromatographic test strip.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The immunochromatographic test strip for NMP22 detection provided by this invention, through further optimization of the sample pad treatment solution, achieves higher sensitivity and specificity compared to mainstream commercially available NMP22 detection reagents, and shortens the interpretation time from 30 minutes to 15 minutes. This invention, while ensuring high sensitivity and specificity, reduces the required interpretation time by 50%, significantly improving detection speed and providing a non-invasive, rapid, and accurate urine detection method for point-of-care testing. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The results show the specificity of the treatment solutions for three different sample pad formulations. Figure 2 The sensitivity test results are for three different formulations of sample pad treatment solutions; Figure 3 The results show the specificity of three further optimized sample pad treatment solutions. Figure 4 The sensitivity detection results for three further optimized sample pad treatment solutions; Figure 5 The sensitivity and specificity detection results for replacing NMP-Ab2 with NMP-Ab3; Figure 6 The results show the specificity of the sample pad treatment solution at different concentrations. Figure 7 The results show the sensitivity detection of sample pad treatment solutions of different concentrations; Figure 8 The results show the sensitivity detection comparison between the invented reagent and competing reagents. Detailed Implementation

[0020] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0021] In a first aspect, the present invention provides an immunochromatographic test strip for detecting NMP22, the immunochromatographic test strip comprising a base plate and a sample pad, a conjugation pad, a detection pad and absorbent paper sequentially stacked on the base plate; The binding pad contains a marker-labeled antibody against nuclear matrix protein 22; Along the chromatography direction of the sample to be tested, a detection line and a control line are sequentially arranged on the detection pad; the detection line is coated with a nuclear matrix protein 22-coated antibody; The nuclear matrix protein 22 labeled antibody and the nuclear matrix protein 22 coated antibody recognize different epitopes of nuclear matrix protein 22; The sample pad is a glass fiber membrane or polyester membrane impregnated with a sample pad treatment solution; the sample pad treatment solution includes 3~9 g / L tris(hydroxymethyl)aminomethane, 3~7 g / L sodium caseinate, 5~10 g / L polyvinylpyrrolidone-K10, 5~15 g / L bovine serum albumin, 0.1~0.3 g / L Proclin 300, 5~15 g / L Standapoles-1, 1~4 g / L disodium ethylenediaminetetraacetate dihydrate, and 2~4 g / L Tetronic 1307, with water as the solvent.

[0022] The immunochromatographic test strip for detecting NMP22 provided by this invention has advantages such as high sensitivity, good specificity, and simplicity and speed through optimization of the sample pad treatment solution.

[0023] In some alternative embodiments, the conjugate pad further contains a marker-labeled chicken IgY antibody; The quality control line is coated with goat anti-chicken IgY antibody.

[0024] This invention utilizes chicken IgY antibody and goat anti-chicken IgY antibody to achieve quality control detection of the test samples.

[0025] The present invention does not impose specific limitations on the marker. In some optional embodiments, the marker includes, but is not limited to, latex microspheres, colloidal gold, colloidal selenium, fluorescent particles or quantum dots, preferably latex microspheres.

[0026] In some alternative embodiments, the method for labeling latex microspheres with matrix nuclear protein 22-labeled antibodies and chicken IgY antibodies is as follows: ① Cleaning with the original solution of latex microspheres; ② The latex microspheres were reconstituted by sonication using a labeled buffer solution; ③ Add the antibody to the reconstituted microsphere solution, mix well, and then add the reactant EDC (carbodiimide). ④ After the reaction is complete, centrifuge and discard the supernatant, add blocking solution and sonicate to reconstitute the microspheres for sealing; ⑤ After the sealing is completed, centrifuge and discard the supernatant, add microsphere preservation solution, and sonicate to reconstitute the microspheres to obtain latex microsphere-antibody conjugate.

[0027] In some optional embodiments, the nuclear matrix protein 22 coated antibody is NMP-Ab1, purchased from Hangzhou Jingjie Biotechnology Co., Ltd., catalog number: ABT-102146; The nuclear matrix protein 22 labeled antibody is either NMP-Ab2 or NMP-Ab3; NMP-Ab2 was purchased from Hangzhou Jingjie Biotechnology Co., Ltd., catalog number: ABT-102144; NMP-Ab3 was purchased from Hangzhou Jingjie Biotechnology Co., Ltd., catalog number: ABT-102148. It should be noted that this invention does not impose specific limitations on the selection of the labeling antibody and the coating antibody; both can be antibodies that recognize different epitopes of nuclear matrix protein 22.

[0028] In some optional embodiments, the diluent for the nuclear matrix protein 22-coated antibody and the goat anti-chicken IgY antibody on the detection pad is a mixture of PB buffer and trehalose solution or a mixture of PBS buffer and trehalose solution.

[0029] In some alternative embodiments, the detection pad is a nitrocellulose membrane.

[0030] In some alternative embodiments, the bonding pad is a hydrophilic microporous material, which is a glass fiber membrane or a polyester membrane.

[0031] In some optional embodiments, the sample pad treatment solution comprises 5-8 g / L tris(hydroxymethyl)aminomethane, 3-7 g / L sodium caseinate, 7-10 g / L polyvinylpyrrolidone-K10, 3-8 g / L bovine serum albumin, 0.1-0.3 g / L Proclin 300, 7-10 g / L Standapoles-1, 2-3 g / L disodium ethylenediaminetetraacetate dihydrate, and 2-3 g / L Tetronic 1307.

[0032] In some optional embodiments, the sample pad treatment solution comprises 7 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 7.5 g / L polyvinylpyrrolidone-K10, 5 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L tandapoles-1, 2.5 g / L disodium ethylenediaminetetraacetate dihydrate, and 3 g / L Tetronic 1307.

[0033] In some alternative embodiments, the hydrophilic microporous material is a glass fiber membrane or a polyester membrane.

[0034] In some alternative embodiments, the width of the test strip is 3-4 mm.

[0035] Secondly, the present invention provides the application of the above-mentioned immunochromatographic test strip in the preparation of bladder cancer detection products.

[0036] The immunochromatographic test strip provided by this invention can be used to detect the bladder cancer marker nuclear matrix protein 22, and the detection results of nuclear matrix protein 22 can be used to assist in the diagnosis of bladder cancer.

[0037] Thirdly, the present invention provides a kit comprising the above-described immunochromatographic test strip.

[0038] This kit can be used to assist in the diagnosis of bladder cancer.

[0039] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0040] Example 1 I. Immunochromatographic test strips: The device includes a base plate and a sample pad, a conjugate pad, a test pad, and absorbent paper stacked sequentially on the base plate. The conjugate pad contains NMP-Ab2 (manufacturer: Hangzhou Jingjie Biotechnology Co., Ltd., catalog number: ABT-102144) labeled with latex microspheres and chicken IgY antibody labeled with latex microspheres. Along the chromatography direction of the sample to be tested, the test pad has a test line and a control line arranged sequentially. The test line is coated with NMP-Ab1 (manufacturer: Hangzhou Jingjie Biotechnology Co., Ltd., catalog number: ABT-102146). The control line is coated with goat anti-chicken IgY antibody.

[0041] The preparation method is as follows: 1) Microsphere ultrasound The original latex microsphere solution was ultrasonically dispersed, and care was taken to carry out the process in an ice-water bath to avoid generating heat during the ultrasonication.

[0042] 2) Microsphere cleaning Take the sonicated microspheres, dilute them with labeling buffer to 1% solid content, vortex to mix, centrifuge and discard the supernatant; add labeling buffer again and sonicate to resuspend to 1% solid content.

[0043] 3) Antibody conjugation Add 0.25~0.5 mg / mL of antibody (NMP-Ab2, chicken IgY antibody) to the resuspended microsphere solution and mix immediately. Then add 0.01~0.15% EDC reagent and mix immediately for 4~24 h.

[0044] 4) Microsphere encapsulation Centrifuge the reacted microspheres and discard the supernatant; then add blocking solution, sonicate to resuspend, and block for 4-8 hours.

[0045] 5) Microsphere collection Centrifuge the sealed microspheres and discard the supernatant; then add preservation solution and sonicate to resuspend until the microsphere content is 1%, thus obtaining the antibody-labeled conjugate.

[0046] 6) Preparation of the binding pad Using a conventional binding pad (glass fiber membrane), NMP-Ab2 labeled with latex microspheres and chicken IgY antibody labeled with latex microspheres were sprayed onto the surface.

[0047] 7) Preparation of the test pad Using a standard test pad (nitrocellulose membrane), the test line is coated with NMP-Ab1; the control line is coated with goat anti-chicken IgY antibody.

[0048] 8) Preparation of sample pads Prepare the sample pad treatment solution and pour it onto a glass fiber membrane. Gently scrape the glass fiber with tweezers to guide the treatment solution to be evenly absorbed by the glass fiber membrane. After the glass fiber membrane has absorbed the solution evenly, hold the two diagonal corners with tweezers and quickly transfer the glass fiber to the draining mesh rack. Place the draining mesh rack in an oven at 37°C and dry for 4 hours until excess moisture evaporates. The relative humidity of the oven should be ≤30%.

[0049] II. Detection Methods: 1) Before testing, equilibrate the test strip, buffer solution, and sample at room temperature (15°C to 30°C); 2) Remove a test strip from the sealed aluminum foil bag and place it on a clean, flat surface; 3) Hold the sample collection tube upright and open the cap. Add 3 drops of urine sample to the sample well. Avoid creating air bubbles when adding the sample; 4) Wait for the red line to appear, and read the test result after 15 minutes. Results after 20 minutes are invalid.

[0050] 5) Interpretation of test results: Positive: Two clear colored lines appear. One should be located in the control line area (C), and the other should be located in the test line area (T). The intensity of the color in the test line area (T) may vary depending on the antigen concentration in the sample. Therefore, any color appearing in the test line area (T) should be considered positive.

[0051] Negative: Only one color band appears in the control line area (C).

[0052] Invalid: Control line not appeared. Insufficient sample volume or improper operation are the most likely reasons for the control line not appearing. Double-check the operation procedure and repeat the test using a new test strip.

[0053] III. Sample Pad Treatment Solution Formulation Screening The following three sample pad treatment solutions were prepared: NMP-SP1: 9 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 5 g / L polyvinylpyrrolidone-K10, 10 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Tetronic 1307, 2 g / L disodium ethylenediaminetetraacetate dihydrate; NMP-SP2: 9 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 5 g / L polyvinylpyrrolidone-K10, 10 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Standapoles-1, 2 g / L disodium ethylenediaminetetraacetate dihydrate; NMP-SP3: 9 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 5 g / L polyvinylpyrrolidone-K10, 10 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Tween 20, 2 g / L disodium ethylenediaminetetraacetate dihydrate.

[0054] Immunochromatographic test strips prepared from sample pads treated with the above three processing solutions were used to detect positive and negative clinical samples. The concentration of NMP22 in positive samples ranged from 15.625 to 1000 ng / mL. Specificity results were as follows: Figure 1 As shown, the sensitivity results are as follows: Figure 2 As shown.

[0055] Specificity is calculated as follows: Specificity = (Number of negative samples / Total number of negative samples) * 100%; Sensitivity is calculated as follows: Sensitivity = (Number of positive samples / Total number of positive samples) * 100%. The same applies below.

[0056] The results showed that among the sample pads treated with three different solutions, the NMP-SP1 sample pad had the highest sensitivity, reaching 97.3%, but its specificity was the worst among the three sample pads, at only 88.2%. The NMP-SP2 sample pad had a slightly lower sensitivity than the NMP-SP1 sample pad, at 95.5%, but its specificity was the best among the three sample pads, at 100%. The NMP-SP3 sample pad's sensitivity and specificity were between the other two sample pads.

[0057] IV. Optimizing Sensitivity Based on NMP-SP2 Based on NMP-SP2, the following three sample pad treatment solutions were designed: (1) NMP-SP2-1: 9 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 5 g / L polyvinylpyrrolidone-K10, 10 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Standapoles-1, 2 g / L disodium ethylenediaminetetraacetate dihydrate, 3 g / L sodium dodecyl sulfate; (2) NMP-SP2-2: 9 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 5 g / L polyvinylpyrrolidone-K10, 10 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Standapoles-1, 2 g / L disodium ethylenediaminetetraacetate dihydrate, 3 g / L Tetronic 1307; (3) NMP-SP2-3: 9 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 5 g / L polyvinylpyrrolidone-K10, 10 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Standapoles-1, 2 g / L disodium ethylenediaminetetraacetate dihydrate, 3 g / L sodium carbonate.

[0058] Immunochromatographic test strips prepared with the above-mentioned sample pad treatment solution were used to test positive and negative clinical samples, and the specificity results are as follows: Figure 3 As shown, the sensitivity results are as follows: Figure 4 As shown.

[0059] The results showed that adding sodium dodecyl sulfate, Tetronic 1307, or sodium carbonate to the NMP-SP2 base had the following effects: sodium dodecyl sulfate and Tetronic 1307 had no effect on specificity, but sodium dodecyl sulfate significantly reduced sensitivity by 5.5%, while Tetronic 1307 enhanced sensitivity by 4.5%. Sodium carbonate had no significant effect on sensitivity or specificity. Therefore, the NMP-SP2-2 sample pad was selected.

[0060] Furthermore, based on NMP-SP2, replacing NMP-Ab2 in the test strip with NMP-Ab3 (manufacturer: Hangzhou Jingjie Biotechnology Co., Ltd., product number: ABT-102148) yielded similar sensitivity and specificity to NMP-Ab2. The specificity and sensitivity results are as follows: Figure 5 As shown.

[0061] V. Confirmation of the concentration of each component in the NMP-SP2-2 sample pad treatment solution Prepare the following 9 sample pad treatment solutions: (1) NMP-SP2-2-1: 1.5 g / L tris(hydroxymethyl)aminomethane, 1 g / L sodium caseinate, 3 g / L polyvinylpyrrolidone-K10, 3 g / L bovine serum albumin, 0.05 g / L Proclin 300, 3 g / L Standapoles-1, 0.5 g / L disodium ethylenediaminetetraacetate dihydrate, 1 g / L Tetronic 1307; (2) NMP-SP2-2-2: 3 g / L tris(hydroxymethyl)aminomethane, 3 g / L sodium caseinate, 5 g / L polyvinylpyrrolidone-K10, 5 g / L bovine serum albumin, 0.1 g / L Proclin 300, 5 g / L Standapoles-1, 1 g / L disodium ethylenediaminetetraacetate dihydrate, 2 g / L Tetronic 1307; (3) NMP-SP2-2-3: 4 g / L tris(hydroxymethyl)aminomethane, 4 g / L sodium caseinate, 6 g / L polyvinylpyrrolidone-K10, 6 g / L bovine serum albumin, 0.1 g / L Proclin 300, 6 g / L Standapoles-1, 1.5 g / L disodium ethylenediaminetetraacetate dihydrate, 3 g / L Tetronic 1307; (4) NMP-SP2-2-4: 5 g / L tris(hydroxymethyl)aminomethane, 5 g / L sodium caseinate, 7 g / L polyvinylpyrrolidone-K10, 7 g / L bovine serum albumin, 0.1 g / L Proclin 300, 7 g / L Standapoles-1, 2 g / L disodium ethylenediaminetetraacetate dihydrate, 3 g / L Tetronic 1307; (5) NMP-SP2-2-5: 6 g / L tris(hydroxymethyl)aminomethane, 5 g / L sodium caseinate, 8 g / L polyvinylpyrrolidone-K10, 8 g / L bovine serum albumin, 0.2 g / L Proclin 300, 8 g / L Standapoles-1, 2 g / L disodium ethylenediaminetetraacetate dihydrate, 4 g / L Tetronic 1307; (6) NMP-SP2-2-6: 6 g / L tris(hydroxymethyl)aminomethane, 5 g / L sodium caseinate, 10 g / L polyvinylpyrrolidone-K10, 10 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Standapoles-1, 3 g / L disodium ethylenediaminetetraacetate dihydrate, 4 g / L Tetronic 1307; (7) NMP-SP2-2-7: 7 g / L tris(hydroxymethyl)aminomethane, 5 g / L sodium caseinate, 7.5 g / L polyvinylpyrrolidone-K10, 10 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Standapoles-1, 3 g / L disodium ethylenediaminetetraacetate dihydrate, 4 g / L Tetronic 1307; (8) NMP-SP2-2-8: 9 g / L tris(hydroxymethyl)aminomethane, 7 g / L sodium caseinate, 10 g / L polyvinylpyrrolidone-K10, 15 g / L bovine serum albumin, 0.3 g / L Proclin 300, 15 g / L Standapoles-1, 4 g / L disodium ethylenediaminetetraacetate dihydrate, 4 g / L Tetronic 1307; (9) NMP-SP2-2-9: 12 g / L tris(hydroxymethyl)aminomethane, 10 g / L sodium caseinate, 15 g / L polyvinylpyrrolidone-K10, 20 g / L bovine serum albumin, 0.5 g / L Proclin 300, 20 g / L Standapoles-1, 8 g / L disodium ethylenediaminetetraacetate dihydrate, 5 g / L Tetronic 1307; Immunochromatographic test strips prepared using the above nine sample pad treatment solutions were used to detect positive and negative clinical samples. The specificity results are as follows: Figure 6 As shown, the sensitivity results are as follows: Figure 7 As shown in the results, the NMP-SP2-2-1 sample pad reduces both sensitivity and specificity. While the NMP-SP2-2-9 sample pad does not reduce sensitivity, it lowers specificity. The NMP-SP2-2-2 to NMP-SP2-2-8 sample pads, however, achieve 100% sensitivity and specificity for both positive and negative clinical samples, demonstrating superior overall performance. Therefore, the optimal concentration range for each component of this sample pad is: 3–9 g / L tris(hydroxymethyl)aminomethane, 3–7 g / L sodium caseinate, 5–10 g / L polyvinylpyrrolidone-K10, 5–15 g / L bovine serum albumin, 0.1–0.3 g / L Proclin 300, 5–15 g / L Standapoles-1, 1–4 g / L disodium ethylenediaminetetraacetate dihydrate, and 2–4 g / L Tetronic 1307.

[0062] VI. Sensitivity Comparison with Competing Reagents The competing reagent used for comparison is the NMP22 immunochromatographic qualitative rapid test kit manufactured by Abbott Diagnostics (USA) Scarborough Inc.

[0063] Invented reagent addition method: Use a 40μL dropper to add 3 drops of positive or negative clinical samples into the sample well of the reagent card, interpret the results after 15-20 minutes, and the results are invalid after 20 minutes.

[0064] Competitor reagent sample addition method: Use the dropper provided with the reagent to add 4 drops of positive and negative clinical samples into the sample addition well of the reagent card, interpret the results after 30 minutes, and the results are invalid after 50 minutes.

[0065] Sensitivity and specificity results are as follows Figure 8 As shown in the results, the specificity of the invented reagent is basically the same as that of the competing products, but the sensitivity is higher than that of the competing reagents. Moreover, compared with the 30-minute interpretation time required by the competing products, the interpretation time of the present invention is shortened by 50%. While ensuring the detection sensitivity and specificity, the detection efficiency is significantly improved, making it more suitable for point-of-care testing scenarios.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immunochromatographic test strip for detecting NMP22, characterized in that, The immunochromatographic test strip includes a base plate and a sample pad, a conjugation pad, a detection pad, and absorbent paper that are stacked sequentially on the base plate. The binding pad contains a marker-labeled antibody against nuclear matrix protein 22; The test pad is equipped with test lines and quality control lines; The detection line is coated with an antibody-coated nuclear matrix protein 22. The nuclear matrix protein 22 labeled antibody and the nuclear matrix protein 22 coated antibody recognize different epitopes of nuclear matrix protein 22; The sample pad is a glass fiber membrane or polyester membrane impregnated with a sample pad treatment solution; the sample pad treatment solution includes 3~9 g / L tris(hydroxymethyl)aminomethane, 3~7 g / L sodium caseinate, 5~10 g / L polyvinylpyrrolidone-K10, 5~15 g / L bovine serum albumin, 0.1~0.3 g / L Proclin 300, 5~15 g / L Standapoles-1, 1~4 g / L disodium ethylenediaminetetraacetate dihydrate, and 2~4 g / L Tetronic 1307.

2. The immunochromatographic test strip according to claim 1, characterized in that, The conjugate pad also contains marker-labeled chicken IgY antibodies.

3. The immunochromatographic test strip according to claim 2, characterized in that, The quality control line is coated with goat anti-chicken IgY antibody.

4. The immunochromatographic test strip according to any one of claims 1-3, characterized in that, The markers include latex microspheres, colloidal gold, colloidal selenium, fluorescent particles, or quantum dots.

5. The immunochromatographic test strip according to claim 1, characterized in that, The detection pad is a nitrocellulose membrane.

6. The immunochromatographic test strip according to claim 1, characterized in that, The bonding pad is a hydrophilic microporous structure material, which is a glass fiber membrane or a polyester membrane.

7. The immunochromatographic test strip according to claim 1, characterized in that, The sample pad treatment solution comprises 5-8 g / L tris(hydroxymethyl)aminomethane, 3-7 g / L sodium caseinate, 7-10 g / L polyvinylpyrrolidone-K10, 3-8 g / L bovine serum albumin, 0.1-0.3 g / L Proclin 300, 7-10 g / L Standapoles-1, 2-3 g / L disodium ethylenediaminetetraacetate dihydrate, and 2-3 g / L Tetronic 1307.

8. The immunochromatographic test strip according to claim 7, characterized in that, The sample pad treatment solution comprises 7 g / L tris(hydroxymethyl)aminomethane, 3.5 g / L sodium caseinate, 7.5 g / L polyvinylpyrrolidone-K10, 5 g / L bovine serum albumin, 0.2 g / L Proclin 300, 10 g / L Standapoles-1, 2.5 g / L disodium ethylenediaminetetraacetate dihydrate, and 3 g / L Tetronic 1307.

9. The use of the immunochromatographic test strip according to any one of claims 1-8 in the preparation of bladder cancer detection products.

10. A reagent kit, characterized in that, Includes the immunochromatographic test strip according to any one of claims 1-8.

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