A fluorescent immunochromatographic test strip for detecting bisphenol A, its preparation method and application

Abstract

This invention provides a fluorescent immunochromatographic test strip for detecting bisphenol A (BPA), its preparation method, and its application. The test strip is obtained by sequentially overlapping and pasting a sample pad, a fluorescently labeled pad, a coating pad, and absorbent paper onto a PVC base plate. The fluorescently labeled pad is a glass fiber membrane coated with fluorescent microsphere-labeled mouse anti-BPA monoclonal antibody. The coating pad is a nitrocellulose membrane coated with goat anti-mouse IgG polyclonal antibody as a control line and coated with a BPA antigen complex as a detection line. The test strip is simple to operate and can be used for self-testing. It has high sensitivity for detecting BPA, reaching 0.05 ng / mL; it is negative for the detection of bisphenol A, bisphenol S, hydroquinone, and o-hydroxybenzoic acid, indicating good specificity. The test strip also has good stability and can be effectively stored for 14 months; each test can be completed within 10 minutes, enabling rapid sample testing and suitable for rapid on-site detection.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a fluorescent immunochromatographic test strip for detecting bisphenol A, its preparation method, and its application. Background Technology

[0002] Bisphenol A (BPA), also known as diphenol propane, is chemically named 2,2-bis(4-hydroxyphenyl)propane. It is formed by the condensation of two molecules of phenol and one molecule of acetone. It is a monomer used in the production of plastics, primarily polycarbonate (PC) and epoxy resins, among other polymers. BPA's structure is similar to that of the synthetic estrogen diethylstilbestrol (DES), giving it estrogenic activity; it is a typical exogenous estrogen. Due to its widespread use, the possibility of human exposure to BPA is very high. People can come into contact with BPA through the skin, respiratory tract, and digestive tract in daily life. The harmful effects of BPA are widely recognized through extensive research. After entering the body, BPA binds to intracellular estrogen receptors, producing estrogen-like or anti-estrogenic effects through various mechanisms. This can lead to endocrine disorders, interfere with the reproductive system, and induce precocious puberty in children. Studies have shown that BPA has certain embryotoxic and teratogenic effects, and can significantly increase the incidence of ovarian cancer, prostate cancer, leukemia, and other cancers in animals. Although there are multiple routes of exposure, the most common way for humans to ingest bisphenol A (BPA) is through its migration from food contact materials into food. When heated or exposed to acidic or alkaline substances, BPA molecules linked by ester bonds in polycarbonate and epoxy resins undergo accelerated hydrolysis, allowing small amounts of BPA to migrate from food contact materials or containers into the food medium. Banning BPA has gradually become a global consensus, with various countries issuing stringent limits on BPA in food contact materials and packaging containers. For example, BPA is prohibited in infant bottles, nipples, teething toys, and food contact materials and articles for children under 3 years old. These increasingly stringent BPA limits not only place severe quality management pressure on manufacturers but also impose stricter testing and regulatory requirements on testing institutions and customs. Exported products that fail testing and are deemed non-compliant with the exporting country's regulations will be forcibly returned, destroyed, or recalled, causing financial losses to domestic enterprises and damaging the country's reputation. The detection standards for bisphenol A have expanded from high performance liquid chromatography (HPLC) to gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS). However, these methods are relatively complex, time-consuming, and require specialized technical personnel. For the testing of export products, there is an urgent need to provide a reagent that can quickly and accurately detect bisphenol A.

[0003] Immunological detection technology is widely used in the field of drug residue detection, offering advantages such as high sensitivity, high specificity, speed, and ease of operation. Therefore, immunoassay provides a new approach for bisphenol A (BPA) content detection. However, because BPA is a small molecule and lacks antigenic properties, preparing antibody-based detection reagents is challenging. Currently, there are no reports on BPA haptens, artificial antigens, antibodies, their preparation methods, or applications. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a fluorescent immunochromatographic test strip for detecting bisphenol A, its preparation method, and its application. This test strip is suitable for detecting bisphenol A, can complete sample detection within 10 minutes, and has advantages such as high sensitivity, good stability, portability, and ease of use.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A fluorescent immunochromatographic test strip for detecting bisphenol A is obtained by sequentially overlapping and pasting a sample pad, a fluorescently labeled pad, a coated pad, and absorbent paper onto a PVC base plate. The fluorescently labeled pad is a glass fiber membrane coated with fluorescent microspheres labeled with mouse anti-bisphenol A monoclonal antibody, and the coated pad is a nitrocellulose membrane coated with goat anti-mouse IgG polyclonal antibody as a control line and coated with bisphenol A antigen complex as a detection line.

[0007] Preferably, the bisphenol A antigen complex in the fluorescent immunochromatographic test strip described above is obtained by the following preparation method: bisphenol A, NHS, and EDC are dissolved in DMF and reacted in the dark to obtain solution A. Solution A is added dropwise to solution BSA, stirred evenly, and reacted in the dark at room temperature for 12 hours to obtain solution B. Solution B is centrifuged at 6000 r / min for 5 min, and the supernatant is placed in a dialysis bag and placed in 0.01 mol / L PBS at pH 7.4. Dialysis is performed at 4°C for 72 hours, with the solution changed every 12 hours. After 48 hours, the bisphenol A antigen complex is obtained.

[0008] In the fluorescent immunochromatographic test strip described above, preferably, the fluorescent label pad is made by adding 0.5-1 mg of mouse anti-bisphenol A monoclonal antibody to 1 mL of fluorescent microspheres for labeling.

[0009] In the fluorescent immunochromatographic test strip described above, preferably, the coating concentration of the goat anti-mouse IgG polyclonal antibody is 2-3 mg / mL; and the coating concentration of the bisphenol A antigen complex is 0.5-2 mg / mL.

[0010] Further, it is preferred that the coating concentration of the goat anti-mouse IgG polyclonal antibody is 2.2 mg / mL; and it is preferred that the coating concentration of the bisphenol A antigen complex is 1.3 mg / mL.

[0011] A method for preparing a fluorescent immunochromatographic test strip for detecting bisphenol A, comprising the following steps:

[0012] S1. After activating the carboxyl groups of the fluorescent microspheres, add labeling buffer and mouse anti-bisphenol A monoclonal antibody to react, then block, centrifuge and wash to obtain fluorescent microsphere labeled antibody.

[0013] S2. Spray the fluorescent microsphere-labeled antibody onto a glass fiber membrane, dry it, and obtain a fluorescently labeled pad.

[0014] S3. Dilute the goat anti-mouse IgG polyclonal antibody and bisphenol A antigen complex to the working concentration using coating buffer to prepare the control line working solution and the test line working solution. Use a coating machine to apply the control line and test line working solutions onto a nitrocellulose membrane, dry them, and obtain the coating pad.

[0015] S4. Place the sample pad, fluorescent marker pad, coating pad, and absorbent paper along the length of the PVC base plate, and then cut them into strips 0.3-0.5 cm wide to obtain the fluorescent immunochromatographic test strip for detecting bisphenol A.

[0016] In the preparation method described above, preferably, in step S1, the carboxyl activation is performed using EDC and NHS, the labeling buffer is a borate buffer with a pH of 8.5 with 0.05% TX-100 added, the fluorescent microspheres and mouse anti-bisphenol A monoclonal antibody are labeled at a ratio of 1 mL to 0.5-1 mg, the blocking is performed using BSA with a reaction concentration of 1 mg / mL, after centrifugation the precipitate is washed with borate buffer containing 0.05% Tween-20, and then the fluorescent microsphere-labeled antibody is diluted with 5% trehalose.

[0017] In the preparation method described above, preferably, in step S2, drying is performed in a drying oven at 37°C for 3 to 4 hours.

[0018] In the preparation method described above, preferably, in step S3, the concentration of the control working solution for the goat anti-mouse IgG polyclonal antibody is 2.2 mg / mL, and the concentration of the detection working solution for the bisphenol A antigen complex is 1.3 mg / mL.

[0019] Extensive experimental studies have revealed that if the test strips are not prepared according to the preferred concentrations described above, non-specific bands will appear, and false positives will occur in the blank control solution test.

[0020] The application of the fluorescent immunochromatographic test strip for detecting bisphenol A as described above in the detection of bisphenol A.

[0021] The beneficial effects of this invention are as follows:

[0022] The fluorescent immunochromatographic test strip for detecting bisphenol A provided by this invention is simple to operate and can be used independently. It exhibits high sensitivity for detecting bisphenol A, reaching 0.05 ng / mL, and shows no detection for BVA, bisphenol S, hydroquinone, and o-hydroxybenzoic acid, indicating good specificity. The test strip prepared by this invention also has good stability and can be effectively stored for 14 months. Each test can be completed within 10 minutes, enabling rapid sample detection and making it suitable for rapid on-site testing. Attached Figure Description

[0023] Figure 1 This is a diagram illustrating the assembly of the test strips;

[0024] Figure 2 The results of the sensitivity test of the test strips and the fitted working curve;

[0025] Figure 3 This is the result of the repeatability test of the test strip. Detailed Implementation

[0026] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0027] Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art. All materials and reagents used in the examples are commercially available unless otherwise specified. Specifically, the fluorescent microspheres (Bangs), bisphenol A standard, BVA, bisphenol S, BSA, hydroquinone, and o-hydroxybenzoic acid standard used in the examples were purchased from Sinopharm Group Pharmaceutical Co., Ltd.; the mouse anti-bisphenol A monoclonal antibody was purchased from Shanghai Aixin Biotechnology Co., Ltd.; NHS, EDC, DMF, and goat anti-mouse IgG polyclonal antibody were purchased from Sigma-Aldrich; the labeling pad was from Ahlstrom, the NC membrane from Sartorius, the sample pad from Shanghai Jieyi Biotechnology Co., Ltd., and the absorbent pad from Shanghai Jieyi Biotechnology Co., Ltd. The instruments used in the following examples include: a YG10 fluorescence immunochromatographic immunoassay analyzer (provided by the China Academy of Inspection and Quarantine Sciences); a coating machine (Shanghai Jinbiao Biotechnology Co., Ltd.); and a drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.). Unless otherwise specified, all percentages (%) in the following examples refer to weight percentages.

[0028] Example 1: Preparation of Bisphenol A Antigen Complex

[0029] Because bisphenol A (BSA) is a small molecule, it lacks immunogenicity and cannot induce antibody production in organisms. Furthermore, since it does not contain a carboxyl group, a carboxyl group must be introduced chemically before it is coupled with a large molecular carrier such as a protein to obtain a complete antigen. This invention introduces three straight-chain carbon atom linkers and a carboxyl group into BSA, and then couples BSA with BSA via the carbodiimide method to synthesize the artificial antigen BSA-BSA. Specific steps: Dissolve 1 mg of BSA, 3.3 mg of NHS, and 5.0 mg of EDC in 250 μL of DMF (dimethylformamide), and react at 18°C, 200 rpm, and in the dark for 18 h to obtain solution A. Dissolve 15 mg of BSA (bovine serum albumin) in 2 mL of PBS at pH 8.5 to obtain solution BSA. Add solution A dropwise to solution BSA, stir well, and react at room temperature in the dark for 12 h to obtain solution B. Centrifuge solution B at 6000 rpm for 5 min, collect the supernatant and put it into a dialysis bag in 0.01 mol / L PBS at pH 7.4. Dialyze at 4°C for 72 hours, changing the medium every 12 hours. After 48 hours, a complex containing bisphenol A antigen is obtained.

[0030] Example 2: Preparation of fluorescent immunochromatographic test strips for bisphenol A detection

[0031] (1) Preparation of fluorescently labeled pads

[0032] First, take an appropriate amount of fluorescent microspheres (with carboxyl groups). Activate the microspheres by diluting EDC and NHS with MES buffer. Specifically, add 100 μg each of EDC and NHS diluted in MES buffer to 1 mL of fluorescent microspheres. Activate in the dark for 30 min. Centrifuge the fluorescent microspheres at 12000 rpm for 10 min and discard the supernatant. Reconstitute 0.5 mL of microspheres with labeling buffer and simultaneously add 0.75 mg of mouse anti-bisphenol A monoclonal antibody. Stir and react for 45 min. Add 0.5 mL of BSA aqueous solution containing 2 mg / mL to the labeling buffer after the reaction. Block for 30 min, centrifuge at 12000 rpm for 10 min and discard the supernatant. Wash the labeled mouse anti-bisphenol A monoclonal antibody-containing fluorescent microspheres multiple times with borate washing buffer to obtain fluorescent microsphere-labeled antibody. Dilute 50 times with labeling diluent and spray onto a glass fiber membrane as a fluorescent labeling pad. Dry in a drying oven at 37℃ for 4 hours and store dry. The labeling buffer was borate buffer (BBS, an aqueous solution containing 0.015 mol / L sodium borate and 0.15 mol / L sodium chloride, pH 8.5) with 0.05% TX-100 added by volume. The addition of TX-100 helps to disperse the fluorescent microspheres and prevent them from agglomerating, which is beneficial for the full reaction of the fluorescent microspheres. The borate washing solution was BBS with 0.05% Tween-20 added by volume. The labeling dilution solution was borate buffer with trehalose added by mass percentage. Trehalose helps to protect the mouse anti-bisphenol A monoclonal antibody and maintain its antibody activity. Without trehalose, false positives are likely to occur in the test results.

[0033] (2) Preparation of the padding

[0034] Goat anti-mouse IgG polyclonal antibody 2.2 mg / mL and bisphenol A antigen complex were diluted to a working concentration of 1.3 mg / mL with coating buffer to prepare control line working solution (C) and test line working solution (T). The control line and test line working solutions were applied to nitrocellulose membranes using a coating machine and dried in a drying oven at 37°C for 8 hours. The dried membranes were then stored to obtain the coated pads. The coating buffer was 0.01 M phosphate buffer with pH 8.0 and 2-4% trehalose added.

[0035] Based on the sensitivity of the reagent reaction, the optimization experiment was mainly carried out from the pH value of the buffer and the concentration of trehalose. Furthermore, it is preferred to add 2.5% trehalose to 0.01M phosphate buffer, which has high sensitivity.

[0036] (3) Sample pad preparation

[0037] Cut the sample pad to the required size, dry and store for later use.

[0038] (4) Assembly

[0039] The sample pad, fluorescent marker pad, coating pad, and absorbent paper are sequentially glued onto the PVC base plate. A 4mm wide reagent strip is then cut and inserted into the cartridge. Figure 1 As shown.

[0040] Add 200 μL of the sample to be tested to the sample well. After 10 minutes, place it in a YG10 fluorescence immunochromatographic analyzer and read the T and C fluorescence signal values ​​(excitation wavelength 365 nm, emission wavelength 610 nm). Under chromatography, bisphenol A in the sample, as an antigen, binds to mouse anti-bisphenol A monoclonal antibody-fluorescent microspheres, forming a fluorescently labeled mouse anti-bisphenol A monoclonal antibody-antigen (bisphenol A) conjugate. The conjugate precipitates along the nitrocellulose membrane. The bisphenol A in the sample and the bisphenol A antigen complex coated on the detection line compete for the specific antibody labeled with gold by the fluorescent microspheres. The higher the bisphenol A content in the sample, the fewer fluorescently labeled mouse anti-bisphenol A monoclonal antibodies bind to the detection line, resulting in a weak signal value at the T line. Other fluorescent microsphere mouse anti-bisphenol A monoclonal antibodies that do not bind to the T line reach the C line and bind to goat anti-mouse secondary antibody, resulting in a signal value at the C line. If the test solution does not contain bisphenol A, both the T and C lines will show signal values. If the test solution contains bisphenol A, the T line will show no signal value or a weak signal value for a positive result. When the test result is valid, the C line will show a certain signal value. At this time, the signal intensity on the T line is related to the concentration of the sample; the higher the concentration, the lower the T value.

[0041] Example 3: Performance Testing of Fluorescent Immunochromatographic Test Strips for Bisphenol A Detection

[0042] 1) Limit of Detection (CUT OFF) and Working Curve Fitting of Fluorescent Immunochromatographic Reagents: Bisphenol A (BPA) standard was diluted with sample diluent (PBS solution with pH 8.0) to concentrations of 8 ng / mL, 4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, 0.1 ng / mL, and 0.05 ng / mL. Three batches of fluorescent immunochromatographic assay reagents prepared according to the method in Example 2 were used for sequential detection, with each concentration repeated five times. The diluent was used as a blank control. The fitting curve ranged from 0.05 ng / mL to 8 ng / mL. The BPA standard concentration was plotted on the x-axis, and the signal T value read by the fluorescent immunoassay analyzer was plotted on the y-axis to establish the fitting curve. The equation is: y = 1163.7x 2 -13802x+40338, R 2 =0.9906. The result is shown below. Figure 2 As shown.

[0043] Therefore, it can be concluded that the fluorescent immunochromatographic test strip prepared in this invention can detect all 0.05 ng / mL bisphenol A standard, that is, the limit of detection is 0.05 ng / mL.

[0044] The content of bisphenol A can be obtained by measuring the fluorescence value of the T line in the sample solution according to the standard curve.

[0045] Simultaneously, the bisphenol A antigen complex prepared in Example 1 was used to prepare colloidal gold immunochromatographic test strips. The preparation method employed conventional procedures: colloidal gold particles were first labeled with mouse anti-bisphenol A monoclonal antibody; the detection band on the nitrocellulose membrane was coated with the bisphenol A antigen complex; and the quality control band was coated with goat anti-mouse IgG polyclonal antibody. The obtained colloidal gold immunochromatographic test strips were tested using bisphenol A standard concentrations of 8 ng / mL, 4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, 0.1 ng / mL, and 0.05 ng / mL. The results showed that only bisphenol A standard concentrations greater than or equal to 1 ng / mL were detectable, indicating a positive result. This demonstrates that the detection limit of the prepared colloidal gold is 1 ng / mL, indicating that the sensitivity of the fluorescent immunochromatographic reagent prepared in this invention is higher than that of colloidal gold immunochromatographic reagents prepared using existing technologies.

[0046] 2) For the repeatability testing of fluorescent immunochromatographic reagents, three batches of reagents prepared according to the method in Example 2 were repeatedly extracted and tested for bisphenol A standards at concentrations of 8 ng / mL, 2 ng / mL, and 0.05 ng / mL. Each concentration point was tested in parallel 15 times. The test results are as follows: Figure 3 As shown in the figure. The results demonstrate that the test strip of the present invention has good repeatability.

[0047] 3) Three batches of fluorescent immunochromatographic reagents prepared according to the method in Example 2 were specifically extracted for the detection of BVA (bisphenol A), bisphenol S (4,4-dihydroxydiphenyl sulfone), hydroquinone, and o-hydroxybenzoic acid standards, diluted to 1000 ppb, with each sample tested three times. According to the standard curve, the results showed that the reactions with BVA, bisphenol S, hydroquinone, and o-hydroxybenzoic acid standards were undetectable. The results indicate that when three batches of fluorescent immunochromatographic test strips were extracted to detect BVA, bisphenol S, hydroquinone, and o-hydroxybenzoic acid standards diluted to 1000 ppb, with each sample tested three times, the results were all negative, demonstrating that the fluorescent immunochromatographic test strips showed no cross-reactivity with these standards and exhibited good specificity.

[0048] 4) Stability: The fluorescent immunochromatographic reagent prepared and packaged according to the method in Example 2 was stored at room temperature. The concentration of bisphenol A standard was tested periodically within 14 months. The concentration of bisphenol A standard was recorded as the target value. Each concentration was tested 3 times. The average value and relative average deviation of the 3 detected concentrations were calculated by referring to the standard curve. The results are shown in Table 1.

[0049] Table 1 Stability test results

[0050]

[0051] The results showed that when two batches of test strips were prepared according to the method of Example 2, and bisphenol A standard at the target concentration was tested periodically, the relative average deviation between the test results (average value) and the theoretical value (target value) was less than ±15%, indicating that the stability of the fluorescent chromatography test strips prepared in this invention is 14 months.

[0052] Therefore, it can be seen that the test strip prepared in this invention is negative for the detection of bisphenol A (BVA), bisphenol S, hydroquinone, and o-hydroxybenzoic acid, indicating good specificity. The test strip also has good stability and can be effectively stored for 14 months; each test can be completed within 10 minutes, enabling rapid sample detection and making it suitable for rapid on-site testing.

Claims

1. A fluorescent immunochromatographic test strip for detecting bisphenol A, characterized in that, It is obtained by sequentially overlapping and pasting a sample pad, a fluorescent marker pad, a coating pad, and absorbent paper onto a PVC base plate. The fluorescent marker pad is a glass fiber membrane coated with fluorescent microspheres labeled with mouse anti-bisphenol A monoclonal antibody. The coating pad is a nitrocellulose membrane coated with goat anti-mouse IgG polyclonal antibody as a quality control line and coated with bisphenol A antigen complex as a detection line. The bisphenol A antigen complex was obtained by the following method: bisphenol A, NHS, and EDC were dissolved in DMF and reacted in the dark to obtain solution A. Solution A was added dropwise to BSA solution, stirred evenly, and reacted at room temperature in the dark for 12 hours to obtain solution B. Solution B was centrifuged at 6000 rpm for 5 minutes, and the supernatant was placed in a dialysis bag in 0.01 mol / L PBS (pH 7.4) and dialyzed at 4°C. The solution was changed every 12 hours. After 48 hours, the bisphenol A antigen complex was obtained. The fluorescently labeled pad was obtained by the following method: 100 μg each of EDC and NHS diluted in MES buffer was added to 1 mL of fluorescent microspheres and activated in the dark for 30 minutes. The fluorescent microspheres were centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, and 0.5 mL of labeling buffer was added to reconstitute the microspheres. Simultaneously, 0.75 mg of mouse anti-bisphenol A monoclonal antibody was added and the mixture was stirred for 45 minutes. The reaction mixture was then added to the labeling buffer after the reaction. Add 0.5 mL of a 2 mg / mL BSA aqueous solution, block for 30 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, and wash the labeled mouse anti-bisphenol A monoclonal antibody fluorescent microspheres multiple times with borate washing buffer to obtain fluorescent microsphere labeled antibody. Dilute 50 times with labeling diluent and spray onto a glass fiber membrane as a fluorescent labeling pad. Dry in a drying oven at 37℃ for 4 hours to obtain the fluorescent labeling pad. The labeling buffer is borate buffer, which is an aqueous solution containing sodium borate and sodium chloride at a final concentration of 0.015 mol / L and 0.15 mol / L, with 0.05% TX-100 added at pH 8.

5. The coating concentration of the goat anti-mouse IgG polyclonal antibody was 2.2 mg / mL; the coating concentration of the bisphenol A antigen complex was 1.3 mg / mL; the coating buffer used was a 0.01 M phosphate buffer with a pH of 8.0 and containing 2.5% trehalose by mass, to dilute the goat anti-mouse IgG polyclonal antibody and the bisphenol A antigen complex, respectively.

2. A method for preparing a fluorescent immunochromatographic test strip for detecting bisphenol A, characterized in that, It includes the following steps: S1. After activating the carboxyl groups of the fluorescent microspheres, add labeling buffer and mouse anti-bisphenol A monoclonal antibody to react, then block, centrifuge and wash to obtain fluorescent microsphere labeled antibody. S2. Spray the fluorescent microsphere-labeled antibody onto a glass fiber membrane, dry it, and obtain a fluorescently labeled pad. S3. Dilute the goat anti-mouse IgG polyclonal antibody and bisphenol A antigen complex to the working concentration using coating buffer to prepare the control line working solution and the test line working solution. Use a coating machine to apply the control line and test line working solutions onto a nitrocellulose membrane, dry them, and obtain the coating pad. The bisphenol A antigen complex was obtained by the following preparation method: Bisphenol A, NHS, and EDC were dissolved in DMF and reacted in the dark to obtain solution A. Solution A was added dropwise to solution BSA and stirred evenly. The mixture was then reacted in the dark at room temperature for 12 hours to obtain solution B. Solution B was centrifuged at 6000 r / min for 5 min, and the supernatant was placed in a dialysis bag and placed in 0.01 mol / L PBS at pH 7.

4. Dialysis was performed at 4℃, and the solution was changed every 12 hours. After 48 hours, the bisphenol A antigen complex was obtained. The working solution concentration of the goat anti-mouse IgG polyclonal antibody control line is 2.2 mg / mL, and the working solution concentration of the bisphenol A antigen complex detection line is 1.3 mg / mL. The coating buffer was a 0.01M phosphate buffer with a pH of 8.0 and containing 2.5% trehalose by mass. S4. Place the sample pad, fluorescent marker pad, coating pad and absorbent paper along the length of the PVC base plate, and then cut them into strips 0.3-0.5 cm wide to obtain the fluorescent immunochromatographic test strip for detecting bisphenol A. The carboxyl activation was performed using EDC and NHS. The labeling buffer was a borate buffer with a pH of 8.5 containing 0.05% TX-100. Fluorescent microspheres were labeled with mouse anti-bisphenol A monoclonal antibody at a ratio of 1 mL to 1.5 mg. Blocking was performed using BSA at a concentration of 1 mg / mL. After centrifugation, the precipitate was washed with borate buffer containing 0.05% Tween-20. Then, the fluorescent microsphere-labeled antibody was diluted with 5% trehalose.

3. The preparation method according to claim 2, characterized in that, In step S2, drying is performed in a drying oven at 37°C for 3 to 4 hours.

4. The application of the bisphenol A fluorescent immunochromatographic test strip according to claim 1 in the detection of bisphenol A.

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