Lateral flow immunochromatography test paper based on phenylboronic acid directional coupling antibody mediation and detection method thereof

Abstract

The invention discloses lateral flow immunochromatography test paper based on phenylboronic acid directional coupling antibody mediation and a detection method of the lateral flow immunochromatography test paper. According to the lateral flow immunochromatography test paper, the phenylboronic acid directional coupling antibody is adopted, the detection sensitivity of LFIA is improved, specific coupling is conducted on the Fc segment of the antibody, the antigen capture rate is increased, and signal amplification is achieved; meanwhile, the nano-particles are combined, and the detection line is colored by utilizing the color of the nano-particles, so that the portability and the sensitivity of the whole detection are greatly improved; the detection method based on the test paper can realize qualitative analysis on a target object by observing the color depth on the detection line with naked eyes or quantitative analysis on the content of the target object by measuring the RGB value of the detection line, has the advantages of strong specificity on the target object, high sensitivity, convenience in use, simple and rapid detection process and safe and reliable detection result, and has a great application prospect.

Description

Technical Field

[0001] The invention relates to a lateral flow chromatography test paper and a detection method thereof, and in particular to a lateral flow immunochromatography test paper and a detection method thereof based on phenylboronic acid directional coupling antibody mediation. Background Art

[0002] Lateral flow immunoassay (LFIA) is a rapid immunoassay method based on immunochemical reaction and chromatography, which has many advantages such as low cost, simple operation, on-site response, and intuitive results. Colloidal gold nanoparticles are usually used as markers to couple with capture antibodies to synthesize capture probes. Antibody-nanoparticle conjugates have been widely used in bioanalysis, imaging, diagnosis and other fields.

[0003] However, the preparation of high-performance antibody-nanoparticle conjugates remains a huge challenge, because traditional antibodies are fixed on the surface of nanoparticles by modifying their amino acid residues containing free amino or carboxyl groups on the surface of nanoparticles or directly adsorbing and fixing on the surface of nanoparticles through physical action by adjusting the pH. These conjugation methods are not site-selective, because all available amino or carboxyl groups on the antibody molecule may participate in the conjugation reaction and the physical interactions are usually weak and sensitive to changes in conditions such as pH, temperature or salt concentration. These random conjugations can lead to incorrect orientation of antibodies on the surface of nanomaterials and reduce the biological activity of antibody conjugates. Therefore, the development of promising site-specific antibody conjugation strategies is crucial to avoid randomly oriented antibodies and effectively maintain antibody activity. Summary of the invention

[0004] Purpose of the invention: The present invention aims at the problems of lateral flow immunochromatography in the prior art and provides a lateral flow immunochromatography test paper based on phenylboronic acid directed coupled antibody mediation which is green, efficient, highly sensitive, easy to operate and low in cost, and can realize the combined detection of multiple targets in a short time. A detection method of the lateral flow chromatography is also provided.

[0005] Technical solution: The lateral flow immunochromatography test paper based on phenylboronic acid directional coupling antibody mediation described in the present invention includes a flat plate, a sample pad, a nitrocellulose membrane and an absorption pad. The sample pad, the nitrocellulose membrane and the absorption pad are sequentially pasted on the single side surface of the flat plate. From one end of the sample pad, the surface of the nitrocellulose membrane is sequentially coated with a first detection line, a second detection line and a quality control line; the first detection line and the second detection line are respectively coated with detection antibodies of two targets, and the quality control line is coated with a secondary antibody; the target is combined with a nanoparticle directional coupling antibody detection probe to form an immune complex, which is captured by the detection antibodies on the first detection line and the second detection line, and the nanoparticles are aggregated on the detection line for color development, and the excess detection probes are combined with the secondary antibody on the quality control line and aggregated.

[0006] Wherein, the target includes disease biomarkers, drugs or pathogenic microorganisms; preferably alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA).

[0007] Among them, the directional coupled antibody detection probe is obtained by co-incubating mercaptophenylboronic acid with a monoclonal antibody that recognizes the target. The preparation method is: taking a gold-platinum nanoparticle solution, reacting it with a mercaptophenylboric acid solution to form a conjugate system, and then co-incubating it with a monoclonal antibody that recognizes the target to form a gold-platinum nanoparticle-mercaptophenylboric acid-antibody mixture, and adding bovine serum albumin to obtain a gold-platinum nanoparticle directional coupled antibody detection probe.

[0008] The method for preparing the directional coupled antibody detection probe is as follows:

[0009] (1) Au@Pt NPs directional coupling labeled detection probe for identifying the first target: taking an Au@Pt NPs solution, reacting it with a mercaptophenylboronic acid solution, and then co-incubating it with a monoclonal antibody that recognizes the first target, and then adding bovine serum albumin to obtain an Au@Pt NPs directional coupling labeled detection probe for identifying the first target; the concentration of the Au@Pt NPs solution is 1-2 mg / mL, the concentration of mercaptophenylboronic acid is 1-2 mg / mL, the concentration of the monoclonal antibody that recognizes the first target is 1-3 mg / mL, and the incubation time is 4-6 h;

[0010] (2) Au@Pt NPs directional coupling labeled detection probe for recognizing the second target: Take an Au@Pt NPs solution, react it with a mercaptophenylboronic acid solution, and then incubate it with a monoclonal antibody that recognizes the second target, and then add bovine serum albumin to obtain an Au@Pt NPs directional coupling labeled detection probe for recognizing the second target; the concentration of the Au@Pt NPs solution is 1-2 mg / mL, the concentration of mercaptophenylboronic acid is 1-2 mg / mL, the concentration of the monoclonal antibody that recognizes the second target is 1-3 mg / mL, and the incubation time is 4-6 h.

[0011] The mass ratio of the gold-platinum nanoparticles to mercaptophenylboric acid is 1:1-1000, the mass ratio of the monoclonal antibody to the conjugate system is 1:1-250, the mass ratio of bovine serum albumin to the gold-platinum nanoparticles-mercaptophenylboric acid-antibody mixture is 1:1-100, and the incubation time is 4-6 hours.

[0012] The first detection line and the second detection line are respectively prepared with the detection antibody of the first target and the detection antibody of the second target to prepare the spray liquid, which are sprayed on the nitrocellulose membrane at intervals to obtain two detection lines with a distance of 3-5 mm.

[0013] Among them, the quality control line uses a spray liquid with a concentration of 0.8-1.2 mg / mL of secondary antibody, which is sprayed on the nitrocellulose membrane. The distance between the quality control line and the second detection line is 3-5 mm, and the distance between the quality control line and the end of the nitrocellulose membrane is 5-10 mm.

[0014] The sample pad and the absorption pad overlap the interface of the nitrocellulose membrane by 2-4 mm respectively.

[0015] The above-mentioned detection method based on lateral flow immunochromatographic test paper mediated by phenylboronic acid directional coupling antibody comprises the following steps:

[0016] (1) preparing directed coupled antibody detection probes for recognizing the first target and the second target respectively, adding the probes to the sample to be tested, mixing them evenly, and standing them to obtain immune complexes formed by the target and the corresponding detection probes;

[0017] (2) The immune complex is added dropwise to the sample pad of the test strip. The sample liquid moves to the absorption pad under the action of capillary force. The target object can undergo specific antigen-antibody recognition with the detection antibody coated on the detection line on the nitrocellulose membrane, and the nanoparticles are aggregated on the detection line. The target object is qualitatively analyzed by observing the color depth on the detection line with the naked eye; or the RGB value of the detection line is measured to quantitatively analyze the target content.

[0018] Wherein, in step (1), the detection probe concentration is 1-5 mg / mL, and the standing time is 5-20 min.

[0019] Wherein, in step (2), the quantitative analysis uses Color Picker software to analyze the T-line RGB value, and based on the comparison between the RGB value-target concentration standard curve and the standard curve, a quantitative judgment of the target concentration in the sample to be tested is achieved.

[0020] Principle of the invention: The lateral flow immunochromatography test paper based on phenylboronic acid directional coupling antibody of the present invention adopts phenylboronic acid directional coupling antibody to improve the detection sensitivity of LFIA, and specifically couples the Fc segment of the antibody to improve the antigen capture rate and realize signal amplification. At the same time, combined with nanoparticles, Au@Pt NPs can use its own color to make the detection line show color, greatly improving the portability and sensitivity of the overall detection.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0022] (1) The lateral flow immunochromatographic test paper based on phenylboronic acid directional coupling antibody mediation of the present invention is simple to assemble, easy to use, can quickly and efficiently complete qualitative and quantitative detection, and has high sensitivity and high specificity; it can detect multiple targets at the same time, realizes the instant detection of a wide spectrum of targets, greatly saves the detection cost and reduces the detection limit, and has universal applicability in the field of target detection;

[0023] (2) The lateral flow immunochromatography detection method based on phenylboronic acid-directed coupled antibody of the present invention, taking alpha-fetoprotein and carcinoembryonic antigen as examples, can detect the target with a minimum concentration of 2 ng / mL. All detection can be completed within 25 minutes, and the test results can be observed by naked eyes. The present invention has strong specificity for the target, high sensitivity, and is easy to use. The detection process is simple and fast, and the detection results are safe and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of a lateral flow chromatography test strip of the present invention;

[0025] Figure 2 is a transmission electron microscopy image of Au@Pt NPs of the present invention;

[0026] Figure 3 Schematic diagram of antigen binding rate;

[0027] Figure 4 The results of the colorimetric detection of alpha-fetoprotein and carcinoembryonic antigen using immunochromatographic test strips based on the phenylboronic acid-directed coupled antibody detection method were 2ng / mL for alpha-fetoprotein and 2ng / mL for carcinoembryonic antigen;

[0028] Figure 5 The standard curve diagram of the detection results of the phenylboronic acid directional coupling antibody detection method, wherein (A) is the standard curve diagram of the method for detecting AFP, and (B) is the standard curve diagram of the method for detecting CEA;

[0029] Figure 6 The results of the colorimetric detection of alpha-fetoprotein and carcinoembryonic antigen by immunochromatographic test strips based on the electrostatically coupled antibody detection method were 20 ng / mL for alpha-fetoprotein and 10 ng / mL for carcinoembryonic antigen;

[0030] Figure 7 The standard curve diagrams are based on the detection results of electrostatically coupled antibodies, wherein (A) is the standard curve diagram of the method for detecting AFP, and (B) is the standard curve diagram of the method for detecting CEA;

[0031] Among them, there are a plate 1, a sample pad 2, a nitrocellulose membrane 3, an absorption pad 4, a first detection line 5, a second detection line 6, and a quality control line 7. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0033] Example 1

[0034] The lateral flow immunochromatographic test paper based on phenylboronic acid directional coupling antibody mediation of the present invention is as follows Figure 1 As shown, it includes a plate 1, a sample pad 2, a nitrocellulose membrane 3 and an absorption pad 4, wherein the sample pad 2, the nitrocellulose membrane 3 and the absorption pad 4 are sequentially pasted on a single side surface of the plate 1, the sample pad 2 and the absorption pad 4 overlap the boundary of the nitrocellulose membrane 3 by 2 mm respectively, the absorption pad 4 is a 20-30 mm wide absorbent paper, and the plate 1 is a polyvinyl chloride plate; from one end of the sample pad 2, the surface of the nitrocellulose membrane 4 is sequentially coated with a first detection line 5, a second detection line 6 and a quality control line 7; the first detection line 5 and the second detection line 6 are respectively coated with detection antibodies of two targets, and the quality control line 7 is coated with a secondary antibody; the target is combined with the nanoparticle-directed coupled antibody detection probe to form an immune complex, which is captured by the detection antibodies on the first detection line 5 and the second detection line 6, the nanoparticles are aggregated on the detection line for color development, and the excess detection probe is combined with the secondary antibody on the quality control line 7 and aggregated.

[0035] The first detection line 5 and the second detection line 6 are respectively prepared with the detection antibody of the first target and the detection antibody of the second target to prepare the spray liquid, which are sprayed on the nitrocellulose membrane at a rate of 1 μL / cm by spraying, to obtain two detection lines with a distance of 3 mm. The quality control line 7 is prepared with a spray liquid with a concentration of 1.2 mg / mL using the secondary antibody, which is sprayed on the nitrocellulose membrane at a rate of 1 μL / cm by spraying, and the distance between the quality control line 7 and the second detection line 6 is 3 mm, and the distance between the quality control line 7 and the end of the nitrocellulose membrane 3 is 5 mm.

[0036] Among them, the preparation method of gold-platinum nanoparticles Au@Pt NPs is as follows: 3.0mL H2PtCl6 solution (20mM) and 3.0mL HAuCl4 solution (20mM) are mixed with Pluronic F127 (0.06g) in a round-bottom flask. Then, 6.0mL (0.1M) AA solution is added and the mixture is sonicated for 15min. After reacting for 24h, the product is collected after centrifugation at 10000 rpm for 20 minutes, and the residual Pluronic F127 is removed by washing / centrifugation with acetone and water three times in a row, and the obtained product is redispersed in pure water. Figure 2 , which is a transmission electron microscopy image of the Au@Pt NPs of the present invention, indicating that the synthesized Au@Pt NPs are uniformly dispersed spherical particles without obvious aggregation, and the particle size is 40-50nm;

[0037] Au@Pt NPs labeled detection probe for recognizing alpha-fetoprotein: 1 mL of 1 mg / mL Au@Pt NPs solution was added with mercaptophenylboronic acid for 2 h, and then 20 μL of phosphate buffered saline containing 20 μg of anti-alpha-fetoprotein monoclonal antibody was added dropwise. The mixed solution was stirred at room temperature for 4 h, and then 10% bovine serum albumin solution was added to block the unreacted sites, and the mixture was centrifuged. The obtained precipitate was added with 1 mL of phosphate buffered saline containing 10% sucrose and 1% serum albumin, and stored at 4°C for later use;

[0038] The detection probe for recognizing carcinoembryonic antigen labeled with Au@Pt NPs was prepared according to the following method: 1 mL of 1 mg / mL Au@Pt NPs solution was added with mercaptophenylboronic acid to react for 2 h, and then 20 μL of phosphate buffered saline solution containing 20 μg of monoclonal antibody against carcinoembryonic antigen was added dropwise, the mixed solution was stirred at room temperature for 4 h, and then 10% bovine serum albumin solution was added to block the unreacted sites, centrifuged, and the resulting precipitate was diluted with 1 mL of phosphate buffered saline solution containing 10% sucrose and 1% bovine serum albumin and stored at 4°C for later use.

[0039] The antigen binding rate of the prepared directional coupled antibody detection probe is as follows Figure 3 As shown, the two probes are directionally coupled, and because the antibodies are directionally fixed, more antigen binding sites can be exposed, and the antigen binding rate is higher.

[0040] Example 2

[0041] The detection method based on phenylboronic acid directional coupling antibody mediated lateral flow immunochromatographic test paper is as follows:

[0042] (1) Take 50 μL of the sample to be tested, prepare directional coupled antibody detection probes for recognizing the first target and the second target respectively, the concentration of the detection probe is 1 mg / mL, take 20 μL and add it to the sample to be tested, mix well, and let it stand for 5 minutes to obtain an immune complex formed by the target and the corresponding detection probe;

[0043] (2) The immune complex is added dropwise to the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. The target can undergo specific antigen-antibody recognition with the detection antibody coated on the detection line on the nitrocellulose membrane, and the nanoparticles are aggregated on the detection line. After the quality control line is colored, the target is qualitatively analyzed by observing the color depth on the detection line with the naked eye. The ColorPicker software is used to analyze the RGB value of the T line, and the RGB value-target concentration standard curve is compared with the standard curve to achieve quantitative judgment of the target concentration in the sample to be tested.

[0044] Table 1. Results of testing the levels of alpha-fetoprotein and carcinoembryonic antigen in serum

[0045]

[0046] As shown in Table 1, the results of detecting the content of alpha-fetoprotein and carcinoembryonic antigen in serum based on the directional coupled antibody immunochromatographic test strip detection platform are shown in Table 1. The detection recovery rate of alpha-fetoprotein is 90.84%-101.87%, the relative standard deviation is 1.82%-2.39%, and the detection recovery rate of carcinoembryonic antigen is 91.34%-107.70%, the relative standard deviation is 1.21%-2.80%. This shows that the method is accurate in detecting alpha-fetoprotein and carcinoembryonic antigen in blood samples.

[0047] like Figure 5 As shown in the figure, the color development results of the immunochromatographic test strip based on the directional coupled antibody for the detection of alpha-fetoprotein and carcinoembryonic antigen. The detection limit is 2ng / mL. Figure 6 As shown in the figure, the standard curve of alpha-fetoprotein and carcinoembryonic antigen detected by the directional coupled antibody immunochromatographic test strip detection platform is shown, indicating that the detection platform has good linearity in the detection of alpha-fetoprotein and carcinoembryonic antigen, with a linear range of 2-200 ng / mL, and the R 2 =0.9725, R of carcinoembryonic antigen 2 =0.9789. Compared with the electrostatic coupling method, the detection limit of alpha-fetoprotein was reduced by 10 times, and the detection limit of carcinoembryonic antigen was reduced by 5 times.

[0048] Comparative Example 1

[0049] Taking gold-platinum nanoparticles (Au@Pt NPs) as an example, the specific method for preparing the detection probe of Au@Pt NPs electrostatically coupled antibodies is as follows:

[0050] (1) Detection probe for Au@Pt NPs electrostatic coupling: 1 mg of anti-alpha-fetoprotein monoclonal antibody was added to 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-alpha-fetoprotein monoclonal antibody solution. 1.5 mg of anti-carcinoembryonic antigen monoclonal antibody was added to 1 mL of phosphate buffered saline to prepare a 1.5 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution for later use.

[0051] (2) Detection probe for recognizing the first target object labeled by electrostatic coupling of Au@Pt NPs: Take an Au@Pt NPs solution, adjust the pH by K2CO3, and then incubate with a monoclonal antibody that recognizes the first target object, and then add bovine serum albumin to obtain a detection probe for recognizing the first target object labeled by electrostatic coupling of Au@Pt NPs; the concentration of the Au@Pt NPs solution is 1 mg / mL, the concentration of the monoclonal antibody that recognizes the first target object is 1 mg / mL, and the incubation time is 4 h.

[0052] (3) Detection probe for recognizing the second target object labeled by electrostatic coupling of Au@Pt NPs: Take an Au@Pt NPs solution, adjust the pH by K2CO3, and then incubate it with a monoclonal antibody that recognizes the second target object, and then add bovine serum albumin to obtain a detection probe for recognizing the second target object labeled by electrostatic coupling of Au@Pt NPs; the concentration of the Au@Pt NPs solution is 1 mg / mL, the concentration of the monoclonal antibody that recognizes the second target object is 1 mg / mL, and the incubation time is 4 h.

[0053] The detection method based on the lateral flow immunochromatographic test paper mediated by electrostatically coupled antibodies is as follows:

[0054] (1) Take 50 μL of the sample to be tested, prepare directional coupled antibody detection probes for recognizing the first target and the second target respectively, the concentration of the detection probe is 1 mg / mL, take 20 μL and add it to the sample to be tested, mix well, and let it stand for 5 minutes to obtain an immune complex formed by the target and the corresponding detection probe;

[0055] (2) The immune complex is added dropwise to the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. The target can undergo specific antigen-antibody recognition with the detection antibody coated on the detection line on the nitrocellulose membrane, and the nanoparticles are aggregated on the detection line. After the quality control line is colored, the target is qualitatively analyzed by observing the color depth on the detection line with the naked eye. The ColorPicker software is used to analyze the RGB value of the T line, and the RGB value-target concentration standard curve is compared with the standard curve to achieve quantitative judgment of the target concentration in the sample to be tested.

[0056] like Figure 6 The figure shows the color development results of the immunochromatographic test strips based on electrostatically coupled antibodies for the detection of alpha-fetoprotein and carcinoembryonic antigen. For alpha-fetoprotein samples with a concentration greater than or equal to 20 ng / mL, the test strip test line is obviously colored after the test, and the detection limit is 20 ng / mL; for alpha-fetoprotein samples with a concentration less than 20 ng / mL, the test strip test line does not show color after the test. For carcinoembryonic antigen samples with a concentration greater than or equal to 10 ng / mL, the test strip test line is obviously colored after the test, and the detection limit is 10 ng / mL; for carcinoembryonic antigen samples with a concentration less than 10 ng / mL, the test strip test line does not show color after the test.

[0057] like Figure 7 As shown in the figure, it is the standard curve of the electrostatically coupled antibody immunochromatographic test strip detection platform for detecting alpha-fetoprotein and carcinoembryonic antigen. The linear range of the detection of alpha-fetoprotein is 20-200ng / mL, R 2 =0.9633, the linear range of carcinoembryonic antigen detection is 10-200ng / mL, R 2 =0.9533.

[0058] Therefore, the lateral flow immunochromatography test paper based on phenylboronic acid directional coupling antibody mediation of the present invention has high sensitivity and high specificity, can detect multiple targets at the same time, realizes the instant detection of broad-spectrum targets, greatly saves the detection cost and reduces the detection limit; the above-mentioned lateral flow immunochromatography detection method is easy to use, the detection process is simple and fast, qualitative or quantitative analysis can be carried out, and the detection results are safe and reliable.

Claims

1. A lateral flow immunochromatography test paper based on phenylboronic acid directional coupling antibody mediation, comprising a flat plate (1), a sample pad (2), a nitrocellulose membrane (3) and an absorption pad (4), wherein the sample pad (2), the nitrocellulose membrane (3) and the absorption pad (4) are sequentially attached to a single side surface of the flat plate (1), characterized in that: From one end of the sample pad (2), the surface of the nitrocellulose membrane (4) is coated with a first detection line (5), a second detection line (6) and a quality control line (7) in sequence; the first detection line (5) and the second detection line (6) are respectively coated with detection antibodies of two targets, and the quality control line (7) is coated with a secondary antibody; the target is combined with the nanoparticle-directed coupled antibody detection probe to form an immune complex, which is captured by the detection antibodies on the first detection line (5) and the second detection line (6), and the nanoparticles are aggregated on the detection line for color development, and the excess detection probe is combined with the secondary antibody on the quality control line (7) and aggregated.

2. The lateral flow chromatography test paper according to claim 1, characterized in that The target includes a disease biomarker, a drug or a pathogenic microorganism.

3. The lateral flow chromatography test paper according to claim 1, characterized in that The directional coupled antibody detection probe is obtained by co-incubating mercaptophenylboronic acid with a monoclonal antibody that recognizes a target object. The preparation method is as follows: taking a gold-platinum nanoparticle solution, reacting it with a mercaptophenylboric acid solution to form a conjugate system, and then co-incubating it with a monoclonal antibody that recognizes the target object to form a gold-platinum nanoparticle-mercaptophenylboric acid-antibody mixture, and adding bovine serum albumin to obtain a gold-platinum nanoparticle directional coupled antibody detection probe.

4. The lateral flow chromatography test paper according to claim 3, characterized in that The mass ratio of the gold-platinum nanoparticles to mercaptophenylboric acid is 1:1-1000, the mass ratio of the monoclonal antibody to the conjugate system is 1:1-250, the mass ratio of bovine serum albumin to the gold-platinum nanoparticles-mercaptophenylboric acid-antibody mixture is 1:1-100, and the incubation time is 4-6 hours.

5. The lateral flow chromatography test paper according to claim 1, characterized in that The first detection line (5) and the second detection line (6) are prepared by using a detection antibody for the first target and a detection antibody for the second target to prepare a spray liquid, which are sprayed on the nitrocellulose membrane at intervals to obtain two detection lines with a distance of 3-5 mm.

6. The lateral flow chromatography test paper according to claim 1, characterized in that The quality control line (7) is prepared by using a secondary antibody to form a spray solution with a concentration of 0.8-1.2 mg / mL, which is sprayed on the nitrocellulose membrane by spraying. The distance between the quality control line (7) and the second detection line (6) is 3-5 mm, and the distance between the quality control line (7) and the end of the nitrocellulose membrane (3) is 5-10 mm.

7. The lateral flow chromatography test paper according to claim 1, characterized in that The sample pad (2) and the absorption pad (4) overlap with the nitrocellulose membrane (3) by 2-4 mm respectively.

8. A detection method based on lateral flow immunochromatographic test paper mediated by phenylboronic acid directional coupling antibody according to claim 1, characterized in that: The following steps are involved: (1) preparing directed coupled antibody detection probes for recognizing the first target and the second target respectively, adding the probes to the sample to be tested, mixing them evenly, and standing them to obtain immune complexes formed by the target and the corresponding detection probes; (2) The immune complex is added dropwise to the sample pad of the test strip. The sample liquid moves to the absorption pad under the action of capillary force. The target object can undergo specific antigen-antibody recognition with the detection antibody coated on the detection line on the nitrocellulose membrane, and the nanoparticles are aggregated on the detection line. The target object is qualitatively analyzed by observing the color depth on the detection line with the naked eye; or the RGB value of the detection line is measured to quantitatively analyze the target content.

9. The lateral flow chromatography detection method according to claim 8, characterized in that: In step (1), the detection probe concentration is 1-5 mg / mL, and the standing time is 5-20 min.

10. The lateral flow chromatography detection method according to claim 8, characterized in that: In step (2), the quantitative analysis uses Color Picker software to analyze the T-line RGB value, and based on the comparison of the RGB value-target concentration standard curve with the standard curve, a quantitative judgment of the target concentration in the sample to be tested is achieved.

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