Hapten, artificial antigen for simultaneously detecting bisphenol sardin and bisphenol sardin acetate in weight loss foods and application thereof

Abstract

The present invention discloses haptens and artificial antigens for simultaneously detecting bisphenol salvin and bisphenol salvin acetate in weight-loss foods, and their applications. The present invention designs universal haptens and artificial antigens containing the core structures of bisphenol salvin and bisphenol salvin acetate, and prepares antibodies capable of simultaneously recognizing bisphenol salvin and bisphenol salvin acetate. The antibodies prepared by the present invention have good recognition performance for both bisphenol salvin and bisphenol salvin acetate, with minimum detection limits (LODs) of 0.03 ng / mL and 1.23 ng / mL, respectively, and half inhibitory concentrations (ICs). 50 The concentrations of bisphenol A and bisphenol B were 0.26 ng / mL and 16.85 ng / mL, respectively, and there was no cross-reaction with the functional analogs of bisphenol A and bisphenol B acetate. The present invention can provide a technical basis for establishing a synchronous immunological detection method for bisphenol A and bisphenol B acetate, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, in particular to a hapten, an artificial antigen and applications thereof for simultaneously detecting bisphenol A and bisphenol B acetate in weight-loss foods. Background Art

[0002] Bisoxatin and bisoxatin acetate are laxative chemicals that strongly irritate the intestinal mucosa, promoting bowel movements and relieving constipation. However, excessive consumption can lead to dehydration, gastrointestinal discomfort, dizziness, nausea, and other symptoms, which can be life-threatening in severe cases. As living standards improve, obesity has become a growing health concern. The surge in demand for weight loss has spurred the emergence of a market for slimming products, with products such as slimming coffee, slimming tea, slimming capsules, and slimming enzymes. The Food Safety Law of the People's Republic of China clearly stipulates that drugs may not be added to food. However, some unscrupulous vendors, seeking to enhance product efficacy, add these laxative chemicals to slimming foods, achieving weight loss through laxative effects and seriously endangering human health. Therefore, to strengthen the regulation of bisoxatin and bisoxatin acetate in slimming foods, it is necessary to develop an accurate, convenient, and rapid detection method.

[0003] The reported detection method for this type of drug in weight-loss foods is mainly high-performance liquid chromatography tandem mass spectrometry (Xun Zhiqing et al., Determination of bisphenol A and other laxative drugs in candies and preserves by high-performance liquid chromatography tandem mass spectrometry, Guangdong Chemical Industry, Vol. 48, No. 24, 2021). Although high-performance liquid chromatography tandem mass spectrometry has high accuracy, the sample pre-treatment is cumbersome, the detection time is long, the instrument is expensive, and professional operators are required, which limits the wide application of this method and does not meet the requirements for on-site detection of large quantities of samples. In comparison, immunoassay technology has the advantages of high sensitivity, strong specificity, rapidity, and easy operation. It has been widely used in the field of drug residue detection and has many advantages over high-performance liquid chromatography tandem mass spectrometry. However, there is currently no report on the synthetic structure of bisphenol A and bisphenol A acetate haptens, and there is a lack of antibodies that can be used for detection, which cannot meet the needs of rapid testing for grassroots food safety supervision. Therefore, it is necessary to establish an efficient method for detecting bisphenol A and bisphenol A acetate, and it is imperative to find immunoassay raw materials such as haptens and artificial antigens that can produce antibodies with high specificity for bisphenol A and bisphenol A acetate. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the problem that existing immunoassay methods lack rapid, sensitive and simple detection of bisphenol A and bisphenol A acetate, and to provide haptens, artificial antigens and their applications for simultaneously detecting bisphenol A and bisphenol A acetate in weight-loss foods.

[0005] The first object of the present invention is to provide two bisphenol A and / or bisphenol A acetate haptens.

[0006] The second object of the present invention is to provide a method for preparing the bisphenol salbutamol and / or bisphenol salbutamol acetate hapten.

[0007] The third object of the present invention is to provide the use of the bisphenol salvin and / or bisphenol salvin acetate hapten in the preparation of bisphenol salvin and / or bisphenol salvin acetate artificial antigens.

[0008] The fourth object of the present invention is to provide two bisphenol A and / or bisphenol A acetate artificial antigens.

[0009] The fifth object of the present invention is to provide the use of the bisphenol A and / or bisphenol A acetate artificial antigen in the preparation of bisphenol A and / or bisphenol A acetate antibodies.

[0010] The sixth object of the present invention is to provide a bisphenol A and / or bisphenol A acetate artificial antigen combination.

[0011] The seventh object of the present invention is to provide the use of the bisphenol salbutamol and / or bisphenol salbutamol acetate artificial antigen combination in the preparation of a kit for detecting bisphenol salbutamol and / or bisphenol salbutamol acetate.

[0012] The eighth object of the present invention is to provide a kit for detecting bisphenol A and / or bisphenol A acetate, wherein the kit comprises the artificial antigen combination of bisphenol A and / or bisphenol A acetate.

[0013] In order to achieve the above object, the present invention is implemented through the following scheme:

[0014] A bisphenol sardine and / or bisphenol sardine acetate hapten, characterized in that its structural formula is as shown in formula (I), named BSO-1,

[0015]

[0016] The bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-1 adopts the systematic nomenclature: 2-(4-(2-(4-hydroxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-yl)phenoxy)acetic acid; that is, 2-(4-(2-(4-hydroxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-yl)phenoxy)acetic acid.

[0017] The method for preparing the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-1 having a structural formula as shown in formula (I) comprises the following steps: dissolving bisphenol sartine in methanol and fully reacting with ethyl bromoacetate, cesium carbonate and sodium iodide; fully hydrolyzing the resulting reactant under alkaline conditions; and then adjusting the pH to acidic to obtain the hapten.

[0018] Specifically, the preparation method of the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-1 having a structural formula as shown in formula (I) comprises the following steps: dissolving 1 mol of bisphenol sartine in 20 mL of methanol, then adding 2 mol of ethyl bromoacetate, 6 mol of cesium carbonate and 2 mol of sodium iodide, and stirring at 50°C for 8 hours to obtain a reactant; separating and purifying the reactant by column chromatography using silica gel powder as a stationary phase and a mixed solution of dichloromethane:methanol with a volume ratio of 70:1 as a mobile phase, dissolving the obtained purified product in 5 mL of methanol, then adding 5 mL of 3 mol / L sodium hydroxide aqueous solution and stirring at room temperature for 3 hours, and adjusting the pH to 6-7 after the reaction. The obtained precipitate is the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-1 having a structural formula as shown in formula (I).

[0019] A bisphenol sardine and / or bisphenol sardine acetate hapten, characterized in that its structural formula is as shown in formula (II), and is named BSO-2.

[0020]

[0021] The bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-2 adopts the systematic nomenclature: 4-((4-(2-(4-hydroxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-yl)phenoxy)methyl)benzoic acid; that is, 4-((4-(2-(4-hydroxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-yl)phenoxy)methyl)benzoic acid.

[0022] The method for preparing the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2 having a structural formula as shown in formula (II) comprises the following steps: dissolving bisphenol sartine in methanol and fully reacting with methyl 4-(bromomethyl)benzoate, cesium carbonate and sodium iodide; fully hydrolyzing the resulting reactant under alkaline conditions; and then adjusting the pH to acidic to obtain the hapten.

[0023] Specifically, the preparation method of the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2 having a structural formula as shown in formula (II) comprises the following steps: dissolving 1 mol of bisphenol sartine in 20 mL of methanol, then adding 2 mol of methyl 4-(bromomethyl)benzoate, 6 mol of cesium carbonate and 2 mol of sodium iodide, and stirring at 50°C for 8 hours to obtain a reactant; separating and purifying the reactant by column chromatography using silica gel powder as a stationary phase and a mixed solution of dichloromethane:methanol in a volume ratio of 70:1 as a mobile phase, dissolving the obtained purified product in 5 mL of methanol, then adding 5 mL of a 3 mol / L sodium hydroxide aqueous solution and stirring at room temperature for 3 hours. After the reaction is completed, the pH is adjusted to 6-7, and the obtained precipitate is the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2 having a structural formula as shown in formula (II).

[0024] The use of the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-1 and / or bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2 in the preparation of bisphenol sartine and / or bisphenol sartine acetate artificial antigens should also be within the scope of protection of the present invention.

[0025] An artificial antigen of bisphenol sardin and / or bisphenol sardin acetate, characterized in that it is obtained by coupling the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-1 with a carrier protein, the structural formula of which is shown in formula (III).

[0026]

[0027] Among them, P is the carrier protein.

[0028] Preferably, the carrier protein is chicken ovalbumin, that is, bisphenol A and / or bisphenol A acetate artificial antigen BSO-1-OVA, or the carrier protein is bovine serum albumin, that is, bisphenol A and / or bisphenol A acetate artificial antigen BSO-1-BSA.

[0029] Further preferably, the carrier protein is bovine serum albumin, that is, bisphenol A and / or bisphenol A acetate artificial antigen BSO-1-BSA.

[0030] An artificial antigen of bisphenol sardin and / or bisphenol sardin acetate, characterized in that it is obtained by coupling the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-2 with a carrier protein, the structural formula of which is shown in formula (IV).

[0031]

[0032] Among them, P is the carrier protein.

[0033] Preferably, the carrier protein is chicken ovalbumin, that is, bisphenol A and / or bisphenol A acetate artificial antigen BSO-2-OVA, or the carrier protein is bovine serum albumin, that is, bisphenol A and / or bisphenol A acetate artificial antigen BSO-2-BSA.

[0034] Further preferably, the carrier protein is chicken ovalbumin, that is, bisphenol A and / or bisphenol A acetate artificial antigen BSO-2-OVA.

[0035] The method for preparing the artificial antigen of bisphenol sarcoside and / or bisphenol sarcoside acetate should also be within the scope of protection of the present invention, which is characterized in that the bisphenol sarcoside and / or bisphenol sarcoside acetate hapten BSO-1 (structural formula shown in formula (I)) or the bisphenol sarcoside and / or bisphenol sarcoside acetate hapten BSO-2 (structural formula shown in formula (II)) is coupled to a carrier protein through the active ester method.

[0036] Preferably, the active ester method for coupling the carrier protein comprises the following steps:

[0037] S1. The bisphenol sardine and / or bisphenol sardine acetate hapten BSO-1 or the bisphenol sardine and / or bisphenol sardine acetate hapten BSO-2, N,N-dimethylformamide, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are fully reacted in the dark to obtain solution A;

[0038] S2. dissolving the carrier protein in phosphate buffer to obtain solution B;

[0039] S3. After solution A and solution B fully react, they are dialyzed to obtain bisphenol salbutamol and / or bisphenol salbutamol acetate artificial antigens.

[0040] Preferably, in step S1, the mass volume ratio of the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-1 or the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2, N,N-dimethylformamide, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 8-12 mg:50-100 μL:1-3 mg:2-4 mg.

[0041] More preferably, in step S1, the mass volume ratio of the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-1 or the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2, N,N-dimethylformamide, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 10 mg:100 μL:2 mg:3 mg.

[0042] Further more preferably, in step S1, the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-1 or the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-2 is first dissolved in N,N-dimethylformamide, and then N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added, and stirred in the dark at 25°C to 30°C for 2 to 4 hours.

[0043] More preferably, in step S1, the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-1 or the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-2 is first dissolved in N,N-dimethylformamide, and then N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added, and stirred at 25°C in the dark for 4 hours.

[0044] Preferably, in step S2, the carrier protein is chicken ovalbumin or bovine serum albumin.

[0045] More preferably, in step S2, the mass-to-volume ratio of the carrier protein to the phosphate buffer is 8-12 mg:0.5-1.5 mL.

[0046] More preferably, in step S2, the mass-to-volume ratio of the carrier protein to the phosphate buffer is 10 mg:1 mL.

[0047] Preferably, in step S3, the volume ratio of solution A to solution B is 1-3:5-10.

[0048] Further preferably, in step S3, the volume ratio of solution A to solution B is 1:10.

[0049] More preferably, in step S3, solution A is added dropwise to solution B, and stirred at 3°C to 5°C for 10 h to 14 h.

[0050] Even more preferably, in step S3, solution A is added dropwise to solution B and stirred at 4°C for 12 h.

[0051] More preferably, in step S3, the dialysis method is to dialyze with PBS buffer for 2 days, 4 times a day.

[0052] The use of the bisphenol A and / or bisphenol A acetate artificial antigens in the preparation of bisphenol A and / or bisphenol A acetate antibodies should also be within the scope of protection of the present invention.

[0053] A bisphenol A and / or bisphenol A acetate antibody is prepared by immunizing an animal with the bisphenol A and / or bisphenol A acetate artificial antigen.

[0054] Further more preferably, the bisphenol sarcoside and / or bisphenol sarcoside acetate artificial antigen is obtained by coupling the bisphenol sarcoside and / or bisphenol sarcoside acetate hapten BSO-1 with bovine serum albumin, namely BSO-1-BSA.

[0055] More preferably, the bisphenol A and / or bisphenol A acetate antibody is a monoclonal antibody, which is obtained by immunizing an animal with an artificial antigen of bisphenol A and / or bisphenol A acetate to obtain hybridoma cells, culturing the obtained hybridoma cells and collecting the cell supernatant, and then identifying and purifying the obtained hybridoma cells.

[0056] More preferably, the bisphenol A and / or bisphenol A acetate antibody is a polyclonal antibody, which is obtained by collecting serum from animals immunized with the bisphenol A and / or bisphenol A acetate artificial antigen and purifying it by ammonium sulfate precipitation.

[0057] The use of the bisphenol A and / or bisphenol A acetate antibodies in detecting bisphenol A and / or bisphenol A acetate for non-disease diagnosis purposes should also be within the scope of protection of the present invention.

[0058] Preferably, the bisphenol A and / or bisphenol A acetate antibody is used to detect bisphenol A and / or bisphenol A acetate in weight loss foods.

[0059] More preferably, the bisphenol A and / or bisphenol A acetate antibodies are used to detect bisphenol A and / or bisphenol A acetate in weight loss foods, wherein the weight loss foods are foods with weight loss functions, including but not limited to compressed candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, blended liquors, fruit wines and jellies, etc. with weight loss functions.

[0060] The use of the bisphenol sarcoside and / or bisphenol sarcoside acetate antibody in the preparation of a kit for detecting bisphenol sarcoside and / or bisphenol sarcoside acetate should also be within the scope of protection of the present invention.

[0061] A bisphenol sartine and / or bisphenol sartine acetate artificial antigen combination, characterized in that it comprises an immunogen and a coating agent, wherein the coating agent is obtained by coupling the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-1 or the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2 with chicken egg white albumin or bovine serum albumin; and the immunogen is obtained by coupling the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-1 or the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2 with chicken egg white albumin or bovine serum albumin.

[0062] Preferably, a bisphenol sartin and / or bisphenol sartin acetate artificial antigen combination is characterized in that it comprises an immunogen and a coating agent, wherein the coating agent is obtained by coupling bisphenol sartin and / or bisphenol sartin acetate hapten BSO-1 or bisphenol sartin and / or bisphenol sartin acetate hapten BSO-2 with chicken egg white albumin; and the immunogen is obtained by coupling bisphenol sartin and / or bisphenol sartin acetate hapten BSO-1 or bisphenol sartin and / or bisphenol sartin acetate hapten BSO-2 with bovine serum albumin.

[0063] More preferably, the coating source is obtained by coupling bisphenol salvin and / or bisphenol salvin acetate hapten BSO-2 with chicken ovalbumin, that is, the coating source is bisphenol salvin and / or bisphenol salvin acetate artificial antigen BS O-2-OVA.

[0064] More preferably, the immunogen is obtained by coupling the hapten BSO-1 of bisphenol A and / or bisphenol A acetate to bovine serum albumin, that is, the immunogen is the artificial antigen BS O-1-BSA of bisphenol A and / or bisphenol A acetate.

[0065] Most preferably, the coating agent is obtained by coupling bisphenol sartan and / or bisphenol sartan acetate hapten BSO-2 with chicken ovalbumin; the immunogen is obtained by coupling bisphenol sartan and / or bisphenol sartan acetate hapten BSO-1 with bovine serum albumin; that is, the coating agent is the bisphenol sartan and / or bisphenol sartan acetate artificial antigen BSO-2-OVA, and the immunogen is BSO-1-BSA.

[0066] The use of the bisphenol sarcoside and / or bisphenol sarcoside acetate artificial antigen combination in detecting bisphenol sarcoside and / or bisphenol sarcoside acetate for non-disease diagnosis purposes should also be within the scope of protection of the present invention.

[0067] Preferably, the bisphenol A and / or bisphenol A acetate artificial antigen combination is used to detect bisphenol A and / or bisphenol A acetate in weight loss foods.

[0068] More preferably, the bisphenol sartan and / or bisphenol sartan acetate artificial antigen combination is used to detect bisphenol sartan and / or bisphenol sartan acetate in weight loss foods, wherein the weight loss foods are foods with weight loss functions, including but not limited to compressed candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, blended liquors, fruit wines and jellies with weight loss functions.

[0069] A kit for detecting bisphenol A and / or bisphenol A acetate, comprising an artificial antigen combination of bisphenol A and / or bisphenol A acetate.

[0070] Preferably, the coating source of the bisphenol sartin and / or bisphenol sartin acetate artificial antigen combination is obtained by coupling bisphenol sartin and / or bisphenol sartin acetate hapten BSO-2 with chicken egg white albumin; the immunogen of the bisphenol sartin and / or bisphenol sartin acetate artificial antigen combination is obtained by coupling bisphenol sartin and / or bisphenol sartin acetate hapten BSO-1 with bovine serum albumin.

[0071] More preferably, the kit comprises bisphenol A and / or bisphenol A acetate antibodies, which are obtained by immunizing an animal with an immunogen obtained by coupling the bisphenol A and / or bisphenol A acetate hapten BSO-1 to bovine serum albumin.

[0072] More preferably, the kit further comprises an ELISA plate, a standard substance of bisphenol salvin and / or bisphenol salvin acetate, and a substrate colorimetric solution. The CAS number of the bisphenol salvin is 17692-24-9; the CAS number of the bisphenol salvin acetate is 14008-48-1.

[0073] Even more preferably, the ELISA plate is coated with a coating agent obtained by coupling bisphenol A and / or bisphenol A acetate hapten BSO-2 with chicken ovalbumin.

[0074] Even more preferably, the substrate color developing solution comprises urea peroxide and tetramethylbenzidine.

[0075] More preferably, the kit further comprises a stop solution, a washing solution and a blocking solution, an enzyme conjugate concentrate and an enzyme conjugate diluent.

[0076] Even more preferably, the stop solution is 1 mol / L to 3 mol / L H2SO4.

[0077] Most preferably, the stop solution is 2 mol / L H2SO4.

[0078] Even more preferably, the washing solution is a 0.1-0.3 mol / L phosphate buffer solution containing 0.5-1.0% w / v Tween-20 and 0.01-0.03% w / v sodium azide, with a pH of 7.2-7.6.

[0079] Most preferably, the blocking solution is 0.2 mol / L phosphate buffer containing 2% w / v casein, pH=7.3.

[0080] Even more preferably, the enzyme conjugate concentrate is goat anti-rabbit antibody or goat anti-mouse antibody labeled with horseradish peroxidase.

[0081] Even more preferably, the enzyme conjugate diluent is 0.1-0.3 mol / L phosphate buffer.

[0082] Most preferably, the enzyme conjugate diluent is 0.2 mol / L phosphate buffer.

[0083] Preferably, the kit contains a colloidal gold test strip, which includes a base plate, on which a sample pad, a reaction membrane and a water absorbent pad are overlapped in sequence, and the reaction membrane is a nitrocellulose membrane provided with a detection area and a quality control area; the detection area is coated with the coating antigen in the bisphenol salbutamol and / or bisphenol salbutamol acetate artificial antigen combination, and the quality control area is coated with IgG.

[0084] More preferably, the coating source in the bisphenol sartine and / or bisphenol sartine acetate artificial antigen combination is obtained by coupling the bisphenol sartine and / or bisphenol sartine acetate hapten BSO-2 with chicken ovalbumin.

[0085] More preferably, the IgG is goat anti-rabbit IgG or goat anti-mouse IgG.

[0086] The method of using the colloidal gold test strip is as follows:

[0087] The sample to be tested is evenly mixed with a colloidal gold-labeled antibody, and incubated at room temperature for 5 minutes. The antibody is a polyclonal antibody or a monoclonal antibody. The sample of the bisphenol A and / or bisphenol A acetate colloidal gold test strip is placed in the mixed solution and allowed to stand for 3 minutes. The test strip is then taken out and the sample pad is removed. The result is determined after 3 to 5 minutes.

[0088] The specific determination method is:

[0089] If the control line does not show color, the test result is invalid and needs to be retested; if the control line shows red, the test result is valid, and the test line is further interpreted: if the test line does not show color or the color is very weak, it means that the sample to be tested contains bisphenol salvin and / or bisphenol salvin acetate, and the judgment result is positive or weakly positive; if the test line shows red, it means that the sample to be tested does not contain bisphenol salvin and / or bisphenol salvin acetate, and the judgment result is negative.

[0090] Preferably, the kit is used to detect bisphenol A and / or bisphenol A acetate in weight loss foods.

[0091] More preferably, the kit is used to detect bisphenol A and / or bisphenol A acetate in weight loss foods, wherein the weight loss foods are foods with weight loss functions, including but not limited to compressed candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, blended liquors, fruit wines and jellies, etc. with weight loss functions.

[0092] The use of the colloidal gold test strip in detecting bisphenol A and / or bisphenol A acetate should also be within the scope of protection of the present invention.

[0093] Preferably, the colloidal gold test strip is used to detect bisphenol A and / or bisphenol A acetate in weight loss foods.

[0094] More preferably, the colloidal gold test strip is used to detect bisphenol A and / or bisphenol A acetate in weight loss foods, wherein the weight loss foods are foods with weight loss functions, including but not limited to compressed candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, blended liquors, fruit wines, and jellies with weight loss functions.

[0095] Compared with the prior art, the present invention has the following beneficial effects:

[0096] The present invention provides a universal hapten for bisphenol sarcoside and / or bisphenol sarcoside acetate, and prepares antibodies to bisphenol sarcoside and / or bisphenol sarcoside acetate, which have high titer, strong specificity, and high affinity, providing core raw materials for establishing a specific immunoassay method for bisphenol sarcoside and / or bisphenol sarcoside acetate. The present invention also establishes an immunoassay method for bisphenol sarcoside and / or bisphenol sarcoside acetate with higher specificity and sensitivity, wherein the minimum detection limit LOD of bisphenol sarcoside is 0.03 ng / mL and the half inhibition concentration IC is 0.03 ng / mL. 50 The minimum detection limit LOD of bisphenol salbutamol acetate was 1.23 ng / mL, and the half inhibition concentration IC 50 The method has no cross-reaction with any functional analogue of bisphenol salvin and / or bisphenol salvin acetate and can be used for rapid qualitative and quantitative detection of bisphenol salvin and / or bisphenol salvin acetate in diet foods. It is simple to operate and produces accurate and reliable test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 The invention provides a synthetic route for bisphenol salbutamol and / or bisphenol salbutamol acetate haptens BSO-1 and BSO-2.

[0098] Figure 2 This is a UV scanning identification result diagram of bisphenol A and / or bisphenol A acetate artificial antigens BSO-1-OVA and BSO-1-BSA.

[0099] Figure 3 This is a UV scanning identification result diagram of bisphenol A and / or bisphenol A acetate artificial antigens BSO-2-OVA and BSO-2-BSA.

[0100] Figure 4 Indirect competition ELISA standard curve for monoclonal antibodies against bisphenol A and / or bisphenol A acetate.

[0101] Figure 5This is a schematic diagram of the structure of the bisphenol sarcoside and / or bisphenol sarcoside acetate colloidal gold test strip of Example 8 of the present application; it includes: a PVC plastic base, a sample pad, an NC film, a water-absorbing pad, a test line, and a control line.

[0102] Figure 6 This is a result judgment diagram of the bisphenol sartin and / or bisphenol sartin acetate colloidal gold test strip of Example 8 of the present application; wherein: A is a valid negative sample test result, B is a valid positive sample test result, and C and D are invalid test results. DETAILED DESCRIPTION

[0103] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0104] Example 1 Synthesis and Identification of Universal Haptens of Bisphenol Sardin and / or Bisphenol Sardin Acetate

[0105] 1. Synthesis and Identification of Bisphenol A and / or Bisphenol A Acetate Hapten BSO-1

[0106] 1. Bisphenol A and / or Bisphenol A acetate hapten BSO-1

[0107] The synthetic route of bisphenol sardine and / or bisphenol sardine acetate hapten BSO-1 is as follows Figure 1 The specific steps are as follows:

[0108] 1 mol of bisphenol saltin is dissolved in 20 mL of methanol, and then 2 mol of ethyl bromoacetate, 6 mol of cesium carbonate and 2 mol of sodium iodide are added, and the mixture is stirred at 50°C for 8 hours to obtain a reactant; the reactant is separated and purified by column chromatography using silica gel powder as a stationary phase and a mixed solution of dichloromethane:methanol with a volume ratio of 70:1 as a mobile phase, and the obtained purified product is dissolved in 5 mL of methanol, and then 5 mL of 3 mol / L sodium hydroxide aqueous solution is added and stirred at room temperature for 3 hours. After the reaction is completed, the pH is adjusted to 6-7, and the obtained precipitate is bisphenol saltin and / or bisphenol saltin acetate hapten BSO-1.

[0109] (2) Identification of bisphenol A and / or bisphenol A acetate hapten BSO-1

[0110] The results of the nuclear magnetic resonance hydrogen spectrum of bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-1 are: 1HNMR (600MHz, Methanol-d4) δ7.20-7.16(m,2H),7.07-7.02(m,2H),6.90(dd,J=7.9,1.5Hz,1H),6.81(td,J=7.6,1.6 Hz,1H),6.79-6.77(m,2H),6.77(td,J=7.6,1.5Hz,1H),6.70(dd,J=7.8,1.6Hz,1H),6.63-6.59(m,2H),4.54(s,2H).

[0111] The mass spectrometry results of bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-1 are: MS: C 22 H 17 NO6: 391.38, ESI+[M+H]+: 392.4.

[0112] It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results correspond to the molecular weight of BSO-1 and the hydrogen spectrum number of nuclear magnetic resonance corresponds to the hydrogen spectrum number on the structure of BSO-1, indicating that bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-1 has been successfully prepared, and its structural formula is shown in formula (I).

[0113]

[0114] The hapten BSO-1 and / or hapten BSO-1 is named as 2-(4-(2-(4-hydroxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-yl)phenoxy)aceticacid using the systematic nomenclature, that is, 2-(4-(2-(4-hydroxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-yl)phenoxy)acetic acid.

[0115] 3. Synthesis and identification of bisphenol sardine and / or bisphenol sardine acetate hapten BSO-2

[0116] (1) Synthesis of bisphenol sardin and / or bisphenol sardin acetate hapten BSO-2

[0117] 1 mol of bisphenol saltin is dissolved in 20 mL of methanol, and then 2 mol of methyl 4-(bromomethyl)benzoate, 6 mol of cesium carbonate, and 2 mol of sodium iodide are added, and the mixture is stirred at 50° C. for 8 hours to obtain a reactant; the reactant is separated and purified by column chromatography using silica gel powder as a stationary phase and a mixed solution of dichloromethane:methanol with a volume ratio of 70:1 as a mobile phase; the obtained purified product is dissolved in 5 mL of methanol, and then 5 mL of a 3 mol / L sodium hydroxide aqueous solution is added, and the mixture is stirred at room temperature for 3 hours. After the reaction is completed, the pH is adjusted to 6-7, and the obtained precipitate is bisphenol saltin and / or bisphenol saltin acetate hapten BSO-2.

[0118] (2) Identification of bisphenol A and / or bisphenol A acetate hapten BSO-2

[0119] The results of hydrogen nuclear magnetic resonance spectrum of bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-2 were: 1HNMR (600 MHz, Methanol-d4) δ8.04–8.00 (m, 2H), 7.52 (d, J = 8.2 Hz, 2H), 7.30–7.24 (m, 2H), 7.18–7.13 (m, 2H), 7.00 (dd, J = 7.9, 1.5 Hz, 1H), 6.96–6.92 (m, 2H), 6.89 (dtd, J = 21.6, 7.5, 1.6 Hz, 2H), 6.80 (dd, J = 7.7, 1.7 Hz, 1H), 6.73–6.69 (m, 2H), 5.13 (s, 2H).

[0120] The mass spectrometry results of bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-4C are: MS: C 28 H 21 NO6: 467.48, ESI+[M+H]+: 468.47.

[0121] It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results correspond to the molecular weight of BSO-2 and the hydrogen spectrum number of nuclear magnetic resonance corresponds to the hydrogen spectrum number on the structure of BSO-2, indicating that bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-4C has been successfully prepared, and its structural formula is shown in formula (II).

[0122]

[0123] The bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-2 is named using the systematic nomenclature: 4-((4-(2-(4-hydroxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-yl)phenoxy)methyl)benzoic acid; that is, 4-((4-(2-(4-hydroxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-yl)phenoxy)methyl)benzoic acid.

[0124] Example 2 Synthesis and Identification of Bisphenol Sardin and / or Bisphenol Sardin Acetate Artificial Antigens

[0125] The bisphenol sardin and / or bisphenol sardin acetate hapten BSO-1 and the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-2 prepared in Example 1 were coupled to chicken ovalbumin (OVA) and bovine serum albumin (BSA) respectively by the active ester method.

[0126] 1. Synthesis and Identification of Bisphenol A and / or Bisphenol A Acetate Coating Agents

[0127] 1. Synthesis of bisphenol sardine and / or bisphenol sardine acetate coating

[0128] 10 mg of bisphenol salbutamol and / or bisphenol salbutamol acetate hapten BSO-1 (whose structural formula is shown in Formula (I)) prepared in Example 1 was dissolved in 100 μL of N,N-dimethylformamide (DMF), 2 mg of N-hydroxysuccinimide (NHS) and 3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added, and the mixture was stirred in the dark at 25° C. for 4 h to obtain Solution A.

[0129] Dissolve 10 mg of OVA in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain solution B. The formula of the PBS buffer used is: Na2HPO4·12H2O 2.90 g, NaCl 8.50 g, KCl 0.20 g, KH2PO4 0.20 g, and distilled water is added to make up to 1000 mL.

[0130] 100 μL of solution A was added dropwise to 1 mL of solution B, and stirred at 4°C for 12 h. The solution was dialyzed against PBS buffer for two days, four times a day, to obtain bisphenol A and / or bisphenol A acetate-coated original BSO-1-OVA, which was divided into centrifuge tubes and stored at -20°C for use.

[0131] According to the above method, the only difference is that the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-2 is used instead of the bisphenol sardin and / or bisphenol sardin acetate hapten BSO-1 to prepare the bisphenol sardin and / or bisphenol sardin acetate coated original BSO-2-OVA.

[0132] 2. Identification of bisphenol sardine and / or bisphenol sardine acetate coating

[0133] OVA, BSO-1 and BSO-1-OVA were respectively subjected to UV scanning determination (200-400 nm). The results are shown in the figure. Figure 2 As shown in (a), the ultraviolet absorption peak of the original BSO-1-OVA coated with bisphenol sardin and / or bisphenol sardin acetate is significantly shifted compared with the ultraviolet absorption peak of the hapten BSO-1, and the original BSO-1-OVA coated with bisphenol sardin and / or bisphenol sardin acetate has the characteristic absorption peaks of both the hapten BSO-1 and OVA, indicating that the coupling of the original BSO-1-OVA coated with bisphenol sardin and / or bisphenol sardin acetate is successful. Its structural formula is shown in formula (III).

[0134]

[0135] Among them, P is the carrier protein OVA.

[0136] OVA, BSO-2 and BSO-2-OVA were respectively subjected to UV scanning (200-400 nm). The results are shown in the figure. Figure 3 As shown in (a), the ultraviolet absorption peak of the original BSO-2-OVA coated with bisphenol sardin and / or bisphenol sardin acetate is significantly shifted compared with the ultraviolet absorption peak of the hapten BSO-2, and the original BSO-2-OVA coated with bisphenol sardin and / or bisphenol sardin acetate has the characteristic absorption peaks of both the hapten BSO-2 and OVA, indicating that the coupling of the original BSO-2-OVA with bisphenol sardin and / or bisphenol sardin acetate is successful. Its structural formula is shown in formula (IV).

[0137]

[0138] Among them, P is the carrier protein OVA.

[0139] 2. Synthesis and Identification of Bisphenol A and / or Bisphenol A Acetate Immunogens

[0140] 1. Synthesis of Bisphenol A and / or Bisphenol A Acetate Immunogens

[0141] The preparation method is similar to the synthesis method of the above-mentioned bisphenol sarcoside and / or bisphenol sarcoside acetate coating agent, except that BSA is used instead of OVA, and the bisphenol sarcoside and / or bisphenol sarcoside acetate hapten BSO-1 and the bisphenol sarcoside and / or bisphenol sarcoside acetate hapten BSO-2 prepared in Example 1 are used to synthesize the bisphenol sarcoside and / or bisphenol sarcoside acetate immunogens BSO-1-BSA and BSO-2-BSA.

[0142] 2. Identification of bisphenol A and / or bisphenol A acetate immunogens

[0143] BSA, BSO-1 and BSO-1-BSA were respectively subjected to UV scanning determination (200-400 nm). The determination results are as follows: Figure 2 As shown in (b), the ultraviolet absorption peak of bisphenol sartin and / or bisphenol sartin acetate immunogen BSO-1-BSA is significantly shifted compared with the ultraviolet absorption peak of the hapten BSO-1, and the bisphenol sartin and / or bisphenol sartin acetate immunogen BSO-1-BSA has the characteristic absorption peaks of both the hapten BSO-1 and BSA, indicating that the bisphenol sartin and / or bisphenol sartin acetate immunogen BSO-1-BSA is successfully coupled. Its structural formula is shown in formula (III).

[0144]

[0145] Wherein, P is the carrier protein BSA.

[0146] BSA, BSO-2 and BSO-2-BSA were respectively subjected to UV scanning (200-400 nm). The results are shown in the following table. Figure 3 As shown in (b), the ultraviolet absorption peak of bisphenol sartin and / or bisphenol sartin acetate immunogen BSO-2-BSA is significantly shifted compared with the ultraviolet absorption peak of the hapten BSO-2, and the bisphenol sartin and / or bisphenol sartin acetate immunogen BSO-2-BSA has the characteristic absorption peaks of both the hapten BSO-2 and BSA, indicating that the bisphenol sartin and / or bisphenol sartin acetate immunogen BSO-2-BSA is successfully coupled. Its structural formula is shown in formula (IV).

[0147]

[0148] Wherein, P is the carrier protein BSA.

[0149] Example 3 Preparation of antibodies for simultaneous detection of bisphenol A and / or bisphenol A acetate

[0150] 1. Preparation of polyclonal antibodies

[0151] The artificial antigen BSO-1-BSA (its structural formula is shown in Formula (III), where P represents the carrier protein BSA) prepared in Example 2 was used as an immunogen and emulsified evenly with an immune adjuvant (incomplete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for subsequent booster immunizations) at a volume ratio of 1:1. New Zealand white rabbits weighing 2.5-3 kg were immunized using multiple subcutaneous injections in the neck and back. Four weeks later, a second immunization was performed, and subsequent booster immunizations were performed every three weeks. One week after the third booster immunization, blood was collected from the ear vein, and the serum titer was determined using an indirect competitive ELISA.

[0152] When the titer stopped rising, booster immunization was performed on rabbits via ear vein injection. One week later, cardiac blood was collected from the rabbits. The collected blood was incubated at 37°C for 0.5 hours and then allowed to stand at 4°C overnight. The separated serum was then pipetted out and centrifuged at 5000 rpm for 10 minutes at 4°C. The supernatant was collected to obtain the antiserum. The antiserum was purified by ammonium sulfate precipitation to obtain polyclonal antibodies, which were then frozen at -20°C for future use.

[0153] 2. Preparation of Monoclonal Antibodies

[0154] The artificial antigens BSO-1-BSA (whose structural formula is shown in Formula (III), P is the carrier protein BSA) and BSO-2-BSA (whose structural formula is shown in Formula (IV), P is the carrier protein BSA) prepared in Example 2 were used as immunogens and were emulsified evenly with an immune adjuvant (complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for subsequent booster immunizations) at a volume ratio of 1:1 for immunization of female Balb / c mice.

[0155] Babizi mice were immunized by multiple subcutaneous injections in the abdomen. One week after each booster immunization, blood was collected from the tail vein of the mice to detect the serum titer. When the antibody titer no longer increased, another booster immunization was performed. Seven days later, the mouse spleen cells were taken and fused with mouse myeloma cells to obtain fusion cells.

[0156] After hybridoma cells are screened from the fused cells using HAT medium, they are cultured in complete medium. The hybridoma supernatant is tested using IC-ELISA, and cells from wells with strongly positive results are cloned and cultured using limiting dilution. One week later, the cells are retested, selected, and cloned again. After three rounds of cloning and culture, hybridoma cells producing monoclonal antibodies are obtained. After amplification and culture, the hybridoma cells are inoculated into the peritoneal cavity of mice to produce antibody-containing ascites. The ascites is purified using caprylic acid-ammonium sulfate precipitation to obtain the monoclonal antibody, which is then frozen at -20°C for future use.

[0157] Example 4 Combination of Immunogen and Coating Gen

[0158] 1. Experimental Methods

[0159] The artificial antigens BSO-1-OVA (whose structural formula is shown in Formula (III), where P is the carrier protein OVA) and BSO-2-OVA (whose structural formula is shown in Formula (IV), where P is the carrier protein OVA) prepared in Example 2 were used as coating agents, and antisera prepared in Example 3 using BSO-1-BSA (whose structural formula is shown in Formula (III), where P is the carrier protein BSA) and BSO-2-BSA (whose structural formula is shown in Formula (IV), where P is the carrier protein BSA) as immunogens were used. The optimal combination of immunogen and coating agent was selected by testing the titer and inhibition rate of the antisera. The specific steps are as follows:

[0160] 1. Dilute the original coating agents BSO-1-OVA and BSO-2-OVA to 1 μg / mL with coating solution (0.05 M carbonate buffer, pH = 9.6). Coat a 96-well ELISA plate at a rate of 100 μL / well. Incubate in a 37°C constant temperature water bath for 12 h. Discard the coating solution and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0161] 2. Add 120 μL of blocking solution (1 wt% fish skin collagen, diluted with PBST) to each well, block at 37 °C for 3 h, discard the blocking solution, and dry in a drying oven at 37 °C for later use;

[0162] 3. Dilute the antiserum obtained from immunizing female Balb / c mice with PBST to 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000. Set up blank control wells (using PBST instead). Dilute the 1 mg / mL bisphenol salvin and / or bisphenol salvin acetate standard 1000-fold with PBST to a concentration of 1 μg / mL to obtain a bisphenol salvin and / or bisphenol salvin acetate standard dilution solution.

[0163] 4. Titer column setup: first add 50 μL PBST to each well, then add 50 μL of antiserum at different dilution ratios to each well, and finally add 50 μL PBST to the last well instead of antiserum;

[0164] 5. Inhibition column setup: first add 50 μL of bisphenol A and / or bisphenol A acetate standard dilution to each well, then add 50 μL of antiserum at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of antiserum;

[0165] 6. Incubate at 37°C for 40 min and wash five times;

[0166] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37°C for 30 minutes, and wash 5 times;

[0167] 8. Add color developing solution and incubate at 37°C for 10 minutes. The color developing solution is prepared by mixing urea peroxide and tetramethylbenzidine in a volume ratio of 1:1.

[0168] 9. Add 10% v / v H2SO4 to terminate the reaction and read the OD value at 450nm; calculate the titer and inhibition rate. The titer is OD 450 The dilution factor of the antiserum corresponding to about 1.0, inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0169] 2. Experimental Results

[0170] The results of ELISA tests with different combinations of immunogens and coating agents are shown in Table 1.

[0171] Table 1 ELISA test results of immunogen and coating

[0172]

[0173] a It represents the inhibition rate of mouse antiserum against bisphenol A and / or bisphenol A acetate.

[0174] As can be seen in Table 1, the antisera generated using the artificial antigen BSO-1-BSA, used as an immunogen for female Balb / c mice, exhibited varying degrees of inhibitory activity against the target analytes, BSO-1-BSA and BSO-2-OVA. The combination of the immunogen BSO-1-BSA and the coating agent BSO-2-OVA achieved the highest inhibition rate against BSO-1-BSA and BSO-2-OVA, demonstrating the ability to simultaneously identify the target analytes, BSO-1-BSA and BSO-2-OVA. Therefore, BSO-1-BSA was selected as the optimal immunogen, and BSO-2-OVA as the optimal coating agent.

[0175] Example 5 Establishment of indirect competitive ELISA detection method for bisphenol salbutamol and / or bisphenol salbutamol acetate

[0176] 1. An indirect competitive ELISA detection method for bisphenol salbutamol and / or bisphenol salbutamol acetate, comprising the following steps:

[0177] 1. The artificial antigen BSO-2-OVA (its structural formula is shown in Formula (IV), where P is the carrier protein OVA) prepared in Example 2 was used as a coating source and diluted to 250 ng / mL with a coating solution (0.05 M carbonate buffer, pH = 9.6). 100 μL / well of the solution was added to coat a 96-well ELISA plate and incubated at 37°C overnight (12 h).

[0178] 2. Discard the coating solution, wash twice with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), and pat dry;

[0179] 3. Add 120 μL of blocking solution (i.e., 1 wt% fish skin collagen, diluted in PBST) to each well and block at 37°C for 3 h;

[0180] 4. Discard the blocking solution, dry at 37℃ for 30min, then take out and store in a ziplock bag for later use;

[0181] 5. The monoclonal antibody prepared in Example 3 was diluted with PBST at a volume ratio of 1:4000, and the bisphenol A and / or bisphenol A acetate standard was diluted to concentrations of 10000 ng / mL, 2400 ng / mL, 400 ng / mL, 66.67 ng / mL, 11.11 ng / mL, 1.85 ng / mL, 0.3086 ng / mL, 0.05144 ng / mL, 0.00857, and 0.00143 ng / mL;

[0182] 6. Add 50 μL / well of different concentrations of bisphenol A and / or bisphenol A acetate standards (three sets in parallel), then add 50 μL / well of diluted monoclonal antibody, incubate at 37°C for 40 min, and wash five times;

[0183] 7. Add 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000-fold with PBST), incubate at 37°C for 30 min, and wash five times;

[0184] 8. Add 100 μL / well color development solution and develop for 10 minutes. The color development solution is prepared by mixing carbamide peroxide and tetramethylbenzidine in a volume ratio of 1:1.

[0185] 9. Add 50 μL of 10% H2SO4 solution to terminate the reaction and read the OD value at 450 nm.

[0186] 10. ELISA standard curve drawing: take B / B0 as the vertical axis (B is the absorbance value OD of the standard of different concentrations of bisphenol salbutamol and / or bisphenol salbutamol acetate) 450 , B0 is the absorbance value OD of the blank control well 450), the logarithm of the standard concentration was used as the horizontal axis, and the logistic function was used for curve fitting to obtain the formula of the standard curve and prepare the standard curve.

[0187] 2. Test Results

[0188] The standard curve of the indirect competition ELISA for the detection of bisphenol A and / or bisphenol A acetate is as follows Figure 4 As shown, the half inhibitory concentration (IC 50 ) was 0.26 ng / mL, and the limit of detection (LOD) was 0.03 ng / mL; the half inhibition concentration (IC 50 ) was 16.85 ng / mL, and the minimum detection limit (LOD) was 1.23 ng / mL; this indicated that the antibodies prepared by the present invention for simultaneously identifying bisphenol saltin and / or bisphenol saltin acetate can meet the detection requirements, and the indirect competitive ELISA detection method for bisphenol saltin and / or bisphenol saltin acetate established by the present invention has high recognition ability and sensitivity for bisphenol saltin and / or bisphenol saltin acetate.

[0189] Example 6 Specificity of Antibodies for Simultaneous Detection of Bisphenol A and / or Bisphenol A Acetate

[0190] 1. Experimental Methods

[0191] Rhein, aloe-emodin, rhein, sodium picosulfate and bisacodyl are all functional analogs of bisphenol A and / or bisphenol A acetate, and are often added to weight loss products.

[0192] This example uses a cross-reaction experiment to determine the specificity of the antibody prepared by the present invention for detecting bisphenol A and / or bisphenol A acetate.

[0193] The specificity of the antibody against bisphenol salvin and / or bisphenol salvin acetate was detected according to the “Indirect competitive ELISA method for detecting bisphenol salvin and / or bisphenol salvin acetate” of Example 5, except that the bisphenol salvin and / or bisphenol salvin acetate standard in step 5 was replaced with standard samples of emodin (CAS No.: 518-82-1), aloe-emodin (CAS: 481-72-1), rhein (CAS: 478-43-3), sodium picosulfate (CAS: 10040-45-6) and bisacodyl (CAS: 603-50-9), and the IC values of each functional analogue were obtained. 50 value.

[0194] The cross-reactivity rate (CR) was calculated according to the following formula: CR (%) = IC 50 (Bisphenol Sardin) / IC 50(Functional analogue)×100%. The smaller the cross-reaction rate, the stronger the specificity.

[0195] 2. Experimental Results

[0196] Table 2 Cross-reaction results of bisphenol salbutamol and / or bisphenol salbutamol acetate monoclonal antibodies with bisphenol salbutamol and / or bisphenol salbutamol acetate and their functional analogs

[0197]

[0198] Note: NR means no reaction, which means the antibody does not recognize the analog.

[0199] As shown in Table 2, the cross-reactivity rates of the monoclonal antibodies used to detect bisphenol sarcoside and / or bisphenol sarcoside acetate to bisphenol sarcoside and / or bisphenol sarcoside acetate were 100% and 1.5%, respectively. 50 The concentrations of the antibodies for detecting bisphenol salvin and / or bisphenol salvin acetate were 0.26 ng / mL and 16.85 ng / mL respectively, and there was no crosstalk with emodin, aloe-emodin, rhein, sodium picosulfate and bisacodyl, which are functional analogues of bisphenol salvin and / or bisphenol salvin acetate. This indicates that the antibodies for detecting bisphenol salvin and / or bisphenol salvin acetate have high recognition ability and strong specificity for bisphenol salvin and / or bisphenol salvin acetate, can effectively eliminate the interference of emodin, aloe-emodin, rhein, sodium picosulfate and bisacodyl, which are functional analogues of bisphenol salvin and / or bisphenol salvin acetate, and can be specifically used for the detection of bisphenol salvin and / or bisphenol salvin acetate.

[0200] Example 7 An ELISA kit for simultaneous detection of bisphenol salbutamol and / or bisphenol salbutamol acetate

[0201] 1. Composition

[0202] (1) ELISA plate coated with a coating agent:

[0203] The ELISA plate is prepared by the following method:

[0204] The artificial antigen BSO-2-OVA (its structural formula is shown in Formula (IV), where P is the carrier protein OVA) prepared in Example 2 was used as a coating source. The coating stock solution (0.05 M carbonate buffer solution, pH = 9.6) was diluted to 250 ng / mL and coated on a 96-well ELISA plate at a rate of 100 μL / well. The plate was incubated at 37°C in the dark overnight. The liquid in the wells was poured out, and the plates were washed twice with the washing solution in this kit for 30 seconds each time, and patted dry. The blocking solution in this kit was then added at a rate of 200 μL / well, and the plates were incubated at 25°C in the dark for 2 hours. The liquid in the wells was poured out, patted dry, and after drying, the plates were vacuum-sealed with aluminum film for storage.

[0205] (2) Standards: 10 different concentrations of bisphenol A and / or bisphenol A acetate standards, namely 10000 ng / mL, 2400 ng / mL, 400 ng / mL, 66.67 ng / mL, 11.11 ng / mL, 1.85 ng / mL, 0.3086 ng / mL, 0.05144 ng / mL, 0.00857 ng / mL, and 0.00143 ng / mL;

[0206] (3) Antibodies: the polyclonal or monoclonal antibodies prepared in Example 3 for detecting bisphenol A and / or bisphenol A acetate;

[0207] (4) Enzyme conjugate working solution: horseradish peroxidase-labeled goat anti-rabbit secondary antibody or horseradish peroxidase-labeled goat anti-mouse secondary antibody;

[0208] (5) Substrate color development solution: It consists of solution A and solution B. Solution A is urea peroxide and solution B is tetramethylbenzidine.

[0209] (6) Stop solution 2 mol / L H2SO4;

[0210] (7) Washing solution: pH 7.4, containing 0.8% Tween-20 by volume, 0.02% sodium azide preservative by mass, and 0.2 mol / L phosphate buffer; dilute the washing solution 20 times with water before use (i.e., add 1 part washing solution to 19 parts water, ready for use) to obtain the working solution of washing solution;

[0211] (8) Diluent: 0.2 mol / L phosphate buffer; dilute the diluent 20 times with water before use (i.e., add 1 part diluent to 19 parts water, ready for use) to obtain the diluent working solution.

[0212] 2. Usage

[0213] (1) Sample testing

[0214] Number the corresponding microwells for the samples and the standards in this kit in sequence. Make two parallel wells for each sample and standard, add them to the ELISA plate coated with the coating agent, and record the location of the standard wells and sample wells. Dilute the antibody with diluent at a volume ratio of 1:40 (i.e., add 1 part antibody to 40 parts diluent, freshly prepared for use) to obtain the antibody working solution. Dilute the enzyme conjugate concentrate with diluent at a volume ratio of 1:10 (i.e., add 1 part enzyme conjugate concentrate to 10 parts diluent, freshly prepared for use) to obtain the enzyme conjugate working solution.

[0215] Add 50 μL of standard or sample to the corresponding microwells, then add 50 μL of antibody working solution to the corresponding microwells, gently shake to mix, cover the plate with a cover film, and react at 25°C in a dark environment for 40 minutes.

[0216] Drain the liquid in the wells and add 250 μL of washing solution per well. Wash thoroughly 4-5 times, with 10 seconds between each wash. Discard the washing solution in the wells and pat dry with absorbent paper (if bubbles are not cleared after patting dry, use an unused pipette tip to pop them).

[0217] Add 100 μL / well of enzyme conjugate working solution to the corresponding microwells, gently shake to mix, cover the plate with a cover film, and then react in a dark environment at 25°C for 30 minutes.

[0218] Drain the liquid in the wells and add 250 μL / well of washing solution. Wash thoroughly 4-5 times, with 10 seconds between each wash. Discard the working solution in the wells and pat dry with absorbent paper (if bubbles are not cleared after patting dry, use an unused pipette tip to pop them).

[0219] Add 50 μL / well of substrate color development solution A, then add 50 μL / well of substrate color development solution B, gently shake to mix, cover the plate with cover film, and then react at 25°C in a dark environment for 10 minutes.

[0220] Add 50 μL / well of stop solution, shake gently to mix, set the microplate reader at 450 nm, and measure the OD value of each well.

[0221] (2) Drawing of standard curve

[0222] Draw the ELISA standard curve according to the above optimal conditions, with B / B0 as the vertical axis (B is the absorbance value OD when the standard is added). 450 , B0 is the absorbance value OD when no standard is added 450 ), the logarithm of the standard concentration was used as the horizontal axis, and the logistic function was used for curve fitting to obtain the formula of the standard curve and prepare the standard curve.

[0223] (3) Calculation of sample concentration

[0224] The absorbance value of the sample is measured as OD 450 Substitute the value into the above calculation formula to calculate the percentage absorbance of the sample; substitute the percentage absorbance of the sample into the formula of the above standard curve to obtain the concentration of the sample, and then multiply it by the corresponding dilution factor to obtain the actual concentration of bisphenol salbutamol and / or bisphenol salbutamol acetate in the test sample.

[0225] Example 8 A colloidal gold test strip for detecting bisphenol salbutamol and / or bisphenol salbutamol acetate

[0226] 1. Assembly of colloidal gold test strips

[0227] like Figure 5 As shown in FIG, the colloidal gold test strip is composed of a NC membrane (nitrocellulose membrane), a sample pad, an absorbent pad and a PVC plastic base.

[0228] The coating agent (BSO-2-OVA, whose structural formula is shown in Formula (IV), where P is the carrier protein OVA) was sprayed onto the NC membrane at a spray rate of 0.8 μL / cm using an XYZ three-dimensional spray dot streak apparatus to serve as the test line (T line). Goat anti-rabbit IgG or goat anti-mouse IgG was sprayed onto the NC membrane using the same method and dosage to serve as the control line (C line). The test line (T line) and the control line (C line) were located in the middle of the NC membrane and separated by 6 mm. After drying at 37°C for 12 h, a cellulose membrane was attached to the middle of the backing board, with the sample pad overlapping the T-line end of the NC membrane by 1 mm. An absorbent pad was attached to the upper side of the cellulose membrane and overlapped the cellulose membrane 3 by 1 mm. The assembled test paper was cut into 3.5 mm wide test strips using a chopper.

[0229] 2. Preparation of gold-labeled antibodies

[0230] A colloidal gold suspension with an average diameter of 30 nm was prepared by reducing chloroauric acid with trisodium citrate. The specific method is as follows:

[0231] Take 1 mL of colloidal gold solution, add 0.2 mol / L K2CO3 solution to adjust the pH to about 8.0, add 10 μg of the polyclonal antibody or monoclonal antibody prepared in Example 3 and incubate for 30 min, then add 10% wt BSA solution and incubate for 30 min, centrifuge at 4°C 10000 rpm for 20 min, remove the supernatant, and resuspend with 200 μL 0.2 mol / L pH 7.4 phosphate buffer solution (containing 0.5% v / v Tween-20, 0.5% wt LF, 5% wt sucrose, 0.3% wt polyvinylpyrrolidone (PVP) and 0.03% v / v procline-300) and store at 4°C.

[0232] 3. Preparation of test sample solution

[0233] Preparation of compressed candy, beverage, fruit and vegetable powder, tea substitute, candied fruit, blended alcohol, and wine samples: Accurately weigh 1.0 g of sample into a 10 mL centrifuge tube, add 5 mL of 50% v / v methanol solution (containing 0.1% formic acid), ultrasonically extract for 5 minutes, cool to room temperature, centrifuge at 4000 rpm for 5 minutes, aspirate 1.0 mL of supernatant, dilute fourfold with 0.02 M PB, vortex evenly, and filter through a microporous filter to obtain the sample solution for testing.

[0234] Preparation of jelly samples: Accurately weigh 1.0 g of sample into a 10 mL centrifuge tube, add 5 mL of water, incubate in an 80°C water bath until the sample is dissolved, cool to room temperature, add 5 mL of methanol, ultrasonically extract for 5 minutes, centrifuge at 4000 rpm for 5 minutes, aspirate 1.0 mL of supernatant, dilute fourfold with 0.02 M PB, vortex evenly, and filter with a microporous filter membrane to obtain the sample solution for testing.

[0235] 4. Detection steps

[0236] Take 120 μL of sample solution and 5 μL of gold-labeled antibody and mix them evenly by repeated pipetting. Incubate at room temperature for 5 minutes. Then insert the test strip into the sample solution and react for 3 minutes. Then take out the test strip and remove the sample pad. Determine the result after 3 to 5 minutes.

[0237] 5. Determination of test results

[0238] like Figure 6 As shown in the figure, if the sample does not contain the analyte bisphenol A and / or bisphenol A acetate, the gold-labeled antibody will bind to the coating on the T line (test line) on the rapid test paper, making the test line show a clear red line, indicating that the test sample is negative (such as Figure 6 A); If the sample contains bisphenol A and / or bisphenol A acetate, the bisphenol A and / or bisphenol A acetate will bind to the gold-labeled antibody and cannot be captured by the test line on the rapid test strip, and the test line will not develop color, indicating a positive result (such as Figure 6 Similarly, the gold-labeled antibody also binds to the goat anti-mouse IgG on the C line (control line) on the cellulose membrane, making the quality control line appear red. The presence or absence of the control line color indicates whether the test strip is valid or invalid (such as Figure 6 A and B are valid and C and D are invalid).

[0239] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A bisphenol sardine and / or bisphenol sardine acetate hapten, characterized in that: Its structural formula is shown in formula (I), and it is named BSO-1. Formula (I).

2. A bisphenol sardine and / or bisphenol sardine acetate hapten, characterized in that: Its structural formula is shown in formula (II), and it is named BSO-2. Formula (II).

3. Use of the bisphenol saltin and / or bisphenol saltin acetate hapten in the preparation of bisphenol saltin and / or bisphenol saltin acetate artificial antigen according to claim 1 or 2.

4. A bisphenol sarcoside and / or bisphenol sarcoside acetate artificial antigen, characterized in that: The bisphenol sartan and / or bisphenol sartan acetate artificial antigen is obtained by coupling the bisphenol sartan and / or bisphenol sartan acetate hapten described in claim 1 with a carrier protein, and its structural formula is shown in formula (III). Formula (III), Among them, P is the carrier protein.

5. A bisphenol sarcoside and / or bisphenol sarcoside acetate artificial antigen, characterized in that: The bisphenol sartan and / or bisphenol sartan acetate artificial antigen is obtained by coupling the bisphenol sartan and / or bisphenol sartan acetate hapten described in claim 2 with a carrier protein, and its structural formula is shown in formula (IV). Formula (IV), Among them, P is the carrier protein.

6. Use of the artificial antigen of bisphenol salbutamol and / or bisphenol salbutamol acetate according to any one of claims 4 and 5 in the preparation of antibodies against bisphenol salbutamol and / or bisphenol salbutamol acetate.

7. A bisphenol sarcoside and / or bisphenol sarcoside acetate artificial antigen combination, characterized in that: The invention comprises an immunogen and a coating agent, wherein the immunogen is obtained by coupling the bisphenol sardin and / or bisphenol sardin acetate hapten according to claim 1 or claim 2 with bovine serum albumin; and the coating agent is obtained by coupling the bisphenol sardin and / or bisphenol sardin acetate hapten according to claim 1 or claim 2 with chicken ovalbumin.

8. Use of the bisphenol sartan and / or bisphenol sartan acetate artificial antigen combination according to claim 7 in detecting bisphenol sartan and / or bisphenol sartan acetate for non-disease diagnosis purposes.

9. Use of the bisphenol saltin and / or bisphenol saltin acetate artificial antigen combination according to claim 7 in the preparation of a kit for detecting bisphenol saltin and / or bisphenol saltin acetate.

10. A kit for detecting bisphenol salbutamol and / or bisphenol salbutamol acetate, characterized in that: Comprising the bisphenol A and / or bisphenol A acetate artificial antigen combination as described in claim 7.

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