CN115327097A - Bisphenol S competition ELISA kit and method using gold nanoflower to enhance signal - Google Patents
Bisphenol S competition ELISA kit and method using gold nanoflower to enhance signal Download PDFInfo
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- CN115327097A CN115327097A CN202210887841.3A CN202210887841A CN115327097A CN 115327097 A CN115327097 A CN 115327097A CN 202210887841 A CN202210887841 A CN 202210887841A CN 115327097 A CN115327097 A CN 115327097A
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- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
Bisphenol S competition ELISA kits and methods utilizing nanoflower-enhanced signals comprising BPS complete antigen, nanoflowers, and HRP-labeled BPS monoclonal antibody in combination. Using 5-bromomethyl valerate as an intermediate product to prepare a complete antigen of BPS, and then immunizing a mouse with the complete antigen of BPS to obtain a monoclonal antibody with high affinity and high specificity of BPS; the antibody was then labeled with HRP, and then gold nanoflowers with higher specific surface area and biological binding, to which more antibody was bound, creating a BPS competition ELISA with higher sensitivity, with detection ranges: 15.625-2000 ng/mL, and the detection limit is 15.625 ng/mL. The invention can be used for detecting BPS in environmental matrixes, biological samples, foods and medicines. Meanwhile, compared with the conventional chemical method, the method is simpler, more convenient and quicker, and does not need expensive equipment and complex pretreatment process.
Description
Technical Field
The invention belongs to the field of environmental detection, and particularly relates to a bisphenol S competition ELISA kit and a method using gold nanoflowers to enhance signals.
Background
Bisphenol S (BPS) is a substitute for Bisphenol a (BPA) and is widely used in the production of polycarbonate plastics and resins because of its higher thermal and light stability compared to BPA. But with the increasing use of BPS in "BPA-free" products, its potential to enter the environment is also increasing, leading to the detection of BPS in various environmental matrices and biological samples. For example, the average concentration value of BPS in the water body of the Taihu lake is 16 ng/L; the detected concentration of BPS in the chinese adult urine sample (n = 160) was 0.24 μ g/L. It has been shown that the hormonal activity of BPS interferes with the normal endocrine function of the organism. Such as environmental concentrations of BPS exposure, can have toxic effects on zebrafish development and reproduction. In addition, maternal exposure to low concentrations of BPS can lead to damage to the neuronal and endocrine systems of the offspring. In view of the high concentration and toxic effect of BPS in the environment, it is highly desirable to establish a method for detecting BPS.
The existing detection method of the BPS mainly comprises high performance liquid chromatography and gas chromatography-mass spectrometry, and although the method has high precision and accuracy, the complicated sample pretreatment process, expensive equipment and other problems make the method difficult to popularize and limit the detection work of the BPS. The immunological detection method is to detect BPS by utilizing the recognition effect of an antibody on an antigen, wherein Enzyme linked immunosorbent assay (ELISA) is the most widely applied immunological detection method because of the characteristics of high speed, sensitivity, low requirement on equipment, low cost and the like. Although there has been competition for BPS ELISA, it is mainly used for the detection work of BPS in foods and has limited sensitivity. In the environmental sample, the content of the BPS is low, and the detection difficulty is high, so that a more sensitive BPS detection method is required. The selection of an appropriate signal amplification strategy and the preparation of antibodies with high affinity are key to improving the sensitivity of ELISA. The gold nanoflowers have the same bioavailability, stability and nano enzyme activity as the gold nano materials, and the flower-shaped structures of the gold nanoflowers can effectively increase the specific surface area, so that the gold nanoflowers are a signal amplification element with excellent performance. Therefore, the invention further amplifies the detection signal by preparing the monoclonal antibody with high affinity and high specificity of the BPS and sequentially coupling the antibody with gold nanoflower and Horse Radish Peroxidase (HRP), thereby establishing competitive ELISA of the BPS.
Disclosure of Invention
The invention aims to provide a bisphenol S competition ELISA kit and a method for enhancing signals by utilizing gold nanoflowers, wherein the gold nanoflowers are used as a signal amplification original, so that the competition ELISA kit for detecting BPS is established, and the defect of low detection sensitivity of the existing competition ELISA is overcome.
A bisphenol S competition ELISA kit utilizing gold nanoflower enhanced signals is characterized in that the kit comprises BPS complete antigen, gold nanoflowers and BPS monoclonal antibody jointly marked by HRP;
the BPS complete antigen uses methyl 5-bromovalerate as a linker arm; the carrier protein of the BPS complete antigen is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, fibrinogen or gelatin.
The gold nanoflowers and the HRP are combined to label the BPS monoclonal antibody in the BPS monoclonal antibody, and the BPS monoclonal antibody is prepared from the BPS complete antigen.
In the kit, the BPS complete antigen is prepared by the following method:
firstly, weighing 0.25 g of BPS, adding the BPS into 50 mL acetonitrile, stirring until the BPS is completely dissolved, then adding 0.415 g potassium carbonate to react with 1 h, heating and refluxing 0.293 g of 5-bromovaleric acid methyl ester to obtain 24 h, removing an organic solvent, and purifying the obtained solid through a column (petroleum ether/ethyl acetate) to obtain the hapten of the BPS; weighing 10 mg of BPS hapten, 8.9 mg carbonyl diimine (1- (3-dimethylamino propyl) -3-ethyl carboxyl diimine hydrochloride, EDC) and 6.2 mg of N-hydroxysuccinimide (1-hydroxy-5-pyrrolidinedione, NHS) and dissolving in 4 mL of N, N-Dimethylformamide (N, N-Dimethylformamide, DMF), and reacting at room temperature in a dark place for 2 h; adding the solution dropwise into 2 mL phosphate buffer containing 20 mg Ovalbumin (Ovalbumin, OVA) or 15 mg Bovine serum albumin (Bovine albumin, BSA), reacting at 4 deg.C overnight, centrifuging at 6000 r/min for 5 min to obtain supernatant, dialyzing in 0.01M PBS for 32 h, and replacing dialysate every 8 h; centrifuging the obtained solution again to obtain supernatant which is the BPS-complete antigen, wherein when ovalbumin is added, the product is marked as BPS-BSA which is used as an immune antigen; the product was marked as BPS-OVA as coating antigen when bovine serum albumin was added.
In the kit, the BPS monoclonal antibody is jointly labeled by gold nanoflowers and HRP on the BPS monoclonal antibody through the following steps:
firstly, adopting a sodium periodate method to mark HRP on a BPS monoclonal antibody, wherein the specific method is to mix 1 mL of 5 mg/L HRP solution with 0.2 mL of 0.1M NaIO 4 After the solutions are fully mixed, shaking for 20 min at room temperature; then put into 1 mM acetate buffer (pH 4.4) for dialysis overnight at 4 ℃; taking out the liquid, adding 0.5 mL of 0.16M glycol solution, and shaking at room temperature for 30 min; adding 1 mg BPS monoclonal antibody, mixing, shaking at low temperature for 30 min, and dialyzing in 0.05M carbonate buffer solution (pH 9.5) overnight; the liquid was taken out and 0.1 mL of 5 mg/mL NaBH was added 4 Shaking the solution at 4 ℃ for 2 h; then slowlyAdding saturated ammonium sulfate solution with the same volume, mixing, shaking at 4 deg.C for 30 min, centrifuging at 3000 rpm for 30 min, removing supernatant, dissolving the precipitate in 0.15M PBS with pH =7.4, and dialyzing in PBS at 4 deg.C to remove salt; centrifuging at 3000 rpm for 30 min after taking out the liquid, removing precipitates, and collecting supernate, namely the BPS monoclonal antibody marked by HRP; then 8 mu g of HRP-labeled BPS monoclonal antibody is added into 1 mL gold nanoflowers solution, after 1 h is shaken, 12000 r/min is centrifuged for 10 min, and then 0.02M Tris-HCl solution is used for redissolution and precipitation to obtain gold nanoflowers and HRP-labeled BPS monoclonal antibody.
The BPS indirect competition ELISA detection method using the kit is characterized by comprising the following steps:
Advantages of the invention
Currently, high performance liquid chromatography and gas chromatography are commonly used for detecting BPS, but these methods generally require expensive equipment and complicated sample pretreatment processes. Compared with the chemical method, the conventional ELISA method has the advantages of simple operation, low equipment requirement and high detection speed, but has lower sensitivity. At present, the sensitivity of the detection method is mainly improved by adding a signal amplification strategy and preparing an antibody with higher affinity. Therefore, the invention uses the methyl 5-bromovalerate as a connecting product to prepare the BPS hapten, and the methyl 5-bromovalerate has a longer carbon chain, so that antigenic determinants can be exposed more fully, and the BPS monoclonal antibody with higher affinity and specificity can be prepared. Then, signals are further amplified by using the gold nanoflowers, more antibodies marked by the HRP are combined on the gold nanoflowers, the concentration of the combined antibodies is increased, and meanwhile, the gold nanoflowers have the nano enzyme activity, so that the detection ELISA signals can be increased, and the sensitivity of the competitive ELISA established by the invention is improved. By using the gold nanoflower and HRP dual signal enhancement kit, a quick and sensitive detection method is provided for the detection work of BPS.
Description of the drawings:
FIG. 1 is a diagram of the synthesis of BPS hapten.
FIG. 2 is a BPS complete antigen synthesis diagram, wherein FIG. 2a is a BPS immune antigen synthesis diagram and FIG. 2b is a BPS envelope antigen synthesis diagram.
FIG. 3 is a BPS complete antigen infrared spectrum identification result chart.
FIG. 4 is a diagram of the result of BPS complete antigen polyacrylamide gel electrophoresis.
FIG. 5 is a standard curve of the BPS competition ELISA kit.
The specific implementation mode is as follows:
a bisphenol S competition ELISA kit utilizing gold nanoflowers to enhance signals comprises a kit body and a conventional enzyme-labeled antibody, a coating solution, a washing solution, an antibody diluent, a developing solution and a stop solution which are arranged in the kit body, and is characterized by further comprising a BPS complete antigen, gold nanoflowers and a BPS monoclonal antibody jointly marked by HRP.
The invention will be further elucidated by means of the detailed description and the accompanying drawings.
Example 1 preparation of BPS haptens and monoclonal antibodies
(1) Synthesis of BPS haptens
Firstly, BPS hapten is prepared as shown in figure 1, 0.25 g of BPS is weighed and added into 50 mL acetonitrile to be stirred until the BPS hapten is completely dissolved, then 0.415 g potassium carbonate is added to react with 1 h, 0.293 g of 5-bromovaleric acid methyl ester is heated and refluxed to 24 h, organic solvent is removed, and the obtained solid is purified by a column (petroleum ether/ethyl acetate), so that the BPS hapten can be obtained.
(2) Synthesis of complete antigens
BPS complete antigen was prepared as shown in FIG. 2, 10 mg BPS hapten, 8.9 mg EDC, 6.2 mg NHS were weighed out and dissolved in 4 mL DMF and reacted at room temperature in the dark for 2 h. The solution is added dropwise into 2 mL containing 20 mg OVA or 15 mg BSA phosphate buffer solution, 4 ℃ reaction shaking overnight, 6000 r/min centrifugation for 5 min to get supernatant, placed in 0.01M PBS for dialysis 32 h, every 8 h change dialysate. The obtained solution is centrifuged again to obtain the supernatant, namely the complete antigen (BPS-OVA/BPS-BSA). Wherein, BPS-BSA is an immunizing antigen, and BPS-OVA is a coating antigen. The infrared spectrum scanning is carried out on the prepared complete antigen, the result is shown in figure 3, the infrared absorption peak of the complete antigen coupled with BSA and OVA has obvious characteristic peak, the polyacrylamide gel electrophoresis result of figure 4 also shows that the molecular weight of the complete antigen is obviously greater than that of BSA or OVA, and the above results all prove that the complete antigen of BPS is prepared by the invention.
(3) Preparation of BPS-specific antibodies
Selecting 3 healthy Balb/C mice as experimental organisms, emulsifying 100 microgram of BPS-BSA and an equal volume of Freund's complete adjuvant, and injecting the mice into the abdominal cavity. Mice were immunized after full emulsification with the same dose of BPS-BSA with Freund's incomplete adjuvant every 14 days. 3 days after the end of the 4 th immunization, the mice were sacrificed and the spleens were removed for use. Then, the mouse myeloma cells SP2/0 are recovered, and the mouse myeloma cells and the mouse spleen cells are mixed according to the ratio of 1:5, adding 1 mL polyethylene glycol 1500, standing 90 s, centrifuging for 10 min at 1000 r/min, discarding the supernatant, adding a culture medium for resuspension, transferring to a 96-well plate for culture, obtaining a cell strain capable of secreting the BPS antibody by a limiting dilution method, injecting the cell strain into a mouse to obtain ascites, and purifying the antibody by a Protein G affinity chromatography column to obtain the BPS specific antibody.
(4) Preparation of gold nanoflowers
Firstly, preparing colloidal gold, heating 100 mL aqueous solution containing 0.01% chloroauric acid to boil, quickly adding 5 mL of 1% sodium citrate, stopping heating when the solution turns to wine red, and cooling to room temperature for later use. And then taking 70 mL ultrapure water, heating to 55 ℃, adding 0.8 mL colloidal gold solution, 1 mL of 1% chloroauric acid and 2.7 mL of 1% sodium citrate solution, uniformly stirring, rapidly adding 30 mL hydroquinone containing 33 mg, and continuously stirring for 10 min after the solution turns blue to obtain the gold nanoflowers.
(5) Preparation of enzyme-labeled antibody
Firstly, adopting a sodium periodate method to mark HRP on a BPS monoclonal antibodyThe specific method is to mix 1 mL of 5 mg/L HRP solution with 0.2 mL of 0.1M NaIO 4 After the solution was mixed well, it was shaken at room temperature for 20 min. Then put into 1 mM acetate buffer (pH 4.4) for dialysis overnight at 4 ℃; taking out the liquid, adding 0.5 mL of 0.16M glycol solution, and shaking at room temperature for 30 min; adding 1 mg BPS monoclonal antibody, mixing, shaking at low temperature for 30 min, and dialyzing in 0.05M carbonate buffer solution (pH 9.5) overnight; then 0.1 mL of 5 mg/mL NaBH was added 4 Shaking the solution at 4 ℃ for 2 h; slowly adding equal volume of saturated ammonium sulfate solution, mixing, shaking at 4 deg.C for 30 min, centrifuging at 3000 rpm for 30 min, removing supernatant, dissolving the precipitate in small amount of 0.15M PBS with pH =7.4, and placing into dialysis bag to dialyze at 4 deg.C in PBS for desalting; taking out the liquid, centrifuging at 3000 rpm for 30 min, removing the precipitate, and collecting the supernatant, namely the BPS monoclonal antibody marked by HRP. And then 8 mg of the BPS monoclonal antibody marked by the HRP is added into a gold nanoflower solution prepared by 1 mL, 1 h is shaken, 12000 r/min is centrifuged for 10 min, and then 0.02M Tris-HCl solution is used for redissolution and precipitation, so that the BPS monoclonal antibody marked by the gold nanoflower and the HRP in a combined mode is obtained.
Experimental example 2 establishment of competitive ELISA kit for detecting BPS
(1) Determination of optimal dilution times of coating antigen and enzyme-labeled antibody
The optimal dilution of the coating antigen and the enzyme-labeled antibody was determined by checkerboard titration. First, the BPS-OVA, i.e., the envelope antigen, was diluted at a ratio of 1. Adding the mixture into an enzyme label plate according to the amount of 100 mu L/hole, and incubating overnight at 4 ℃; the next day, pour coating solution, using the washing liquid after washing 3 times, adding 200 u L blocking solution (containing 2% BSA, 0.1% Tween-20 PBS), 37 degrees C were incubated 1 h; the enzyme-labeled antibody was diluted according to 1; then 100. Mu.L of TMB developing solution was added to each well, and after development at 37 ℃ for 10 min, 100. Mu.L of 2M H was added 2 SO 4 Terminating the color development reaction; OD at 450 nm was measured for each well using a microplate reader and the results are shown in Table 1 when combinedThe dilution of the BPS monoclonal antibody was 1:200, and the OD of the competitive ELISA was 1:1000 when the dilution of the BPS complete antigen was 1:200 450 Is 1.215, and is the optimal dilution factor.
Table 1 shows the optimal dilution fold results for BPS competition ELISA coated antigen and labeled antibody
(2) Determination of competitive ELISA Standard Curve
The coated antigen was diluted 1000-fold with PBS and added to a 96-well microplate. After incubation overnight at 4 ℃, 200 μ L of blocking solution was added and 1 h was blocked at 37 ℃; mu.L of the standard and 100. Mu.L of the enzyme-labeled antibody were added, and 1 h was incubated at 37 ℃ for color development. As shown in FIG. 5, when the concentration of BPS is 15.625 to 2000 ng/mL, the competitive ELISA has a good linear relationship, the detection limit is 15.625 ng/mL, and the detection method can be used for detecting BPS.
(3) Determination of competitive ELISA specificity
The results of detection by using the competitive ELISA kit established in the invention with BPA, bisphenol F, bisphenol P, bisphenol Z, bisphenol AP and bisphenol AF of different concentrations as standards are shown in Table 2, and the competitive ELISA established in the invention can specifically identify BPS.
Table 2 shows the results of the competitive ELISA specificity
Example 3 use of BPS Competition ELISA kit
The BPS direct competition ELISA kit specifically comprises the following components:
(1) 1 blank enzyme label plate;
(2) BPS complete antigen 1, diluted 1000-fold before use;
(3) The BPS pure product is 1, and is diluted to the required concentration by an antibody (antigen) diluent before use;
(4) Gold nanoflowers and HRP are combined to mark 1 antibody, and the antibody (antigen) is diluted by 200 times before use;
(5) 1 each of coating solution, blocking solution, washing solution, antibody (antigen) diluent, developing solution and stop solution, wherein the coating solution is 50 mM carbonate buffer solution with pH 9.6: 1.59 g Na 2 CO 3 ，2 .93g NaHCO 3 Adding distilled water to 1000 mL; the blocking solution was 2% OVA in phosphate buffer pH 7.4: 0.2 g OVA is dissolved in 10 mL phosphate buffer solution with pH7.4; the washing solution was 150 mM pH7.4 phosphate buffer containing 0.05-vol Tween-20: 8.0 NaCl,0.2 g KCl,2.9 g Na 2 HPO 4 ·12H 2 O，0.2 g KH 2 PO 4 0.5 mL of Tween-20, and adding distilled water to 1000 mL; the antibody (antigen) diluent is 150 mM phosphate buffer solution containing 0.05% Tween-20 and 1% OVA, and the pH value is 7.4; the color developing solution is TMB single-component color developing solution produced by Beijing Nobel science and technology Limited; the stop solution is H of 2M 2 SO 4 An aqueous solution.
The competitive ELISA kit for detecting BPS can be used for detecting BPS in samples. When in use, the method can be divided into the following steps:
(1) Diluting the BPS complete antigen 1000 times by using a coating solution in a kit, adding 100 mu L of the BPS complete antigen into each hole, coating at 4 ℃ overnight, discarding the coating solution and washing for 3 times;
(2) Adding 200 mu L of confining liquid into each hole, incubating 1 h at 37 ℃, discarding the confining liquid and washing for 3 times;
(3) Diluting the pure BPS product to 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000, 2000 ng/mL by using an antibody (antigen) diluent; diluting the enzyme-labeled antibody by 200 times, mixing the diluted enzyme-labeled antibody with a standard substance or a sample to be detected, adding the mixture into a 96-hole enzyme-labeled plate, incubating for 1 hour at 37 ℃, discarding the solution in the hole, and washing for 5 times, wherein each hole has 100 mu L, and the standard substance or the sample to be detected and the enzyme-labeled antigen are 50 mu L respectively;
(4) Adding 100 mu L of color development liquid into a 96-hole enzyme label plate, and reacting for 10 min at 37 ℃ in a dark place;
(5) Adding 100 mu L of stop solution into each hole;
(6) Measuring the light absorption value of each hole under the wavelength of 450 nm by using an enzyme-labeling instrument within 10 minutes after adding the stop solution;
(7) And (3) calculating: concentration with standardLogarithmic value of degree being abscissa, OD 450 The values are plotted as ordinate against OD 450 Calculating the value to obtain a linear regression equation of the standard curve, and determining the OD of the sample 450 Substituting the value into an equation to calculate the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the BPS in the sample.
Claims (5)
1. A bisphenol S competition ELISA kit utilizing gold nanoflowers to enhance signals is characterized in that the kit comprises BPS complete antigen, gold nanoflowers and BPS monoclonal antibody jointly marked by HRP;
the BPS complete antigen uses methyl 5-bromovalerate as a linker arm; the carrier protein of the BPS complete antigen is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, fibrinogen or gelatin.
2. The kit according to claim 1, wherein the BPS monoclonal antibody of the gold nanoflowers and HRP-labeled BPS monoclonal antibody is prepared from the BPS complete antigen of claim 1.
3. The kit according to claim 1, wherein the BPS complete antigen is prepared by the following method:
firstly, weighing 0.25 g of BPS, adding the BPS into 50 mL acetonitrile, stirring until the BPS is completely dissolved, then adding 0.415 g potassium carbonate to react with 1 h, heating and refluxing 0.293 g of 5-bromovaleric acid methyl ester to 24 h, removing an organic solvent, and then passing the obtained solid through a column for purification to obtain the hapten of the BPS; weighing 10 mg of BPS hapten, 8.9 mg carbodiimide and 6.2 mg of N-hydroxysuccinimide, dissolving in 4 mL of N, N-dimethylformamide, and reacting at room temperature in a dark place for 2 h; dropwise adding the solution into 2 mL phosphate buffer containing 20 mg ovalbumin or 15 mg bovine serum albumin, reacting at 4 ℃ and shaking overnight, centrifuging at 6000 r/min for 5 min to obtain supernatant, placing the supernatant in 0.01M PBS for dialysis, namely 32 h, and replacing the dialysate every 8 h; centrifuging the obtained solution again to obtain supernatant which is the BPS complete antigen, wherein the complete antigen prepared when ovalbumin is added is marked as BPS-BSA which is taken as an immune antigen; the complete antigen produced upon addition of bovine serum albumin was designated as BPS-OVA and used as the envelope antigen.
4. The kit according to claim 3, wherein the BPS monoclonal antibody is labeled with gold nanoflowers and HRP by the following steps:
firstly, adopting a sodium periodate method to mark HRP on a BPS monoclonal antibody, wherein the specific method is to mix 1 mL of 5 mg/L HRP solution with 0.2 mL of 0.1M NaIO 4 After the solution is fully mixed, shaking for 20 min at room temperature; then put into 1 mM acetate buffer (pH 4.4) for dialysis overnight at 4 ℃; taking out the liquid, adding 0.5 mL of 0.16M glycol solution, and shaking at room temperature for 30 min; adding 1 mg BPS monoclonal antibody, mixing, shaking at low temperature for 30 min, and dialyzing in 0.05M carbonate buffer solution (pH 9.5) overnight; the liquid was taken out and 0.1 mL of 5 mg/mL NaBH was added 4 Shaking the solution at 4 ℃ for 2 h; slowly adding an equal volume of saturated ammonium sulfate solution, uniformly mixing, shaking at 4 ℃ for 30 min, centrifuging at 3000 rpm for 30 min, removing supernatant, dissolving the precipitate in 0.15M PBS (pH = 7.4), putting into a dialysis bag, and dialyzing at 4 ℃ in PBS to remove salt; centrifuging the liquid at 3000 rpm for 30 min after the liquid is taken out, removing precipitates, and collecting supernatant, namely the BPS monoclonal antibody marked by HRP; then 8 mu g of HRP-labeled BPS monoclonal antibody is added into 1 mL gold nanoflowers solution, after 1 h is shaken, 12000 r/min is centrifuged for 10 min, and then 0.02M Tris-HCl solution is used for redissolution and precipitation to obtain gold nanoflowers and HRP-labeled BPS monoclonal antibody.
5. The indirect competition ELISA detection method for BPS using the kit of claim 1, characterized by comprising the steps of:
step 1, coating the BPS complete antigen in the kit according to claim 1 on an enzyme label plate by using a coating solution; step 2, sealing the ELISA plate by using sealing liquid; and 3, mixing the enzyme-labeled gold nanoflowers and the BPS monoclonal antibody jointly labeled by HRP with the sample to be detected, adding the mixture into the enzyme-labeled plate, mixing the BPS complete antigen coated in the step 1 with the sample to be detected, and when BPS exists in the sample, competitively combining the BPS monoclonal antibody in the sample.
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