Labeled antibody and preparation method thereof, and chromatography test strip

Abstract

This application discloses a labeled antibody, a method for preparing the same, and a chromatographic test strip. The labeled antibody comprises a marker and an antibody loaded on the marker, wherein the marker is a carboxyl polypyrrole nanoparticle. The application uses carboxyl polypyrrole nanoparticles as a marker to prepare the labeled antibody. Carboxyl polypyrrole nanoparticles are easy to prepare, have high storage stability, and are low in cost. Immunochromatographic test strips using carboxyl polypyrrole nanoparticles as markers can detect relatively low concentrations of target proteins, exhibit high sensitivity, and exhibit good anti-interference properties, making them suitable for widespread use.

Description

Technical Field

[0001] The present application relates to the technical field of immunoassays, and in particular to labeled antibodies and preparation methods thereof, and chromatography test strips. Background Art

[0002] Protein determination is currently important in various fields, including clinical diagnostics, treatment monitoring, food safety, and environmental analysis. Commonly used techniques for protein detection include colorimetric immunoassays, electrochemical methods, radioimmunoassays, enzyme-linked immunosorbent assays (ELISAs), western blotting, and fluorescent immunoassays. However, due to instrumentation requirements, these techniques are time-consuming and limited to laboratory use. Therefore, the development of simple and rapid methods for protein detection remains essential and crucial.

[0003] Immunochromatographic test strips are a type of point-of-care test that has attracted widespread attention due to their rapidity and simplicity. Another major advantage of immunochromatographic test strips is that they can be performed on a variety of biological samples, including plasma, sweat, saliva, serum, urine, and whole blood. Currently, various nanoparticles, including gold nanoparticles (AuNPs), quantum dots (QDs), magnetic particles, upconversion phosphors, and Raman-active nanomaterials, have been used as labels for immunochromatographic test strip development.

[0004] How to improve the detection sensitivity of immunochromatographic test strips and reduce the detection cost is a current research hotspot. Summary of the Invention

[0005] One object of the present application is to provide a labeled antibody that can be applied to a chromatographic test strip and has high detection sensitivity and a preparation method thereof.

[0006] Another object of the present application is to provide a chromatography test strip with higher detection sensitivity.

[0007] Another object of the present application is to provide a use of carboxypolypyrrole nanoparticles.

[0008] To achieve the above objectives, the present application provides a labeled antibody, comprising a label and an antibody loaded on the label, wherein the label is carboxyl polypyrrole nanoparticles.

[0009] Furthermore, the average particle size of the carboxyl polypyrrole nanoparticles is 10 to 200 nm.

[0010] Furthermore, the carboxyl polypyrrole nanoparticles are prepared by oxidative copolymerization of pyrrole monomer and carboxyl pyrrole monomer.

[0011] Furthermore, the carboxyl pyrrole monomer is selected from a mixture of one or more of the following: pyrrole-3-carboxylic acid and pyrrole-1-propionic acid.

[0012] Furthermore, the carboxyl polypyrrole nanoparticles are prepared by the following method: chemically modifying polypyrrole nanoparticles with an organic carboxylic acid having a reactive group, wherein the reactive group is suitable for reacting with polypyrrole to graft the carboxyl group onto the polypyrrole main chain.

[0013] Furthermore, the reactive group is suitable for addition reaction with the five-membered heterocycle on the polypyrrole main chain, the reactive group is a thiol group, and the organic carboxylic acid having a reactive group is selected from a mixture of one or more of the following: thioglycolic acid, mercaptopropionic acid, 6-mercaptohexanoic acid, mercaptosuccinic acid, and cysteine.

[0014] The present application also provides a method for preparing a labeled antibody, comprising adding a carboxyl activator to a dispersion of carboxyl polypyrrole nanoparticles, and then adding an antibody to couple the antibody to the carboxyl polypyrrole nanoparticles.

[0015] The present application also provides a chromatography test strip loaded with a labeled antibody suitable for specific binding to a target, characterized in that the labeled antibody is the aforementioned labeled antibody.

[0016] Furthermore, the chromatography test strip includes a base plate and a sample pad, a conjugation pad, a test pad and a water-absorbing pad arranged on the base plate in sequence, the conjugation pad is loaded with the labeled antibody, the test pad is provided with a detection area and a quality control area, the detection area is close to the conjugation pad, the quality control area is close to the water-absorbing pad, the detection area is provided with a coated antibody suitable for specific binding to the target, and the quality control area is provided with a secondary antibody.

[0017] The present application also provides a use of carboxyl polypyrrole nanoparticles, wherein the carboxyl polypyrrole nanoparticles are used as a marker for antibody labeling.

[0018] Compared with the existing technology, the beneficial effects of the present application are: the present application uses carboxyl polypyrrole nanoparticles as a marker to prepare labeled antibodies. Carboxyl polypyrrole nanoparticles are easy to prepare, have high storage stability, and are low in cost. The immunochromatographic test strips using carboxyl polypyrrole nanoparticles as markers can detect lower concentrations of target proteins, have high sensitivity and good anti-interference properties, and are suitable for popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of an embodiment of a chromatography test strip of the present application;

[0020] Figure 2This is an electron microscope photograph of carboxyl polypyrrole nanoparticles prepared in Example 1;

[0021] Figure 3 These are photos of the chromatographic test strips of Example 4 detecting different concentrations of CEA;

[0022] Figure 4 These are photos of the chromatographic test strips of Example 5 detecting different concentrations of CA-125;

[0023] Figure 5 This is a photo of the chromatographic test strip of Example 6 detecting different concentrations of the novel coronavirus antigen N protein;

[0024] In the figure: 1. Sample pad; 2. Conjugate pad; 3. Test pad; 31. Detection area; 32. Quality control area; 4. Absorbent pad. DETAILED DESCRIPTION

[0025] Below, the present application is further described in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0026] The terms "comprises" and "having" and any variations thereof in the specification and claims of this application are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus that includes a series of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, product or apparatus.

[0027] The present application provides a labeled antibody, comprising a label and an antibody loaded on the label, wherein the label is carboxyl polypyrrole nanoparticles.

[0028] The labeled antibody provided herein is suitable for use in immunochromatographic test strips, wherein the antibody is suitable for specific binding to a target. Carboxypolypyrrole nanoparticles can form a visible black area when aggregated in small amounts, and the more aggregated, the darker the color. The use of carboxypolypyrrole nanoparticles as a marker is highly sensitive and can achieve qualitative or semi-quantitative detection of the target. Furthermore, carboxypolypyrrole nanoparticles have high storage stability and low cost, making them suitable for practical use as a marker.

[0029] It is worth mentioning that simple polypyrrole nanoparticles are difficult to load a large amount of antibodies on their surface due to the lack of necessary functional groups. Therefore, this application uses polypyrrole nanoparticles with carboxyl groups, which can be coupled with the amino groups on the antibodies through chemical bonds to establish a double-antibody sandwich immunoassay test strip.

[0030] In some embodiments, pyrrole monomer and carboxyl pyrrole monomer may be polymerized in one step by oxidative polymerization to obtain carboxyl polypyrrole nanoparticles.

[0031] In a specific embodiment, carboxyl polypyrrole nanoparticles are prepared by the following method: a solvent and a stabilizer are mixed in a container, and then pyrrole monomer and carboxyl pyrrole monomer are added. After stirring for a period of time, an oxidant is quickly added. After stirring for a period of time at room temperature, methanol is added to terminate the reaction.

[0032] In the above embodiments, the stabilizer can be, but is not limited to, polyvinyl alcohol (PVA), sodium dodecyl sulfate (SDS), sodium docusate (AOT), polyethylene glycol (PEG), Tween 40 (Polyoxyethylenesorbitan monopalmitate or TWEEN 40), Tween 60 [Poly(ethyleneglycol)sorbitan monostearate or TWEEN 60], and polyvinylpyrrolidone (PVP).

[0033] Furthermore, the molecular weight M of polyvinyl alcohol w =9000 or 13000 or 31000, the molecular weight of polyethylene glycol M n =4000, molecular weight of polyvinylpyrrolidone M n =40000.

[0034] In the above embodiments, the carboxyl pyrrole monomer may be, but is not limited to, pyrrole-3-carboxylic acid and pyrrole-1-propionic acid.

[0035] In the above embodiments, the oxidant may be, but is not limited to, FeCl3, K2S2O8, H2O2, (NH4)S2O8, AgNO3, and CuCl2.

[0036] In the above embodiments, the solvent may be, but is not limited to, water, methanol, or diethyl ether.

[0037] In a specific embodiment, carboxyl polypyrrole nanoparticles were prepared by the following method: 170 mL of water and 0.7 g of a stabilizer were added to a 500 mL conical flask and stirred at room temperature at 900 rpm for 20 min; then 330 mg of pyrrole monomer and 180 mg of carboxyl pyrrole monomer were added and stirred at room temperature at 900 rpm for 20 min; then 30 mL of an oxidant (concentration of 56 mg / mL) was quickly added and stirred at room temperature at 1500 rpm for 6 h; then 20 mL of methanol was added to terminate the reaction, and the supernatant was removed after centrifugation at 15000 rpm for 30 min. The precipitate was dispersed in deionized water and stored at room temperature for later use.

[0038] In other embodiments, polypyrrole nanoparticles may be chemically modified with organic carboxylic acids having reactive groups, thereby modifying the carboxyl groups on the surface of the polypyrrole nanoparticles. That is, the reactive groups are suitable for reacting with polypyrrole to graft the carboxyl groups onto the polypyrrole main chain.

[0039] In one embodiment, polypyrrole nanoparticles are first prepared by oxidative polymerization. An organic carboxylic acid having a thiol group is then added to a solution containing the dispersed polypyrrole nanoparticles to produce carboxylated polypyrrole nanoparticles. It is worth noting that the thiol group is suitable for addition reactions with the five-membered heterocyclic ring on the polypyrrole backbone.

[0040] In the above embodiment, the organic carboxylic acid having a mercapto group may be, but is not limited to, thioglycolic acid, mercaptopropionic acid, 6-mercaptohexanoic acid, mercaptosuccinic acid, and cysteine.

[0041] In a specific embodiment, 170 mL of water and 0.7 g of a stabilizer were added to a 500 mL conical flask and stirred at room temperature for 20 min at 900 rpm; 500 mg of pyrrole monomer was added and stirred at room temperature for 20 min at 900 rpm; 30 mL of an oxidant (at a concentration of 56 mg / mL) was quickly added and stirred at room temperature for 6 h at 1500 rpm; 20 mL of methanol was then added to terminate the reaction, and the mixture was centrifuged at 15000 rpm for 30 min and then dispersed in deionized water and stored at room temperature to obtain polypyrrole nanoparticles; 50 mL of the aforementioned polypyrrole nanoparticles was taken, 3 mg of a thiol reagent was added, stirred at room temperature for 24 hours, and then centrifuged at 15000 rpm for 30 min, the supernatant was removed, and the precipitate was dispersed in deionized water and stored at room temperature for later use. The stability and oxidant selection in this embodiment can be referred to the stabilizer and oxidant described above.

[0042] In some embodiments, the particle size of the carboxyl polypyrrole nanoparticles is 10 nm to 200 nm. Further, in some embodiments, the particle size of the carboxyl polypyrrole nanoparticles is 30 nm to 80 nm.

[0043] The labeled antibody can be prepared by the following method: adding a carboxyl activator to the dispersion of the carboxyl polypyrrole nanoparticles, and then adding the antibody to couple the antibody to the carboxyl polypyrrole nanoparticles.

[0044] The carboxyl activating agent may be, but is not limited to, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and sodium N-hydroxysuccinimide sulfonate (NHS).

[0045] It is worth mentioning that the antibody is coupled to the carboxypyrrole nanoparticles via a chemical bond.

[0046] In one embodiment, the method for preparing the labeled antibody comprises the following steps:

[0047] S1, providing a dispersion of polypyrrole nanoparticles having carboxyl groups;

[0048] S2, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and sodium salt of N-hydroxysuccinimide sulfonate (NHS) to the dispersion, reacting for a period of time, centrifuging, and resuspending the precipitate in phosphate buffer after centrifugation;

[0049] S3, adding the antibody to the solution obtained in step S2, reacting for a period of time, and centrifuging the solution. After centrifugation, the precipitate is resuspended in a storage solution to obtain a composition containing the labeled antibody.

[0050] Furthermore, the storage solution comprises Na3PO4·12H2O, bovine serum albumin, sucrose, Tween-20 and water. In a specific embodiment, the storage solution comprises 304 mg of Na3PO4·12H2O, 2 g of bovine serum albumin, 4 g of sucrose, 0.1 g of Tween-20 and 40 g of water.

[0051] In a specific embodiment, the preparation method of the labeled antibody is as follows: 25 μL of 3 mg / mL 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC) and 25 μL of 3 mg / mL N-hydroxysuccinimide sulfonic acid sodium salt (NHS) are added to 1 mL of carboxypolypyrrole nanoparticle dispersion, and the mixture is shaken at room temperature for 30 minutes; the dispersion is centrifuged at 12000 rpm for 10 minutes, the precipitate is resuspended in 300 μL phosphate buffered saline (PBS), the antibody (10 μL, 1 mg / mL) is added, and the mixture is shaken at room temperature for 4 hours; the dispersion is centrifuged at 12000 rpm for 10 minutes, the precipitate is resuspended in 300 μL 10% bovine serum albumin (BSA) solution and shaken at room temperature for 30 minutes; the dispersion is then centrifuged at 12000 rpm for 10 minutes, and the precipitate is resuspended in 200 μL storage solution.

[0052] like Figure 1 As shown, the present application also provides a chromatography test strip loaded with the labeled antibody suitable for specific binding to the target.

[0053] Furthermore, the chromatography test strip includes a base plate (not shown in the figure) and a sample pad 1, a conjugation pad 2, a test pad 3 and an absorbent pad 4 arranged on the base plate in sequence, the conjugation pad 2 is loaded with the labeled antibody of the present application, and the test pad 3 is provided with a detection area 31 and a quality control area 32, the detection area 31 is close to the conjugation pad 2, and the quality control area 32 is close to the absorbent pad 4, the detection area 31 is provided with a coated antibody suitable for specific binding to the target, and the quality control area 32 is provided with a secondary antibody.

[0054] The method of using the chromatography test strip is as follows: immerse the sample pad 1 in a solution containing the target substance, and the liquid migrates to the absorbent pad 4. Visually evaluate the detection area 31 and the quality control area 32 within a certain period of time. If black lines appear in both the detection area 31 and the quality control area 32, it is a positive test; if no lines appear in the detection area 31 but lines appear in the quality control area 32, it is a negative test; if no lines appear in the quality control area 32, the test is invalid.

[0055] The working principle of the chromatographic test strip is as follows: When the sample pad 1 is immersed in a solution containing the target, the target first reaches the conjugate pad 2 and binds to the labeled antibody. Then, the labeled antibody bound to the target and the labeled antibody unbound to the target reach the test pad 3. At the detection zone 31, the target binds to the coated antibody, causing a certain amount of labeled material to accumulate in the detection zone 31, thus displaying a black line. At the quality control zone 32, the labeled antibody binds to the secondary antibody, causing a certain amount of labeled material to accumulate in the quality control zone 32, thus displaying a black line. In other words, if the detection zone 31 does not display a black line, it indicates that the sample solution does not contain the target, or the concentration of the target is below the minimum detection limit of the chromatographic test strip.

[0056] In some embodiments, the test pad 3 is a nitrocellulose membrane, the overlap between the sample pad 1 and the nitrocellulose membrane is no less than 2 mm, and the overlap between the nitrocellulose membrane and the absorbent pad 4 is no less than 2 mm. It is worth noting that the length direction herein refers to the direction of lateral movement of the liquid.

[0057] In some embodiments, the distance between the detection area 31 and the quality control area 32 is 4 to 6 mm.

[0058] In some embodiments, the sample pad 1 is made of glass fiber.

[0059] The present application also provides a method for preparing the chromatography test strip, comprising the following steps:

[0060] On the PVC base plate, fix the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad in sequence;

[0061] A solution containing the labeled antibody is sprayed on the conjugate pad, the coated antibody is sprayed on the nitrocellulose membrane as a detection area, and the secondary antibody is sprayed on the nitrocellulose membrane as a quality control area, wherein the adsorption amount of the labeled antibody is 8-12 μL / cm, the spraying amount of the coated antibody is 0.5-2 μL / cm; and the spraying amount of the secondary antibody is 0.5-2 μL / cm.

[0062] The present application also provides a use of carboxyl polypyrrole nanoparticles, wherein the carboxyl polypyrrole nanoparticles are used as a marker for labeling antibodies.

[0063] [Example 1]

[0064] Preparation of carboxyl polypyrrole nanoparticle dispersion: add 20 mL of 7.5% PVA (M w To an aqueous solution (187 mg of FeCl₃) (at 9000 rpm), the mixture was mixed thoroughly. Then, 20 mg of pyrrole and 15 mg of pyrrole-3-carboxylic acid were added, and the mixture was stirred at room temperature for 24 hours. The supernatant was removed by centrifugation (12,000 rpm for 20 minutes), and the resulting solid was washed three times with hot water and dispersed in water for later use. The mass fraction of carboxypolypyrrole nanoparticles in the dispersion was 3.0 wt‰.

[0065] Figure 2 The electron microscope photograph of the dispersion of Example 1 is shown. Figure 2 The photographs show that in Example 1, carboxyl polypyrrole nanoparticles with a particle size of about 30 nm to 80 nm were prepared.

[0066] [Example 2]

[0067] Preparation of a carboxypolypyrrole nanoparticle dispersion: 20 mL of water and 1.6 g of PVA (Mw = 31,000) were added to a conical flask and stirred at 900 rpm for 20 minutes at room temperature. 100 mg of pyrrole monomer was then added and stirred at 900 rpm for 20 minutes at room temperature. 10 mL of 56 mg / mL ferric chloride aqueous solution was then quickly added and stirred at 1500 rpm for 6 hours at room temperature. 5 mL of methanol was added to terminate the reaction. The supernatant was removed and the resulting solid was washed three times with hot water and then dispersed in water for later use. The mass fraction of polypyrrole nanoparticles in the dispersion was 2.5 wt‰. To 10 mL of the dispersion, 30 mg of thioglycolic acid was added and the mixture was stirred at 30°C for 24 hours. The mixture was centrifuged at 15,000 rpm for 30 minutes, the supernatant removed, the solution was washed twice with water, and the dispersion was dispersed in water. The mass fraction of carboxypolypyrrole nanoparticles in the dispersion was 3.0 wt‰.

[0068] [Example 3]

[0069] Preparation of a composition containing a labeled antibody: To 1 mL of the carboxypolypyrrole nanoparticle dispersion of Example 1 or Example 2, 25 μL of 3 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 25 μL of 3 mg / mL N-hydroxysuccinimide sulfonic acid sodium salt (NHS) were added and shaken at room temperature for 30 minutes. The dispersion was centrifuged at 12,000 rpm for 10 minutes, and the pellet was resuspended in 300 μL of phosphate buffered saline (PBS). 10 μL of the target antibody (1 mg / mL) was added and shaken at room temperature for 4 hours. The dispersion was then centrifuged at 12,000 rpm for 10 minutes, and the pellet was resuspended in 300 μL of 10% bovine serum albumin (BSA) solution and shaken at room temperature for 30 minutes. Finally, the dispersion was centrifuged at 12,000 rpm for 10 minutes, and the pellet was resuspended in 200 μL of storage solution.

[0070] The stock solution includes 304 mg of Na3PO4·12H2O, 2 g of bovine serum albumin, 4 g of sucrose, 0.1 g of Tween-20, and 40 g of water.

[0071] [Example 4]

[0072] Preparation of a chromatographic test strip for carcinoembryonic antigen (CEA): A sample pad, nitrocellulose membrane, and absorbent pad were affixed sequentially to a PVC base plate (300 × 60 mm), the sample pad 22 mm × 30 cm, and the nitrocellulose membrane 25 mm × 30 cm. The test zone was 3.5 mm long, and the control zone was 3.5 mm long. The absorbent pad and sample pad overlapped the nitrocellulose membrane by 2 mm. A CEA-coated antibody (1 mg / mL, mouse) was sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm2 as the test zone. A goat anti-mouse IgG (1 mg / mL) was sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm2 as the control zone. The control zone was 6 mm apart, and the membrane was dried at 37°C for 30 min after spraying. The PVC base plate and its layers were cut into 3.5 mm wide chromatographic test strips using a strip cutter. It is worth mentioning that the length direction of the present application refers to the direction of lateral movement of the liquid.

[0073] CAE detection: 5 μL of a composition containing a labeled antibody (prepared by the method of Example 3, where the target antibody in Example 3 is an antibody to CEA) was mixed with 100 μL of PBS buffer containing different CEA concentrations. The sample pad of the chromatography test strip was immersed in each mixture, and the liquid migrated to the absorbent pad. The test area and quality control area were visually evaluated within 10 minutes.

[0074] Figure 3From left to right, the photographs are of the test strips immersed in mixed solutions with target concentrations of 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, and 100 ng / mL. From the display results of the test line and the quality control line, it can be concluded that the labeled antibody prepared in this application can be applied to the chromatographic test strips to detect carcinoembryonic antigen at a concentration of 1 ng / mL or above.

[0075] [Example 5]

[0076] Preparation of a chromatographic test strip for detecting cancer antigen 125 (CA-125): A sample pad, nitrocellulose membrane, and absorbent pad were affixed sequentially to a PVC base plate (300 × 60 mm), the sample pad 22 mm × 30 cm, and the nitrocellulose membrane 25 mm × 30 cm. The test zone was 3.5 mm long, and the control zone was 3.5 mm long. The absorbent pad and sample pad overlapped the nitrocellulose membrane by 2 mm. The nitrocellulose membrane was sprayed with a coating antibody against CA-125 (1 mg / mL, mouse) at a rate of 1 μL / cm2 as the test zone. The nitrocellulose membrane was sprayed with goat anti-mouse IgG (1 mg / mL) at a rate of 1 μL / cm2 as the control zone. The control zone was 6 mm apart from the test zone. After spraying, the membrane was dried at 37°C for 30 min. The PVC base plate and its layers were cut into chromatographic test strips 3.5 mm wide using a strip cutter.

[0077] Detection of CA-125: Mix 5 μL of a composition containing a labeled antibody (prepared by the method of Example 3, where the target antibody in Example 3 is an antibody to CA-125) with 100 μL of PBS buffer containing different concentrations of CA-125. Immerse the sample pad of the chromatography test strip in each mixture, allowing the liquid to migrate to the absorbent pad. Visually evaluate the test area and quality control area within 10 minutes.

[0078] Figure 4 From left to right, the photographs are of the test strips immersed in mixed solutions with target concentrations of 0 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 50 ng / mL, and 100 ng / mL. From the display results of the test line and the quality control line, it can be concluded that the labeled antibody prepared in this application is applied to the chromatographic test strips to detect CA-125 with a concentration of more than 1 ng / mL.

[0079] [Example 6]

[0080] Preparation of chromatography test strips for detecting novel coronavirus antigen N protein: On a PVC base plate, fix the sample pad, nitrocellulose membrane and absorbent pad in sequence. The specifications of the PVC base plate are 300×60 mm, the specifications of the sample pad are 22 mm×30 cm, and the specifications of the nitrocellulose membrane are 25 mm×30 cm. The length of the detection area is 3.5 mm, the length of the quality control area is 3.5 mm, and the absorbent pad and the sample pad overlap with the nitrocellulose membrane by 2 mm respectively; spray mouse anti-SARS-CoV-2 monoclonal antibody-1 (1 mg / mL) on the nitrocellulose membrane as the detection area, and the spraying amount is 1 μ L / cm; spray goat anti-mouse IgG (1 mg / mL) on the nitrocellulose membrane as a quality control area at a spraying amount of 1 μL / cm; the quality control area is 6 mm away from the detection area, and the membrane is dried at 37°C for 30 min after spraying; a composition containing a labeled antibody (prepared by the method of Example 3, wherein the labeled antibody, i.e., carboxypolypyrrole nanoparticle-modified mouse anti-SARS-CoV-2 monoclonal antibody-2, has a mass fraction of 15 wt‰) is sprayed on the conjugate pad at a spraying amount of 4 μL / cm; the PVC base plate and the layers thereon are cut into chromatography test strips with a width of 3.5 mm using a strip cutter.

[0081] Detection of SARS-CoV-2 recombinant N protein: Take 100uL of PBS dispersion of different concentrations of the new coronavirus antigen N protein and drop it onto the conjugate pad of the chromatography test strip. The liquid migrates to the absorbent pad. Visually evaluate the test area and quality control area within 10 minutes.

[0082] Figure 5 From left to right are test photos of chromatography test strips with target concentrations of 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL. From the display results of the test line and the quality control line, it can be concluded that the labeled antibody prepared in this application is applied to the chromatography test strips to detect SARS-CoV-2 recombinant N protein with a concentration of more than 0.5 ng / mL.

[0083] The above describes the basic principles, main features, and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-described embodiments. The above-described embodiments and the specification merely illustrate the principles of the present application. Various changes and improvements may be made to the present application without departing from the spirit and scope of the present application. These changes and improvements fall within the scope of the present application for which protection is sought. The scope of protection claimed by the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a labeled antibody, characterized in that: adding a carboxyl activator to a dispersion of carboxyl polypyrrole nanoparticles, and then adding an antibody to couple the antibody to the carboxyl polypyrrole nanoparticles; The average particle size of the carboxyl polypyrrole nanoparticles is 10 to 200 nm; The carboxyl polypyrrole nanoparticles are prepared by the following method: chemically modifying the polypyrrole nanoparticles with an organic carboxylic acid having a reactive group, thereby modifying the carboxyl group on the surface of the polypyrrole nanoparticles; The reactive group is suitable for addition reaction with the five-membered heterocycle on the polypyrrole main chain. The reactive group is a thiol group. The organic carboxylic acid having the reactive group is selected from a mixture of one or more of the following: thioglycolic acid, mercaptopropionic acid, 6-mercaptohexanoic acid, mercaptosuccinic acid, and cysteine.

2. A chromatography test strip loaded with a labeled antibody suitable for specific binding to a target, characterized in that: The labeled antibody is prepared by the method according to claim 1.

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