Colloidal selenium immunochromatography kit for detecting NT-proBNP

Abstract

The invention discloses a colloidal selenium immunochromatography kit for detecting NT-proBNP. The colloidal selenium immunochromatography kit comprises an immunochromatography test strip, the immunochromatography test strip comprises a bottom plate, a sample pad, a selenium label pad, a detection layer and a water absorption pad, wherein the sample pad, the selenium label pad, the detection layer and the water absorption pad are sequentially connected end to end and adhered to the bottom plate; the selenium label pad is coated with a mouse anti-human NT-proBNP mab-Clone-1 antibody marked by colloidal selenium, and a mouse anti-human NT-proBNP mab-Clone-1 antibody marked by colloidal selenium is coated with a mouse anti The detection layer is a cellulose acetate membrane, and a detection line coated with a mouse anti-human NT-proBNP mab-Clone-2 antibody and a quality control line coated with a goat anti-mouse IgG antibody are arranged on the cellulose acetate membrane. The colloidal selenium is adopted as a marker for NT-proBNP detection, detection of low-concentration NT-proBNP is realized by combining a double-antibody sandwich method, and the colloidal selenium-based NT-proBNP detection kit has relatively low detection limit, relatively good specificity and high detection speed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a colloidal selenium immunochromatographic kit for detecting NT-proBNP. Background Art

[0002] Heart failure (HF) is a syndrome in which the heart is unable to pump enough blood and oxygen to other organs to meet their metabolic needs. Approximately 64 million patients worldwide suffer from heart failure, and its incidence is on the rise due to factors such as an aging population, the increasing burden of cardiovascular disease (CVD) comorbidities and risk factors, and longer survival times after myocardial infarction. Common causes include myocardial infarction, cardiomyopathy, and myocarditis. High-risk groups include the elderly over 70 years old, with an incidence rate exceeding 10% and a 5-year mortality rate as high as 50%. Therefore, early detection, diagnosis, and treatment are crucial for slowing the progression of heart failure to the advanced stage and improving the overall prognosis. However, it is estimated that due to the diverse causes of heart failure, there is still no simple test that can determine the presence of heart failure. Therefore, the diagnosis of heart failure mainly involves comprehensively considering the medical history, physical examination, electrocardiogram, and chest X-ray to judge the possibility of heart failure, and then using natriuretic peptide detection and echocardiography to confirm the presence of heart failure. However, due to their high cost and high technical requirements, the use of these auxiliary tools is limited and not suitable as a screening tool for the general public. This has brought many difficulties to the early diagnosis of cardiovascular diseases and seriously affected the recovery of patients.

[0003] Currently, diagnosing heart failure is a huge challenge. To break through the current bottleneck, emerging technologies such as immunochromatography and microfluidic technology have advantages such as low cost, simple instruments, and easy integration, and have developed to a relatively mature level. They play an important role in the early prevention, diagnosis, and treatment of HF. However, considering the application situation, microfluidic technology still has certain limitations. N-terminal pro-brain natriuretic peptide (NT-proBNP) is a biomarker of cardiac function and the first-choice biomarker for the diagnosis and differential diagnosis of heart failure, as well as the assessment of severity and prognosis. It has been recommended as a first-level recommendation by multiple clinical practice guidelines. The "Chinese Guidelines for the Diagnosis and Treatment of Heart Failure 2024" states that NT-proBNP ≤ 300 pg / mL can rule out acute heart failure; ≤ 125 pg / mL can rule out chronic heart failure.

[0004] Among them, immunochromatographic detection is rapid, simple to operate, and low in cost, suitable for on-site detection. It can be observed with the naked eye without special detection instruments and is most widely used at the grass-roots level. Its detection principle is as follows: A kind of antigen or antibody is labeled with gold nanoparticles, latex microspheres or other nanomaterials, and the corresponding paired antigen or antibody is coated on the NC membrane of the reagent. During detection, when the sample contains the corresponding specific antibody or antigen, the particles of the nanomaterial-labeled antibody combine with the ligand in the sample to form a complex, and then chromatograph on the NC membrane and are captured by the coated antigen or antibody, forming a visible detection (T) line with the naked eye. The result is judged by the presence or absence of the detection line. At present, there is no immunochromatographic test strip for detecting HF on the market. The most commonly used lateral immunochromatographic test strip for detecting other diseases is a qualitative test strip labeled with colloidal gold. However, since the preparation of colloidal gold requires high-temperature boiling and the raw material cost is relatively high, it is still necessary to find a simpler preparation method and a more economical nanomaterial for labeling. Summary of the Invention

[0005] The object of the present invention is to provide a colloidal selenium immunochromatographic kit for detecting NT-proBNP, including an immunochromatographic test strip; The immunochromatographic test strip includes a bottom plate, a sample pad, a selenium-labeled pad, a detection layer, and a water absorption pad. The sample pad, the selenium-labeled pad, the detection layer, and the water absorption pad are sequentially and end-to-end adhered to the bottom plate; The selenium-labeled pad is coated with a colloidal selenium-labeled mouse anti-human NT-proBNP mab-Clone-1 antibody; The detection layer is a cellulose acetate membrane, and a detection line coated with a mouse anti-human NT-proBNP mab-Clone-2 antibody and a quality control line coated with a goat anti-mouse IgG antibody are provided on the cellulose acetate membrane.

[0006] Furthermore, the preparation method of the colloidal selenium-labeled mouse anti-human NT-proBNP mab-Clone-1 antibody includes the following steps: Step 1, preparation of colloidal selenium: PEG 20000 is added to water, and then a sodium dodecyl sulfate solution is added. After stirring, a sodium selenite solution is added, and finally an ascorbic acid solution is added. After stirring, colloidal selenium is prepared; Among them, the dosage ratio of water to PEG 20000 is 17 mL: 100 mg, and the dosage ratio of water to the sodium dodecyl sulfate solution, the sodium selenite solution, and the ascorbic acid solution is 1 mL of 0.1 M sodium dodecyl sulfate solution, 1 mL of 0.04 M sodium selenite solution, and 1 mL of 0.32 M ascorbic acid solution added to every 17 mL of water; Step 2, adjusting the pH of the colloidal selenium solution prepared in Step 1 to 8 with 0.1 M K2CO3; Step 3: Add the murine anti-human NT-proBNP mab-Clone-1 antibody to the colloidal selenium solution obtained in Step 2. After incubation, add 10% BSA solution for blocking. Then centrifuge the colloidal selenium-labeled antibody complex, and finally disperse the selenium-labeled antibody precipitate with the selenium-labeled reconstitution solution and store it at 4°C for later use. Among them, 25 μg of the murine anti-human NT-proBNP mab-Clone-1 antibody is added to every 1 mL of the colloidal selenium solution obtained in Step 2, and the selenium-labeled antibody precipitate is dispersed with 50 μL of the selenium-labeled reconstitution solution. Furthermore, the selenium-labeled reconstitution solution is a 0.01M pH 4.5 Tris buffer solution containing 3% BSA, 5% sucrose, 5% trehalose, and 0.1% Proclin 300.

[0007] In an embodiment of the present invention, the method for preparing the immunochromatographic test strip includes the following steps: Step 1: Take the glass cellulose membrane and pretreat it with a buffer solution. After drying, add the colloidal selenium-labeled murine anti-human NT-proBNP mab-Clone-1 antibody solution dispersed with the selenium-labeled reconstitution solution onto the pretreated glass cellulose membrane, and then dry it to obtain the selenium-labeled pad. Every 3 μL of the dispersed colloidal selenium-labeled murine anti-human NT-proBNP mab-Clone-1 antibody solution is added onto a 7 mm × 4 mm pretreated glass cellulose membrane. Step 2: On the nitrocellulose membrane, respectively scratch the murine anti-human NT-proBNP mab-Clone-2 antibody and the goat anti-mouse IgG antibody to form a test line and a quality control line. Step 3: Paste the sample pad, the selenium-labeled pad, the nitrocellulose membrane, and the absorbent pad on the backing in sequence to obtain the immunochromatographic test strip.

[0008] In an embodiment of the present invention, the kit further includes a positive control and a negative control.

[0009] The second object of the present invention is to provide the application of the above-mentioned colloidal selenium immunochromatographic kit in detecting NT-proBNP.

[0010] The present invention innovatively uses colloidal selenium as a marker for NT-proBNP detection and combines the double antibody sandwich method to achieve the detection of low-concentration NT-proBNP.

[0011] The present invention prepares colloidal selenium by a chemical reduction method, which is simple in operation, energy-saving and environmentally friendly. In particular, PEG20000 is added as a stabilizer during the preparation process, which can improve the stability of colloidal selenium, thereby enhancing the repeatability and accuracy of experiments. Through preliminary experiments by the inventor, it is found that false positives are likely to occur without adding SDS. After adding SDS, since SDS is an anionic surfactant, it can reduce the background interference of the kit, thereby reducing the occurrence of false positives.

[0012] As Figure 1 shown, the colloidal selenium immunochromatographic test strip of the present invention is based on the principle of sandwich immunoassay. The C line serves as a reference to verify the validity of the test strip result and always shows red. After the sample solution is premixed with colloidal selenium-NT-proBNP mab-Clone-1 (colloidal selenium-mAb), it moves towards the absorbent pad under capillary action. When there is no NT-proBNP antigen in the sample, the colloidal selenium-mAb cannot form a double-antibody sandwich structure with the NT-proBNP mab-Clone-2 antibody at the T line, and it can be seen with the naked eye that the T area does not show color. The excess colloidal selenium-mAb will be captured by the IgG antibody on the C line, so the C line also shows red. When the sample contains NT-proBNP antigen, the NT-proBNP antigen binds to part of the colloidal selenium-mAb to form a complex structure, flows through the T line, and binds to the anti-NT-proBNP antibody at the T line to form a sandwich structure, and it can be seen with the naked eye that the T area shows red. As the concentration of HF NT-proBNP antigen in the sample increases, the red color of the T line will be deeper, which is proportional to the concentration of HF NT-proBNP antigen in the sample.

[0013] The colloidal selenium immunochromatographic test strip of the present invention has a low detection limit (which can reach 125 pg / mL), meets the diagnostic criteria for chronic heart failure pointed out in the "Chinese Guidelines for the Diagnosis and Treatment of Heart Failure 2024", and has good specificity, fast detection speed, and can be used for home testing of people at high risk of behavior and rapid preliminary screening of potential patients in some medical institutions. Brief Description of the Drawings

[0014] Figure 1 is the detection principle of the colloidal selenium immunochromatographic test strip of the present invention.

[0015] Figure 2 is the detection result of the colloidal selenium immunochromatographic test strip of the present invention for NT-proBNP antigen. Among them, the NT-proBNP concentrations from left to right are 50000, 10000, 5000, 1000, 300, 125, 0 pg / mL in sequence.

[0016] Figure 3This is the test result of the colloidal selenium immunochromatographic test strip of the present invention for real serum. Among them, the NT-proBNP concentrations from left to right are 10192.8, 979.0, 6482.6, 17308.6, 5012.9, 714.4, 1819.7, 684.3, 18228.7, 1218.3 pg / mL in turn.

[0017] Figure 4 This is the specific test result of the colloidal selenium immunochromatographic test strip of the present invention. Detailed implementation manners

[0018] The preferred implementation manners of the present invention will be described in detail below in conjunction with embodiments. It should be understood that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0019] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0020] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0021] Mouse anti-human NT-proBNP mab-Clone-1 antibody, mouse anti-human NT-proBNP mab-Clone-2 antibody and recombinant human NT-proBNP antigen are all obtained from Nanjing Bioeasy Biotechnology Co., Ltd., goat anti-mouse IgG antibody is purchased from Hangzhou Qitai Biotechnology Co., sodium selenite, ascorbic acid and sodium dodecyl sulfate are all purchased from Shanghai Aladdin Pharmaceutical Co., and PEG20000 is purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Example 1

[0022] I. Colloidal selenium-labeled anti-NT-proBNP antibody 1. Preparation of colloidal selenium: First, prepare 0.1 M SDS, 0.32 M ascorbat (Vc), and 0.04 M Na2SeO3, and filter for later use. Pour 17 mL of ultrapure water into a clean beaker equipped with a clean rotor of appropriate size, then add 100 mg of PEG20000 to the small beaker, stir at 600 rpm for 10 minutes at room temperature, then add 1 mL of 0.1 M SDS, stir at room temperature for 20 minutes, then add 1 mL of 0.04 M Na2SeO3, stir at room temperature for 20 minutes, and finally add 1 mL of 0.32 M Vc to create a reducing environment. At this time, the color of the solution in the beaker gradually changes from colorless to orange, and colloidal selenium is obtained after stirring at room temperature for 20 minutes.

[0023] 2. Adjust the pH of the colloidal selenium solution prepared in step 1 to 8 with 0.1 M K2CO3.

[0024] 3. Labeling of mouse anti-human NT-proBNP mab-Clone-1: Add 25 μg of mouse anti-human NT-proBNP mab-Clone-1 antibody to 1 mL of the colloidal selenium solution in step 2, and then incubate for 20 minutes. Then add 100 μL of 10% BSA solution to block for 20 minutes, and then centrifuge the colloidal selenium-labeled antibody complex at 8500 r for 15 minutes. Finally, redisperse the selenium-labeled antibody precipitate with 50 μL of selenium-labeled complex solution (3% BSA, 5% sucrose, 5% trehalose, 0.1% Proclin 300, 0.01 M Tris-HCl, pH 4.5), and store at 4 °C for further experiments.

[0025] II. Preparation of a gold-grown hollow gold immunochromatographic test strip for detecting NT-proBNP antigen 1. Preparation of the selenium-labeled pad: The selenium-labeled pad (glass fiber membrane) is pre-treated with a buffer solution (20 mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose, 0.1% P300, pH 8.5), dried at 40 °C for 2 h, and cut into pieces of 7 mm × 4 mm with a ZQ2002 type guillotine. After centrifugation of the colloidal selenium-binding antibody, discard the supernatant. Take 3 μL of the solution dispersed with 50 μL of selenium-labeled complex solution and evenly add it to the treated selenium-labeled pad, and dry it at 40 °C for 2 h. After drying, it is ready for use.

[0026] 2. Preparation of the test line: Dilute the mouse anti-human NT-proBNP mab-Clone-2 antibody to 2 mg / mL with 0.1 M PBS at pH 7.4 to make the test line antibody; use a membrane scribing instrument to scribe the antibody on the nitrocellulose membrane at a concentration of 1 μL / cm to form the test line.

[0027] 3. Preparation of the quality control line: Dilute the goat anti-mouse IgG antibody to 0.5 mg / mL with 0.1 M PBS at pH 7.4 to make the quality control line antibody; use a membrane scribing instrument to scribe the antibody on the cellulose acetate membrane at a concentration of 1 μL / cm to form the quality control line.

[0028] 4. Place the scribed nitrocellulose membrane in an oven at 37 °C and dry for 3 hours to obtain the treated nitrocellulose membrane.

[0029] 5. Test strip assembly: Paste the sample pad, selenium-labeled pad, nitrocellulose membrane, and absorbent pad on the backing in sequence. Detection line antibody and quality control line antibody are provided on the nitrocellulose membrane; Cut the pasted plastic plate to 3 - 4 mm and place it in the test card; Store it sealed at room temperature in a drying dish containing a large amount of desiccant. Example 2

[0030] Detection of NT-proBNP recombinant antigen standard sample The NT-proBNP antigen standard solution is prepared by the following steps: 1. Use PBS buffer (prepared from PBS buffer, 0.8% NaCl, 0.02% KCl, 0.027% KH2PO4, 0.142% Na2HPO4, pH 7.4) to gradient dilute NT-proBNP to obtain NT-proBNP antigen standard solutions with different concentrations.

[0031] 2. Use the prepared colloidal selenium immunochromatographic test strip, add 80 μL of sample solution to the sample pad. The sample solution moves towards the absorbent pad under capillary action. After mixing with the colloidal selenium-NT-proBNP mab-Clone-1 in the selenium-labeled pad, it continues to move forward, recognizes the antibodies on the T line and C line of the NC membrane, and detects NT-proBNP antigen dilutions with different concentrations (take 14 different concentrations of standard products, which are 0, 125, 300, 1000, 5000, 10000, 50000 pg / mL respectively). After the membrane chromatography reaction ends (10 - 15 min), observe the color development result.

[0032] As Figure 2 shown, the detection limit of this colloidal selenium immunochromatographic test strip is 125 pg / mL, meeting the diagnostic criteria for chronic heart failure pointed out in the "Chinese Guidelines for the Diagnosis and Treatment of Heart Failure 2024". Example 3

[0033] Detection of NT-proBNP antigen in real serum Dilute the real serum of heart failure patients (provided by Taixing People's Hospital) with PBS to the corresponding concentration. The diluted concentration is in the range of 684 pg / mL - 18200 pg / mL, and take 80 μL of the real serum dilution for detection. As Figure 3 shown, this colloidal selenium immunochromatographic test strip can effectively detect NT-proBNP in the serum of heart failure patients. Example 4

[0034] Specificity of the colloidal selenium immunochromatographic test strip Preparation of negative control and positive control diluents: First, dilute the 1 μg / mL NT-proBNP antigen standard solution with serum to 50 ng / mL (positive control), and prepare a blank serum diluent (negative control). Then, take 40 μL of the negative control solution and the positive control solution, and mix them with 40 μL of the sample diluent (prepared with 0.8% NaCl, 0.02% KCl, 0.027% KH2PO4, 0.142% Na2HPO4, pH 7.4) to prepare a negative control sample solution and a 25 ng / mL positive control NT-proBNP antigen sample solution.

[0035] Preparation of interferent diluents: Dilute with blank serum to prepare 50 ng / mL CRP diluent, 50 ng / mL troponin diluent, and 50 ng / mL phospholipase A2 diluent. Then, mix 40 μL of the serum-diluted CRP diluent, troponin diluent, and phospholipase A2 diluent with 40 μL of the sample diluent respectively to prepare 25 ng / mL CRP sample solution, 25 ng / mL troponin sample solution, and 25 ng / mL phospholipase A2 sample solution.

[0036] 1. Take 80 μL of the negative control sample solution, 25 ng / mL positive control NT-proBNP antigen sample solution, 25 ng / mL CRP sample solution, 25 ng / mL troponin sample solution, and 25 ng / mL phospholipase A2 sample solution respectively for sample addition detection.

[0037] 2. Divide the 25 ng / mL positive control NT-proBNP antigen sample solution into three portions, each portion being 70 μL, and mix them evenly with 30 μL of the above-prepared three interferents to obtain a 100 μL mixed solution. Take 80 μL for sample addition detection.

[0038] The experimental results are as Figure 4 shown. The interferents CRP protein, troponin, and phospholipase A2 in the relevant serum do not interfere with the detection of NT-proBNP antigen in the serum, and the colloidal selenium test strip has good specificity.

Claims

1. A colloidal selenium immunochromatography kit for detecting NT-proBNP, characterized in that: Including immunochromatographic test strips; The immunochromatographic test strip comprises a base plate, a sample pad, a selenium label pad, a detection layer and a water absorbent pad, wherein the sample pad, the selenium label pad, the detection layer and the water absorbent pad are sequentially connected end to end and pasted on the base plate; The selenium label pad is coated with colloidal selenium-labeled mouse anti-human NT-proBNP mab-Clone-1 antibody; The detection layer is a cellulose acetate membrane, on which a detection line coated with mouse anti-human NT-proBNP mab-Clone-2 antibody and a quality control line coated with goat anti-mouse IgG antibody are arranged.

2. The colloidal selenium immunochromatography kit for detecting NT-proBNP according to claim 1, characterized in that: The preparation method of the colloidal selenium-labeled mouse anti-human NT-proBNP mab-Clone-1 antibody comprises the following steps: Step 1, preparation of colloidal selenium: adding PEG 20000 to water, then adding sodium dodecyl sulfate solution, adding sodium selenite solution after stirring, and finally adding ascorbic acid solution, stirring to prepare colloidal selenium; Step 2, adjusting the pH of the colloidal selenium solution prepared in step 1 to 8; Step 3, add mouse anti-human NT-proBNP mab-Clone-1 antibody to the colloidal selenium solution of step 2, add BSA solution for blocking after incubation, then centrifuge the colloidal selenium-labeled antibody complex, and finally disperse the selenium-labeled antibody precipitate with selenium-labeled solution and store for later use.

3. The colloidal selenium immunochromatography kit for detecting NT-proBNP according to claim 2, characterized in that: In step 1, the ratio of water to PEG 20000 is 17 mL:100 mg, and the ratio of water to sodium dodecyl sulfate solution, sodium selenite solution, and ascorbic acid solution is 1 mL of 0.1 M sodium dodecyl sulfate solution, 1 mL of 0.04 M sodium selenite solution, and 1 mL of 0.32 M ascorbic acid solution per 17 mL of water.

4. The colloidal selenium immunochromatography kit for detecting NT-proBNP according to claim 2, characterized in that: In step 3, 25 μg of mouse anti-human NT-proBNP mab-Clone-1 antibody was added to every 1 mL of colloidal selenium solution in step 2, and the resulting selenium-labeled antibody precipitate was dispersed with 50 μL of selenium-labeled reconstitution solution.

5. The colloidal selenium immunochromatography kit for detecting NT-proBNP according to claim 4, characterized in that: The selenium labeling solution is a 0.01M pH 4.5 Tris buffer solution containing 3% BSA, 5% sucrose, 5% trehalose and 0.1% Proclin 300.

6. The colloidal selenium immunochromatography kit for detecting NT-proBNP according to claim 2, characterized in that: The method for preparing the immunochromatographic test strip comprises the following steps: Step 1, pre-treat a glass cellulose membrane with a buffer solution, dry it, take a colloidal selenium-labeled mouse anti-human NT-proBNP mab-Clone-1 antibody solution dispersed with a selenium labeling solution and add it to the pre-treated glass cellulose membrane, and dry it to obtain a selenium labeling pad; Step 2, respectively scratching mouse anti-human NT-proBNP mab-Clone-2 antibody and goat anti-mouse IgG antibody on the nitrocellulose membrane to form a detection line and a quality control line; Step 3, pasting the sample pad, selenium label pad, nitrocellulose membrane and water absorbent pad on the backing in order to obtain the immunochromatographic test strip.

7. The colloidal selenium immunochromatography kit for detecting NT-proBNP according to claim 6, characterized in that: The concentration of the detection line is 1 μL / cm, and the concentration of the quality control line is 1 μL / cm.

8. The colloidal selenium immunochromatography kit for detecting NT-proBNP according to claim 1, characterized in that: The kit also includes a positive control and a negative control.

9. Use of the colloidal selenium immunochromatography kit according to any one of claims 1 to 8 in detecting NT-proBNP.

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