Bisphenol S competitive ELISA kit and method using gold nanoflowers to enhance signal

Abstract

A bisphenol S competitive ELISA kit and method for enhancing the signal using gold nanoflowers, the kit comprises a BPS complete antigen, a gold nanoflower and a BPS monoclonal antibody co-labeled with HRP. The complete antigen of BPS was prepared using 5-bromovalerate methyl ester as an intermediate product, and then a monoclonal antibody with high affinity and high specificity for BPS was obtained by immunizing mice with the BPS complete antigen; the antibody was then labeled with HRP, and then the gold nanoflower had a higher specific surface area and biological binding, and more antibodies were used to bind thereto, and a BPS competitive ELISA with higher sensitivity was established, with a detection range of 15.625-2000 ng / mL and a detection limit of 15.625 ng / mL. The present invention can be used for BPS detection in environmental matrices, biological samples, foods and medicines. At the same time, compared with the currently commonly used chemical methods, the method is simpler and faster, and does not require expensive equipment and complicated pre-treatment processes.

Description

Technical Field

[0001] The invention belongs to the field of environmental detection, and particularly relates to a bisphenol S competitive ELISA kit and method for enhancing signals by using gold nanoflowers. Background Art

[0002] Bisphenol S (BPS) is a substitute for bisphenol A (BPA). It is widely used in the production of polycarbonate plastics and resins because of its higher thermal and light stability compared to BPA. However, as BPS is increasingly used in "BPA-free" products, its potential to enter the environment is also increasing, resulting in BPS being detected in various environmental matrices and biological samples. For example, the average concentration of BPS in Taihu Lake water is 16 ng / L; the detection concentration of BPS in Chinese adult urine samples (n=160) is 0.24 µg / L. Studies have shown that BPS has hormonal activity that interferes with the normal endocrine function of organisms. For example, exposure to environmental concentrations of BPS can have toxic effects on zebrafish development and reproduction. In addition, exposure of the mother to low concentrations of BPS can cause damage to the nervous and endocrine systems of the offspring. Given that BPS has high concentrations and toxic effects in the environment, it is urgent to establish a detection method for BPS.

[0003] At present, the detection methods of BPS are mainly based on high-performance liquid chromatography and gas chromatography-mass spectrometry. Although the above methods have high precision and accuracy, their complex sample pretreatment process and expensive equipment make it difficult to popularize the above methods, limiting the detection of BPS. The immunological detection method is to detect BPS by using the recognition of antibodies to antigens. Among them, enzyme-linked immunosorbent assay (ELISA) is the most widely used immunological detection method because of its rapidity, sensitivity, low equipment requirements, and low cost. Although there are competitive ELISAs for BPS, they are mainly used for the detection of BPS in food, and the sensitivity is limited. In environmental samples, the content of BPS is low and the detection is difficult, so a BPS detection method with higher sensitivity is needed. Selecting a suitable signal amplification strategy and preparing antibodies with high affinity are the key to improving the sensitivity of ELISA. Among them, gold nanoflowers have the same bioavailability, stability and nanozyme activity as gold nanomaterials, and their flower-like structure can effectively increase the specific surface area, making them a signal amplification element with superior performance. Therefore, the present invention prepares a monoclonal antibody with high affinity and specificity for BPS, and sequentially couples the antibody with gold nanoflowers and horseradish peroxidase (HRP) to further amplify the detection signal, thereby establishing a competitive ELISA for BPS. Summary of the invention

[0004] The purpose of the present invention is to provide a bisphenol S competitive ELISA kit and method using gold nanoflowers to enhance the signal, and to use gold nanoflowers as signal amplification elements to establish a competitive ELISA kit for detecting BPS to make up for the shortcoming of low sensitivity of existing competitive ELISA detection.

[0005] A bisphenol S competitive ELISA kit using gold nanoflowers to enhance signals, characterized in that the kit comprises BPS complete antigen, gold nanoflowers and HRP-co-labeled BPS monoclonal antibodies;

[0006] The BPS complete antigen uses 5-bromovalerate methyl ester as a connecting arm; the carrier protein of the BPS complete antigen is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, fibrinogen or gelatin.

[0007] The BPS monoclonal antibody in the BPS monoclonal antibody jointly labeled with gold nanoflowers and HRP is prepared from the BPS complete antigen.

[0008] In the kit, the BPS complete antigen is prepared by the following method:

[0009] First, 0.25 g of BPS was weighed and added to 50 mL of acetonitrile and stirred until completely dissolved. Then, 0.415 g of potassium carbonate was added and reacted for 1 h. After 0.293 g of methyl 5-bromovalerate was heated and refluxed for 24 h, the organic solvent was removed and the obtained solid was purified by column (petroleum ether / ethyl acetate) to obtain the hapten of BPS. 10 mg of BPS hapten, 8.9 mg of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6.2 mg of N-hydroxysuccinimide (1-hydroxy-5-pyrrolidinedione, NHS) were weighed and dissolved in 4 mL of N,N-dimethylformamide (DMF) and reacted at room temperature in the dark for 2 h. The above solution was added dropwise into 2 mL of phosphate buffer containing 20 mg of ovalbumin (OVA) or 15 mg of bovine serum albumin (BSA). After shaking the reaction at ℃ overnight, the supernatant was taken by centrifugation at 6000 r / min for 5 min and dialyzed in 0.01 M PBS for 32 h, with the dialyzate replaced every 8 h. The resulting solution was centrifuged again to obtain the supernatant, which was the BPS-complete antigen. When ovalbumin was added, the product was recorded as BPS-BSA, which was used as the immune antigen; when bovine serum albumin was added, the product was recorded as BPS-OVA, which was used as the coating antigen.

[0010] In the kit, the BPS monoclonal antibody is co-labeled with gold nanoflowers and HRP by the following steps:

[0011] First, HRP was labeled on the BPS monoclonal antibody using the sodium periodate method. The specific method was as follows: 1 mL of 5 mg / L HRP solution was fully mixed with 0.2 mL of 0.1 M NaIO4 solution, and then the mixture was shaken at room temperature for 20 min; then the mixture was dialyzed in 1 mM acetate buffer (pH 4.4) at 4°C overnight; after the liquid was taken out, 0.5 mL of 0.16 M ethylene glycol solution was added, and the mixture was shaken at room temperature for 30 min; 1 mg of BPS monoclonal antibody was added and mixed, and the mixture was shaken at low temperature for 30 min, and then the mixture was dialyzed in 0.05 M carbonate buffer (pH 9.5) overnight; after the liquid was taken out, 0.1 mL of 5 mg / mL NaBH4 solution was added and shaken at 4 °C for 2 h; then an equal volume of saturated ammonium sulfate solution was slowly added, and the mixture was shaken at 4 °C for 30 min after mixing, and centrifuged at 3000 rpm for 30 min, the supernatant was removed, and the precipitate was dissolved in 0.15 M PBS (pH=7.4) and placed in a dialysis bag in PBS for 4 °C for desalting; after the liquid was taken out, it was centrifuged at 3000 rpm for 30 min to remove the precipitate, and the supernatant was collected as the HRP-labeled BPS monoclonal antibody; then 8 μg of HRP-labeled BPS monoclonal antibody was added to 1 mL of gold nanoflower solution, shaken for 1 h, and centrifuged at 12000 r / min for 10 min. Then, the precipitate was re-dissolved with 0.02 M Tris-HCl solution to obtain BPS monoclonal antibody jointly labeled with gold nanoflowers and HRP.

[0012] The BPS indirect competitive ELISA detection method using the above kit is characterized by comprising the following steps:

[0013] Step 1, coating the BPS complete antigen in the kit according to claim 1 on an ELISA plate with a coating solution; Step 2, blocking the ELISA plate with a blocking solution; Step 3, mixing the enzyme-labeled gold nanoflowers and the BPS monoclonal antibody co-labeled with HRP with the sample to be tested and adding them to the ELISA plate, so that the BPS complete antigen coated in step 1 is mixed with the sample to be tested, and when BPS exists in the sample, the BPS in the sample competitively binds to the BPS monoclonal antibody.

[0014] Advantages of the invention

[0015] At present, high performance liquid chromatography and gas chromatography are usually used to detect BPS, but these methods usually require expensive equipment and complex sample pretreatment processes. Although the conventional ELISA method has the advantages of simple operation, low equipment requirements and fast detection speed compared with the chemical method, its sensitivity is relatively low. At present, the sensitivity of the detection method is mainly improved by increasing the signal amplification strategy and preparing antibodies with higher affinity. Therefore, the present invention uses 5-bromovalerate methyl ester as the connection product to prepare the BPS hapten. Since 5-bromovalerate methyl ester has a longer carbon chain, the antigenic determinant can be exposed more fully, thereby preparing a BPS monoclonal antibody with higher affinity and specificity. Then, gold nanoflowers are used to further amplify the signal, so that more HRP-labeled antibodies are bound thereto. While increasing the concentration of the bound antibody, the gold nanoflowers themselves have nanozyme activity, which can increase the detection ELISA signal and improve the sensitivity of the competitive ELISA established by the present invention. Using gold nanoflowers and HRP dual signal enhancement kits, a quick and sensitive detection method is provided for the detection of BPS. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a synthetic diagram of BPS hapten.

[0017] Figure 2 is the complete antigen synthesis diagram of BPS, where Figure 2 a is a synthetic diagram of BPS immune antigen, Figure 2 b is a synthetic diagram of BPS-coated antigen.

[0018] Figure 3 This is the infrared spectrum identification result of BPS complete antigen.

[0019] Figure 4 This is the result of polyacrylamide gel electrophoresis of BPS complete antigen.

[0020] Figure 5 Standard curve for BPS competition ELISA kit. DETAILED DESCRIPTION

[0021] The bisphenol S competitive ELISA kit using gold nanoflowers to enhance the signal includes a box body and conventional enzyme-labeled antibodies, coating solution, washing solution, antibody diluent, color development solution and stop solution in the box body, and is characterized in that it also includes BPS complete antigen, gold nanoflowers and HRP-co-labeled BPS monoclonal antibodies.

[0022] The present invention will be further described below through specific implementations and accompanying drawings.

[0023] Example 1. Preparation of BPS hapten and monoclonal antibody

[0024] (1) Synthesis of BPS hapten

[0025] First, follow Figure 1 To prepare the BPS hapten as shown, 0.25 g of BPS was weighed and added into 50 mL of acetonitrile and stirred until completely dissolved, followed by the addition of 0.415 g of potassium carbonate and the reaction for 1 h. After that, 0.293 g of methyl 5-bromovalerate was heated under reflux for 24 h. After removing the organic solvent, the obtained solid was purified by column (petroleum ether / ethyl acetate) to obtain the BPS hapten.

[0026] (2) Synthesis of complete antigen

[0027] according to Figure 2 Prepare BPS complete antigen as shown, weigh 10 mg BPS hapten, 8.9 mg EDC, and 6.2 mg NHS and dissolve them in 4 mL DMF, and react in the dark for 2 h at room temperature. Add the above solution dropwise into 2 mL phosphate buffer containing 20 mg OVA or 15 mg BSA, shake the reaction at 4°C overnight, centrifuge at 6000 r / min for 5 min, take the supernatant, and dialyze it in 0.01 MPBS for 32 h, replacing the dialysate every 8 h. The resulting solution was centrifuged again to obtain the supernatant, which is the complete antigen (BPS-OVA / BPS-BSA). Among them, BPS-BSA is the immune antigen and BPS-OVA is the coating antigen. The prepared complete antigen was scanned by infrared spectroscopy, and the results are as shown Figure 3 As shown in the figure, the infrared absorption peak of the complete antigen after coupling with BSA and OVA showed obvious characteristic peaks. Figure 4 The polyacrylamide gel electrophoresis results also showed that the molecular weight of the complete antigen was significantly greater than that of BSA or OVA. The above results all confirmed that the present invention prepared the complete antigen of BPS.

[0028] (3) Preparation of BPS-specific antibodies

[0029] Three healthy Balb / C mice were selected as experimental organisms, and 100 μg of BPS-BSA and an equal volume of Freund's complete adjuvant were emulsified and injected into the mouse peritoneum. Every 14 days, the mice were immunized with the same dose of BPS-BSA and Freund's incomplete adjuvant after being fully emulsified. Three days after the fourth immunization, the mice were killed and the spleen was taken for standby. Then, mouse myeloma cells SP2 / 0 were resuscitated, and mouse myeloma cells and mouse spleen cells were mixed at a ratio of 1:5, and 1 mL of polyethylene glycol 1500 was added. After standing for 90 s, the supernatant was discarded, resuspended in culture medium, and transferred to a 96-well plate for culture. The cell line that can secrete BPS antibodies was obtained by limiting dilution method, and the cell line was injected into the mouse to obtain ascites. The antibody was purified using a Protein G affinity column to obtain BPS-specific antibodies.

[0030] (4) Preparation of gold nanoflowers

[0031] First, colloidal gold was prepared. After 100 mL of an aqueous solution containing 0.01% chloroauric acid was heated to boiling, 5 mL of 1% sodium citrate was quickly added. When the solution turned wine red, heating was stopped and cooled to room temperature for use. Then 70 mL of ultrapure water was taken, heated to 55 °C, 0.8 mL of colloidal gold solution, 1 mL of 1% chloroauric acid, and 2.7 mL of 1% sodium citrate solution were added, stirred evenly, and 30 mL of hydroquinone containing 33 mg was quickly added. After the solution turned blue, stirring was continued for 10 min to obtain gold nanoflowers.

[0032] (5) Preparation of enzyme-labeled antibodies

[0033] First, HRP was labeled on the BPS monoclonal antibody using the sodium periodate method. The specific method was to thoroughly mix 1 mL of 5 mg / L HRP solution with 0.2 mL of 0.1 M NaIO4 solution and shake it at room temperature for 20 min. Then, the mixture was dialyzed in 1 mM acetate buffer (pH 4.4) at 4°C overnight. After the liquid was taken out, 0.5 mL of 0.16 M ethylene glycol solution was added and shaken at room temperature for 30 min. 1 mg of BPS monoclonal antibody was added and mixed. After shaking at low temperature for 30 min, the mixture was dialyzed in 0.05 M carbonate buffer (pH 9.5) overnight. 0.1 mL of 5 mg / mL NaBH4 solution was added and shaken at 4°C for 2 h. Then, an equal volume of saturated ammonium sulfate solution was slowly added, mixed and shaken at 4°C for 30 min, centrifuged at 3000 rpm for 30 min, the supernatant was removed, the precipitate was dissolved in a small amount of 0.15 M PBS with pH=7.4 and placed in a dialysis bag and dialyzed in PBS at 4°C for desalination. The liquid was taken out and centrifuged at 3000 rpm for 30 min to remove the precipitate. The supernatant was collected, which was the HRP-labeled BPS monoclonal antibody. Then, 8 mg of HRP-labeled BPS monoclonal antibody was added to 1 mL of the prepared gold nanoflower solution. After shaking for 1 h, the solution was centrifuged at 12000 r / min for 10 min and the precipitate was re-dissolved with 0.02 M Tris-HCl solution to obtain BPS monoclonal antibody co-labeled with gold nanoflowers and HRP.

[0034] Experimental Example 2: Establishment of a competitive ELISA kit for detecting BPS

[0035] (1) Determination of the optimal dilution ratio of coated antigen and enzyme-labeled antibody

[0036] The optimal dilution multiples of the coating antigen and the enzyme-labeled antibody were determined by the chessboard titration method. First, BPS-OVA, i.e., the coating antigen, was diluted at a ratio of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000. 100 μL / well was added to the ELISA plate and incubated at 4°C overnight. The next day, the coating solution was removed and the plate was washed three times with washing solution. Then, 200 μL of blocking solution (PBS containing 2% BSA and 0.1% Tween-20) was added and incubated at 37°C for 1 h. The enzyme-labeled antibody was diluted at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200 and 1:102400 and added to the 96-well plate at 100 μL / well. The plate was incubated at 37°C for 1 h. Then, 100 μL of TMB colorimetric solution was added to each well. After color development at 37°C for 10 min, 100 μL of 2 M H2SO4 was added to terminate the color development reaction. The concentration of each well at 450 °C was measured using an ELISA reader. The OD value at nm is shown in Table 1. When the dilution of the co-labeled BPS monoclonal antibody is 1:200 and the dilution of the BPS complete antigen is 1:1000, the OD value of the competitive ELISA is 450 The dilution factor is 1.215, which is the best dilution factor.

[0037] Table 1 shows the optimal dilution multiples of coated antigen and labeled antibody in BPS competitive ELISA

[0038]

[0039] (2) Determination of the competitive ELISA standard curve

[0040] The coated antigen was diluted 1000 times with PBS and added to a 96-well ELISA plate. After incubation at 4°C overnight, 200 μL of blocking solution was added and blocked at 37°C for 1 h. 100 μL of standard and 100 μL of enzyme-labeled antibody were added and incubated at 37°C for 1 h before color development. The results are shown in Figure 2. Figure 5 As shown in the figure, when the BPS concentration is 15.625~2000 ng / mL, the competitive ELISA has a good linear relationship and its detection limit is 15.625 ng / mL, which can be used for the detection of BPS.

[0041] (3) Determination of the specificity of competitive ELISA

[0042] Different concentrations of BPA, bisphenol F, bisphenol P, bisphenol Z, bisphenol AP and bisphenol AF were used as standard samples for detection using the competitive ELISA kit established by the present invention. The results are shown in Table 2. It was found that the competitive ELISA established by the present invention can specifically identify BPS.

[0043] Table 2 shows the specificity results of competitive ELISA

[0044]

[0045] Example 3: Use of BPS competitive ELISA kit

[0046] The specific composition of the BPS direct competitive ELISA kit is as follows:

[0047] (1) 1 blank ELISA plate;

[0048] (2) 1 tube of BPS complete antigen, diluted 1000 times before use;

[0049] (3) 1 tube of pure BPS, diluted to the required concentration with antibody (antigen) diluent before use;

[0050] (4) One gold nanoflower and HRP co-labeled antibody, diluted 200 times with antibody (antigen) diluent before use;

[0051] (5) 1 tube each of coating solution, blocking solution, washing solution, antibody (antigen) diluent, color development solution and stop solution, wherein the coating solution is 50 mM pH 9.6 carbonate buffer: 1.59 g Na2CO3, 2.93 g NaHCO3, distilled water is added to 1000 mL; the blocking solution is pH 7.4 phosphate buffer containing 2% OVA: 0.2 g OVA dissolved in 10 mL pH7.4 phosphate buffer; the washing solution is 150 mM pH7.4 phosphate buffer containing 0.05% Tween-20: 8.0 g NaCl, 0.2 g KCl, 2.9 gNa2HPO4·12H2O, 0.2 g KH2PO4, 0.5 mL Tween-20, distilled water is added to 1000 mL; the antibody (antigen) diluent is 150 mM pH7.4 phosphate buffer containing 0.05% Tween-20 and 1% OVA Phosphate buffer, pH 7.4; the colorimetric solution was TMB single-component colorimetric solution produced by Beijing Noble Technology Co., Ltd.; the stop solution was 2 M H2SO4 aqueous solution.

[0052] The competitive ELISA kit for detecting BPS of the present invention can be used for detecting BPS in a sample. The use thereof can be divided into the following steps:

[0053] (1) Dilute the complete BPS antigen 1000-fold using the coating solution in the kit, add 100 μL to each well, coat overnight at 4°C, discard the coating solution and wash three times;

[0054] (2) Add 200 μL of blocking solution to each well, incubate at 37°C for 1 h, discard the blocking solution and wash three times;

[0055] (3) Dilute the pure BPS to 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000, and 2000 ng / mL with antibody (antigen) diluent; dilute the enzyme-labeled antibody 200 times, mix it with the standard or the sample to be tested, and add it to a 96-well ELISA plate, 100 μL per well, 50 μL each of the standard or the sample to be tested and the enzyme-labeled antigen, incubate at 37°C for 1 hour, discard the solution in the wells, and wash 5 times;

[0056] (4) Add 100 μL of colorimetric solution to a 96-well ELISA plate and incubate in the dark at 37 °C for 10 min.

[0057] (5) Add 100 μL of stop solution to each well;

[0058] (6) Measure the absorbance of each well at 450 nm using an ELISA reader within 10 minutes after adding the stop solution.

[0059] (7) Calculation: The logarithm of the concentration of the standard substance is used as the horizontal axis, OD 450 The standard curve is drawn with the concentration of the standard substance and OD 450 The linear regression equation of the standard curve was calculated and the OD of the samples was 450 Substitute the value into the equation to calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of BPS in the sample.

Claims

1. A bisphenol S competitive ELISA kit using gold nanoflowers to enhance the signal, characterized in that The kit contains BPS complete antigen, gold nanoflowers, and HRP-co-labeled BPS monoclonal antibodies; The BPS complete antigen uses 5-bromovalerate methyl ester as a connecting arm; the carrier protein of the BPS complete antigen is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, fibrinogen or gelatin; The BPS monoclonal antibody in the BPS monoclonal antibody jointly labeled with gold nanoflowers and HRP is prepared from the BPS complete antigen; The BPS complete antigen is prepared by the following method: First, 0.25 g of BPS was weighed and added to 50 mL of acetonitrile and stirred until completely dissolved. Then, 0.415 g of potassium carbonate was added and reacted for 1 h. After 0.293 g of methyl 5-bromovalerate was heated and refluxed for 24 h, the organic solvent was removed and the obtained solid was purified by column to obtain the hapten of BPS. 10 mg of BPS hapten, 8.9 mg of carbodiimide and 6.2 mg of N-hydroxysuccinimide were weighed and dissolved in 4 mL of N,N-dimethylformamide and reacted in the dark at room temperature for 2 h. The above solution was added dropwise to 2 mL of phosphate buffer containing 20 mg of ovalbumin or 15 mg of bovine serum albumin. After shaking at 4 °C overnight, the supernatant was centrifuged at 6000 r / min for 5 min and dialyzed in 0.01 M PBS for 32 h. h, the dialysate was replaced once; the resulting solution was centrifuged again to obtain the supernatant, which was the complete BPS antigen, wherein the complete antigen obtained when ovalbumin was added was recorded as BPS-OVA, which was used as the immune antigen; the complete antigen obtained when bovine serum albumin was added was recorded as BPS-BSA, which was used as the coating antigen; The BPS monoclonal antibody is co-labeled with gold nanoflowers and HRP by the following steps: First, HRP was labeled on the BPS monoclonal antibody using the sodium periodate method. The specific method was as follows: 1 mL of 5 mg / L HRP solution was fully mixed with 0.2 mL of 0.1M NaIO4 solution, and then shaken at room temperature for 20 min; then placed in 1 mM acetate buffer (pH 4.4) and dialyzed overnight at 4°C; after the liquid was taken out, 0.5 mL of 0.16 M ethylene glycol solution was added, and shaken at room temperature for 30 min; 1 mg of BPS monoclonal antibody was added and mixed, and after shaking at low temperature for 30 min, it was dialyzed in 0.05 M carbonate buffer (pH 9.5) overnight; after the liquid was taken out, 0.1 mL of 5 mg / mL NaBH4 solution was added and shaken at 4 °C for 2 h; then an equal volume of saturated ammonium sulfate solution was slowly added, and after mixing, it was shaken at 4 °C for 30 min, centrifuged at 3000 rpm for 30 min, the supernatant was removed, and the precipitate was dissolved in 0.15 M PBS with pH=7.4 and placed in a dialysis bag in PBS for 4 °C for desalting by dialysis; after the liquid was taken out, it was centrifuged at 3000 rpm for 30 min to remove the precipitate, and the supernatant was collected as the HRP-labeled BPS monoclonal antibody; then 8 μg of HRP-labeled BPS monoclonal antibody was added to 1 mL of gold nanoflower solution, shaken for 1 h, and centrifuged at 12000 r / min for 10 min, and then the precipitate was re-dissolved with 0.02 M Tris-HCl solution to obtain gold nanoflowers and HRP-labeled BPS monoclonal antibodies.

2. The BPS indirect competitive ELISA detection method using the kit according to claim 1 is characterized in that The following steps are involved: Step 1, coating the BPS complete antigen in the kit according to claim 1 on an ELISA plate with a coating solution; Step 2, blocking the ELISA plate with a blocking solution; Step 3, mixing the enzyme-labeled gold nanoflowers and the BPS monoclonal antibody co-labeled with HRP with the sample to be tested and adding them to the ELISA plate, so that the BPS complete antigen coated in step 1 is mixed with the sample to be tested, and when BPS exists in the sample, the BPS in the sample competitively binds to the BPS monoclonal antibody.

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