Hapten, artificial antigen for quickly identifying phenolphthalein in weight loss foods and applications thereof

Abstract

The present invention discloses a hapten, an artificial antigen of phenolphthalein in weight loss foods for rapid identification, and their applications. The present invention provides a phenolphthalein hapten that can not only maximize the retention of the characteristic structures of the three benzene rings and lactone ring in phenolphthalein, but also has a linker arm of a certain length, and also provides a method for preparing the phenolphthalein hapten. The phenolphthalein artificial antigen prepared by conjugating the phenolphthalein hapten with a carrier protein can obtain a phenolphthalein antibody with high affinity after immunizing animals, and its half-inhibition concentration IC 50 is 0.19 ng / mL, the lowest detection limit is 0.007 ng / mL, and the quantitative detection range is 0.02 - 1.64 ng / mL. It not only has high detection sensitivity, but also has strong specificity and high accuracy, and has no cross-reaction with a variety of phenolphthalein structural analogs. The detection effect is not affected by the sample type, and it can be popularized for large-scale rapid and accurate screening of phenolphthalein in weight loss foods.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and specifically, to a hapten, an artificial antigen for rapidly identifying phenolphthalein in weight loss foods, and their applications. Background Art

[0002] Phenolphthalein, also known as phenolphthalein, with the chemical name 3,3-bis(4-hydroxyphenyl)-3H-isobenzofuranone, is a medical irritant laxative. It mainly increases the propulsion force of the intestine by stimulating the intestinal mucosa or activating the nerve endings of the smooth muscle in the intestine, and is used to treat habitual and intractable constipation. Phenolphthalein was listed in the list of Group 2B carcinogens by the International Agency for Research on Cancer of the World Health Organization in 2017. The National Medical Products Administration of China has also issued the "Announcement on Canceling the Drug Certificates of Phenolphthalein Tablets and Phenolphthalein Buccal Tablets (No. 6, 2021)", stopping the production, sales and use of phenolphthalein tablets and phenolphthalein buccal tablets, and canceling the drug registration certificates.

[0003] The conventional methods for detecting phenolphthalein mainly include instrumental detection methods, test paper detection methods, and immunological detection methods. Instrumental detection methods include Raman spectroscopy, high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry, ultraviolet-visible spectrophotometry, etc. These methods have expensive equipment, high costs, complex processes, and require professional operation, which do not meet the requirements for on-site detection of a large number of samples. The test paper detection method is based on the principle that phenolphthalein is in a colorless lactone structure in a solution with a pH value < 8.2 and in a red quinone structure in a solution with a pH value of 8.2 - 10. The test paper prepared with a strong base is used for detection, and it is judged whether phenolphthalein is added to the sample according to the color of the test paper. Although it is simple, its accuracy is low and the false detection rate is relatively high. Immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidity, and simple operation. In the prior art, some immunological detection and analysis technologies for phenolphthalein have been established. For example, a hapten is prepared by introducing a linking arm at the lactone ring structure of phenolphthalein and coupled with a protein to prepare an artificial antigen (CN201710096284.2; Journal of Food Safety and Quality, 2019, 10(24): 8479 - 8483). However, this strategy destroys the characteristic structure of phenolphthalein and is not conducive to generating highly specific and sensitive antibodies. In addition, the carbonyl diimidazole method is also used to directly couple the hydroxyl group (-OH) on phenolphthalein with the carboxyl group on a macromolecular protein to prepare an artificial antigen. However, the small molecule of phenolphthalein cannot be fully exposed, which affects the recognition of the animal body and is also not conducive to generating high-quality antibodies (CN201910653805.9; Journal of Pharmaceutical and Biomedical Analysis, 2022, 212: 114609; Journal of Materials Chemistry B, 2021, 9(18): 3856 - 3862; Immunological Rapid Detection of Illegally Added Ingredients in Diet Pills and Coffee [D]. Jiangnan University, 2021). Therefore, there is still a lack of a rapid detection method for phenolphthalein with good specificity and high sensitivity at present. Summary of the Invention

[0004] In order to solve the problems of poor antibody specificity and low sensitivity in the immunological detection method for detecting phenolphthalein in the prior art, the present invention provides a hapten, an artificial antigen for rapidly identifying phenolphthalein in weight loss foods, and their applications.

[0005] The first object of the present invention is to provide two phenolphthalein haptens.

[0006] The second object of the present invention is to provide the application of the above-mentioned phenolphthalein hapten in the preparation of a phenolphthalein artificial antigen.

[0007] The third object of the present invention is to provide two phenolphthalein artificial antigens.

[0008] The fourth object of the present invention is to provide the application of the above-mentioned phenolphthalein artificial antigen in the preparation of an antibody against phenolphthalein.

[0009] The fifth object of the present invention is to provide a combination of phenolphthalein artificial antigens.

[0010] The sixth object of the present invention is to provide the application of the above-mentioned combination of phenolphthalein artificial antigens in the preparation of a reagent for detecting phenolphthalein.

[0011] The seventh object of the present invention is to provide a kit for detecting phenolphthalein.

[0012] In order to achieve the above objects, the present invention is realized by the following solutions:

[0013] When establishing an immunological detection method and applying this detection method to rapidly identify the phenolphthalein content in weight loss foods, the key technology lies in being able to obtain antibodies with strong specificity and high sensitivity. To achieve this goal, the prerequisite is to synthesize and prepare a suitable phenolphthalein hapten.

[0014] The hapten for rapidly identifying phenolphthalein in weight loss foods provided by the present invention maximally retains the characteristic structures of the three benzene rings and lactone ring in phenolphthalein. By introducing a spacer arm with a certain length at the hydroxyl position of phenolphthalein and then conjugating it with a macromolecular protein, the phenolphthalein small molecule can be fully exposed and basically not shielded by the macromolecular protein, which is more conducive to the recognition of the animal body and is conducive to preparing phenolphthalein antibodies with higher sensitivity and stronger specificity.

[0015] A phenolphthalein hapten, namely phenolphthalein hapten PT-4C, whose structural formula is shown in formula (Ⅰ),

[0016]

[0017] The phenolphthalein hapten PT-4C is named by systematic nomenclature as: 4-(4-(1-(4-hydroxyphenyl)-3-oxo-1,3-dihydroisobenzofuran-1-yl)phenoxy)butanoic acid; that is, 4-(4-(1-(4-hydroxyphenyl))-3-oxo-1,3-dihydroisobenzofuran-1-yl)phenoxy)butyric acid.

[0018] A phenolphthalein hapten, namely phenolphthalein hapten PT-6C, whose structural formula is shown in formula (Ⅱ),

[0019]

[0020] The phenolphthalein hapten PT-6C is named by the systematic nomenclature as: 6-(4-(1-(4-hydroxyphenyl)-3-oxo-1,3-dihydroisobenzofuran-1-yl)phenoxy)hexanoic acid; that is, 6-(4-(1-(4-hydroxyphenyl))-3-oxo-1,3-dihydroisobenzofuran-1-yl)phenoxy)hexanoic acid.

[0021] The preparation method of the compound shown in the structural formula of formula (Ⅰ) is to fully react phenolphthalein, ethyl 4-bromobutyrate, cesium carbonate, sodium iodide and N,N-dimethylformamide; the obtained reactant is fully hydrolyzed in an alkaline environment, and the pH is adjusted to 6-7 to obtain it.

[0022]

[0023] Specifically, dissolve 1 mol of phenolphthalein in 3 mL of N,N-dimethylformamide, then add 2 mol of ethyl 4-bromobutyrate and 6 mol of potassium carbonate, and stir at room temperature (25 °C) overnight (11 h) to obtain a reactant; separate and purify the reactant by column chromatography (the stationary phase is silica powder, and the mobile phase is dichloromethane:ethyl acetate = 10:1), dissolve the obtained purified product in 5 mL of methanol, then add 5 mL of 3 mol / L sodium hydroxide aqueous solution and stir at room temperature for 3 h. After the reaction is completed, adjust the pH to 6-7, and the obtained precipitate is the hapten 1.

[0024] The preparation method of the compound shown in the structural formula of formula (Ⅱ) is to fully react phenolphthalein, ethyl 6-bromohexanoate, cesium carbonate, sodium iodide and N,N-dimethylformamide; the obtained reactant is fully hydrolyzed in an alkaline environment, and the pH is adjusted to 6-7 to obtain it.

[0025]

[0026] Specifically, dissolve 1 mol of phenolphthalein in 3 mL of N,N-dimethylformamide, then add 2 mol of ethyl 6-bromohexanoate and 6 mol of potassium carbonate, and stir at room temperature (25 °C) overnight (11 h) to obtain a reactant; separate and purify the reactant by column chromatography (the stationary phase is silica powder, and the mobile phase is dichloromethane:ethyl acetate = 10:1), dissolve the obtained purified product in 5 mL of methanol, then add 5 mL of 3 mol / L sodium hydroxide aqueous solution and stir at room temperature for 3 h. After the reaction is completed, adjust the pH to 6-7, and the obtained precipitate is the hapten 2.

[0027] The application of the phenolphthalein hapten PT-4C or the phenolphthalein hapten PT-6C in the preparation of the phenolphthalein artificial antigen should also be within the protection scope of the present invention.

[0028] Preferably, the phenolphthalein artificial antigen is used to identify phenolphthalein in weight loss foods.

[0029] The weight-loss food involved in the present invention is a food with weight-loss function, including but not limited to tablet candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, formulated wines, fruit wines and jellies with weight-loss function.

[0030] A phenolphthalein artificial antigen is obtained by conjugating a phenolphthalein hapten PT-4C with a carrier protein, and its structural formula is shown as formula (Ⅲ),

[0031]

[0032] wherein P is the carrier protein, and the carrier protein is ovalbumin or bovine serum albumin, that is, the phenolphthalein artificial antigen PT-4C-OVA or PT-4C-BSA.

[0033] Preferably, the carrier protein is ovalbumin, that is, the phenolphthalein artificial antigen PT-4C-OVA.

[0034] A phenolphthalein artificial antigen is obtained by conjugating a phenolphthalein hapten PT-6C with a carrier protein, and its structural formula is as shown in formula (Ⅳ)

[0035]

[0036] wherein P is the carrier protein, and the carrier protein is ovalbumin or bovine serum albumin, that is, the phenolphthalein artificial antigen PT-6C-OVA or PT-6C-BSA.

[0037] Preferably, the carrier protein is bovine serum albumin, that is, the phenolphthalein artificial antigen PT-6C-BSA.

[0038] A method for preparing the above-mentioned phenolphthalein artificial antigen is to conjugate the above-mentioned phenolphthalein hapten with a carrier protein by the active ester method.

[0039] Preferably, the active ester method includes the following steps:

[0040] S1. React the phenolphthalein hapten C, N, N-dimethylformamide, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the dark sufficiently to obtain solution A; dissolve the carrier protein in phosphate buffer to obtain solution B;

[0041] S2. After solution A and solution B react sufficiently, dialyze to obtain the phenolphthalein artificial antigen.

[0042] More preferably, in step S1, the mass-volume ratio of the phenolphthalein hapten, N,N-dimethylformamide, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is (8 mg to 12 mg):(50 μL to 100 μL):(1 mg to 3 mg):(2 mg to 4 mg).

[0043] Further preferably, in step S1, the mass-volume ratio of the phenolphthalein hapten, N,N-dimethylformamide, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 10 mg:100 μL:2 mg:3 mg.

[0044] More preferably, in step S1, the mass-volume ratio of the carrier protein to the phosphate buffer solution is (8 mg to 12 mg):(0.5 mL to 1.5 mL).

[0045] Further preferably, in step S1, the mass-volume ratio of the carrier protein to the phosphate buffer solution is 10 mg:1 mL.

[0046] More preferably, in step S1, first dissolve the phenolphthalein hapten in N,N-dimethylformamide, then add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and stir in the dark at 25°C to 30°C for 2 to 4 h.

[0047] Further preferably, in step S1, dissolve 10 mg of the phenolphthalein hapten in 100 μL of N,N-dimethylformamide, then add 2 mg of N-hydroxysuccinimide and 3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and stir in the dark at 25°C for 4 h.

[0048] More preferably, in step S2, the volume ratio of solution A to solution B is (1 to 3):(5 to 10).

[0049] Further preferably, in step S2, the volume ratio of solution A to solution B is 1:10.

[0050] More preferably, in step S2, add solution A dropwise to solution B and stir at 3°C to 5°C for 10 h to 14 h.

[0051] Further preferably, in step S2, add solution A dropwise to solution B and stir at 4°C for 12 h.

[0052] More preferably, in step S2, the dialysis method is to dialyze with PBS buffer solution for 2 days, 4 times a day.

[0053] The phenolphthalein artificial antigen prepared by any of the above methods should also be within the protection scope of the present invention.

[0054] The use of any of the above-mentioned phthalein artificial antigens in the preparation of phthalein antibodies should also be within the protection scope of the present invention.

[0055] The use of any of the above-mentioned phthalein artificial antigens in the detection of phthalein should also be within the protection scope of the present invention, and the detection is for non-disease treatment diagnosis purposes.

[0056] Preferably, the phthalein artificial antigen is used to identify phthalein in weight loss foods.

[0057] The weight loss foods involved in the present invention are foods with weight loss functions, including but not limited to tablet candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, formulated wines, fruit wines, and jellies with weight loss functions.

[0058] A phthalein artificial antigen combination includes a coating antigen and an immunogen. The coating antigen is obtained by conjugating phthalein hapten PT-4C or phthalein hapten PT-6C with chicken ovalbumin; the immunogen is obtained by conjugating phthalein hapten PT-4C or phthalein hapten PT-6C with bovine serum albumin.

[0059] Preferably, the coating antigen is obtained by conjugating phthalein hapten PT-4C with chicken ovalbumin, that is, the coating antigen is phthalein artificial antigen PT-4C-OVA.

[0060] Preferably, the immunogen is obtained by conjugating phthalein hapten PT-6C with bovine serum albumin, that is, the immunogen is phthalein artificial antigen PT-6C-BSA.

[0061] More preferably, the coating antigen is obtained by conjugating phthalein hapten PT-4C with chicken ovalbumin; the immunogen is obtained by conjugating phthalein hapten PT-6C with bovine serum albumin; that is, the coating antigen is phthalein artificial antigen PT-4C-OVA, and the immunogen is PT-6C-BSA.

[0062] A phthalein antibody is prepared by immunizing an animal with the above-mentioned immunogen.

[0063] Preferably, the phthalein antibody is a monoclonal antibody. Hybridoma cells are obtained by immunizing an animal with any of the above-mentioned immunogens, the obtained hybridoma cells are cultured and the cell supernatant is collected, and after identification and purification, the phthalein monoclonal antibody is obtained.

[0064] Preferably, the phthalein antibody is a polyclonal antibody. An animal is immunized with any of the above-mentioned immunogens, the serum is collected, and the polyclonal antibody is obtained after purification by ammonium sulfate precipitation method.

[0065] More preferably, the immunogen is obtained by conjugating phthalein hapten PT-6C with bovine serum albumin, that is, the immunogen is PT-6C-BSA.

[0066] An immunoassay method for phenolphthalein, using any of the above phenolphthalein artificial antigen combinations for detection, and using the phenolphthalein antibody as the detection antibody for detection; the immunoassay method is for non-disease treatment diagnosis purposes.

[0067] Preferably, in the phenolphthalein artificial antigen combination, the coating antigen is obtained by conjugating the phenolphthalein hapten PT-4C with chicken ovalbumin; the phenolphthalein antibody is prepared by immunizing an animal with an immunogen obtained by conjugating the phenolphthalein hapten PT-6C with bovine serum albumin.

[0068] The application of the above phenolphthalein artificial antigen combination in the preparation of a reagent for detecting phenolphthalein should also be within the protection scope of the present invention.

[0069] Preferably, the reagent is used to identify phenolphthalein in weight loss foods.

[0070] The weight loss foods involved in the present invention are foods with weight loss functions, including but not limited to tablet candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, formulated wines, fruit wines, and jellies with weight loss functions.

[0071] A kit for detecting phenolphthalein, comprising any of the above phenolphthalein artificial antigen combinations.

[0072] Preferably, the coating antigen is obtained by conjugating the phenolphthalein hapten PT-4C with chicken ovalbumin; the immunogen is obtained by conjugating the phenolphthalein hapten PT-6C with bovine serum albumin.

[0073] More preferably, the kit comprises an antibody, and the antibody is obtained by immunizing an animal with an immunogen obtained by conjugating the phenolphthalein hapten PT-6C with bovine serum albumin.

[0074] Preferably, the kit further comprises an enzyme-linked immunosorbent assay (ELISA) plate, a phenolphthalein standard, and a substrate chromogenic solution. The CAS number of the phenolphthalein standard: 77-09-8.

[0075] More preferably, the ELISA plate is coated with a coating antigen obtained by conjugating the phenolphthalein hapten PT-4C with chicken ovalbumin.

[0076] More preferably, the substrate chromogenic solution contains urea peroxide and tetramethylbenzidine.

[0077] More preferably, the kit further comprises a stop solution, a washing solution, a blocking solution, an enzyme conjugate concentrate, and an enzyme conjugate diluent.

[0078] Further preferably, the stop solution is 1 mol / L to 3 mol / L H 2 SO 4 .

[0079] Most preferably, the stop solution is 2 mol / L H 2 SO4 。

[0080] Further preferably, the washing solution is a 0.1 mol / L - 0.3 mol / L phosphate buffer containing 0.5% w / v - 1.0% w / v Tween-20 and 0.01% w / v - 0.03% w / v sodium azide, with a pH of 7.2 - 7.6.

[0081] Most preferably, the washing solution is a 0.2 mol / L phosphate buffer containing 0.8% w / v Tween-20 and 0.02% w / v sodium azide, with a pH of 7.4.

[0082] Further preferably, the blocking solution is a 0.1 mol / L - 0.3 mol / L phosphate buffer containing 1% w / v - 3% w / v casein, with a pH of 7.1 - 7.5.

[0083] Most preferably, the blocking solution is a 0.2 mol / L phosphate buffer containing 2% w / v casein, with a pH of 7.3.

[0084] Further preferably, the enzyme conjugate concentrate is horseradish peroxidase-labeled goat anti-rabbit antibody or goat anti-mouse antibody.

[0085] Further preferably, the enzyme conjugate diluent is a 0.1 mol / L - 0.3 mol / L phosphate buffer.

[0086] Most preferably, the enzyme conjugate diluent is a 0.2 mol / L phosphate buffer.

[0087] Preferably, the kit further comprises an immunochromatographic test strip, which includes a bottom plate, on which a sample pad, a reaction membrane, and a water absorption pad are sequentially overlapped. 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 above-mentioned phenolphthalein artificial antigen combination, and the quality control area is coated with IgG.

[0088] Preferably, the phenolphthalein artificial antigen is obtained by conjugating the phenolphthalein hapten PT-4C with chicken ovalbumin.

[0089] Preferably, the IgG is goat anti-rabbit IgG or goat anti-mouse IgG.

[0090] The usage method of the phenolphthalein colloidal gold rapid detection test strip is as follows:

[0091] The sample to be tested is mixed evenly with the colloidal gold-labeled antibody, incubated at 25°C for 5 min. The antibody is a polyclonal antibody or a monoclonal antibody; the sample pad 2 of the phenolphthalein colloidal gold rapid detection test strip is left standing in the mixed solution for 3 min, then taken out and the sample pad 2 is removed for result determination;

[0092] The specific determination method is as follows:

[0093] If the control line does not show color, the detection effect is invalid and re-detection is required; if the control line shows red, the detection effect is valid, and the test line is further interpreted: if the test line does not show color or shows very weak color, it indicates that the sample to be tested contains phenolphthalein, and the interpretation result is positive or weakly positive; if the test line shows red, it indicates that the sample to be tested does not contain phenolphthalein, and the interpretation result is negative.

[0094] Preferably, the kit is used to identify phenolphthalein in weight loss foods.

[0095] The weight loss foods involved in the present invention are foods with weight loss functions, including but not limited to tablet candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, formulated wines, fruit wines and jellies with weight loss functions.

[0096] Compared with the prior art, the present invention has the following beneficial effects:

[0097] The present invention provides a phenolphthalein hapten that can not only retain the characteristic structures of the three benzene rings and lactone ring in phenolphthalein to the greatest extent, but also has a connecting arm of a certain length, and also provides a method for preparing the phenolphthalein hapten. The phenolphthalein artificial antigen prepared by conjugating the phenolphthalein hapten with a carrier protein can obtain a phenolphthalein antibody with high affinity after immunizing animals. Its half-inhibitory concentration IC 50 is 0.19 ng / mL, the lowest detection limit is 0.007 ng / mL, and the quantitative detection range is 0.02 - 1.64 ng / mL. It not only has high detection sensitivity, but also has strong specificity, high accuracy, no cross-reaction with a variety of phenolphthalein structural analogs, the detection effect is not affected by the sample type, and it can be popularized for large-scale rapid and accurate screening of phenolphthalein in weight loss foods. Description of the Drawings

[0098] Figure 1 It is the identification result of the phenolphthalein coating antigen in Example 2; a is the ultraviolet scanning diagram of OVA, PT-4C and PT-4C-OVA; b is the ultraviolet scanning diagram of OVA, PT-6C and PT-6C-OVA.

[0099] Figure 2 It is the identification result of the phenolphthalein immunogen in Example 2; a is the ultraviolet scanning diagram of BSA, PT-4C and PT-4C-BSA; b is the ultraviolet scanning diagram of BSA, PT-6C and PT-6C-BSA.

[0100] Figure 3 It is the indirect competitive ELISA standard curve of the antibody for detecting phenolphthalein.

[0101] Figure 4Schematic structural diagram of the phenolphthalein colloidal gold rapid test strip; 1 - PVC plastic base plate; 2 - sample pad; 3 - base film; 4 - absorbent pad; 5 - test line; 6 - control line.

[0102] Figure 5 Result determination diagram of the phenolphthalein colloidal gold rapid test strip of Example 8; A is the negative test result, B is the positive test result, and C and D are the invalid test results. Detailed implementation manners

[0103] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0104] Example 1 Preparation and identification of phenolphthalein hapten

[0105] I. Synthesis and identification of phenolphthalein hapten PT-4C

[0106] 1. Synthesis of phenolphthalein hapten PT-4C

[0107] Dissolve 1 mol of phenolphthalein in 3 mL of N,N-dimethylformamide, then add 2 mol of ethyl 4-bromobutyrate and 6 mol of potassium carbonate, and stir at room temperature (25 °C) overnight (11 h) to obtain a reaction product; separate and purify the reaction product by column chromatography (the stationary phase is silica gel powder, and the mobile phase is dichloromethane:ethyl acetate = 10:1). Dissolve the obtained purified product in 5 mL of methanol, then add 5 mL of 3 mol / L sodium hydroxide aqueous solution and stir at room temperature for 3 h. After the reaction is completed, adjust the pH to 6 - 7, and the obtained precipitate is the hapten PT-4C.

[0108] 2. Identification of phenolphthalein hapten PT-4C

[0109] The nuclear magnetic resonance hydrogen spectrum result of phenolphthalein hapten PT-4C is as follows: 1 H NMR(600MHz,Methanol-d4)δ7.88(dt,J=7.7,1.0Hz,1H),7.73(td,J=7.6,1.1Hz,1H),7.61–7.54(m,2H),7.21–7.15(m,2H),7.11–7.06(m,2H),6.88–6.81(m,2H),6.78–6.72(m,2H),3.97(t,J=6.2Hz,2H),2.46(t,J=7.3Hz,2H),2.06–1.98(m,2H).

[0110] The mass spectrometry identification result of the phenolphthalein hapten PT-4C is: MS: C 24 H 20 O 6 : 404.42, ESI+[M-H]+: 405.44.

[0111] According to the results of nuclear magnetic resonance hydrogen spectrum and mass spectrometry, it can be seen that the mass spectrometry result can correspond to the molecular weight of the hapten PT-4C, and the number of hydrogen spectra in the nuclear magnetic resonance can correspond to the number of hydrogen spectra in the structure of the hapten PT-4C, indicating that the target product phenolphthalein hapten PT-4C has been successfully prepared in this invention, and its structural formula is shown in Formula (Ⅰ):

[0112]

[0113] The phenolphthalein hapten PT-4C is named by systematic nomenclature as: 4-(4-(1-(4-hydroxyphenyl)-3-oxo-1,3-dihydroisobenzofuran-1-yl)phenoxy)butanoic acid; that is, 4-(4-(1-(4-hydroxyphenyl))-3-oxo-1,3-dihydroisobenzofuran-1-yl)phenoxy)butyric acid.

[0114] II. Synthesis and Identification of Phenolphthalein Hapten PT-6C

[0115] 1. Synthesis of Phenolphthalein Hapten PT-6C

[0116] Dissolve 1 mol of phenolphthalein in 3 mL of N,N-dimethylformamide, then add 2 mol of ethyl 6-bromohexanoate and 6 mol of potassium carbonate, and stir overnight (11 h) at room temperature (25 °C) to obtain the reactant; separate and purify the reactant by column chromatography (the stationary phase is silica powder, and the mobile phase is dichloromethane:ethyl acetate = 10:1), dissolve the obtained purified product in 5 mL of methanol, then add 5 mL of 3 mol / L sodium hydroxide aqueous solution and stir at room temperature for 3 h. After the reaction is completed, adjust the pH to 6-7, and the obtained precipitate is the hapten PT-6C.

[0117] 2. Identification of Phenolphthalein Hapten PT-6C

[0118] The nuclear magnetic resonance hydrogen spectrum result of the phenolphthalein hapten PT-6C is: 11H NMR (600 MHz, Methanol-d4) δ 7.87 (dt, J = 7.7, 1.0 Hz, 1H), 7.72 (td, J = 7.6, 1.1 Hz, 1H), 7.61–7.53 (m, 2H), 7.20–7.15 (m, 2H), 7.11–7.06 (m, 2H), 6.86–6.81 (m, 2H), 6.77–6.72 (m, 2H), 3.91 (t, J = 6.4 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.77–1.69 (m, 2H), 1.64 (p, J = 7.4 Hz, 2H), 1.50–1.42 (m, 2H).

[0119] The mass spectrometry identification result of the phenolphthalein hapten PT-6C is: MS: C 26 H 24 O 6 : 432.47, ESI+ [M - H]+: 433.49.

[0120] It can be seen from the 1H NMR and mass spectrometry results that the mass spectrometry result can correspond to the molecular weight of the hapten PT-6C, and the 1H NMR data can correspond to the hydrogen spectrum numbers on the structure of the hapten PT-6C, indicating that the target product phenolphthalein hapten PT-6C has been successfully prepared in this invention, and its structural formula is shown as formula (Ⅱ):

[0121]

[0122] The phenolphthalein hapten PT-6C is named by the systematic nomenclature as: 6-(4-(1-(4-hydroxyphenyl)-3-oxo-1,3-dihydroisobenzofuran-1-yl)phenoxy)hexanoic acid; that is, 6-(4-(1-(4-hydroxyphenyl))-3-oxo-1,3-dihydroisobenzofuran-1-yl)phenoxy)hexanoic acid.

[0123] Example 2 Synthesis and Identification of Phenolphthalein Artificial Antigen

[0124] The phenolphthalein haptens PT-4C and PT-6C prepared in Example 1 were respectively coupled to chicken ovalbumin (OVA) and bovine serum albumin (BSA) by the active ester method.

[0125] I. Synthesis and Identification of Phenolphthalein Coating Antigen

[0126] 1. Synthesis of Phenolphthalein Coating Antigen

[0127] Dissolve 10 mg of the phenolphthalein hapten PT-4C of Example 1 in 100 μL of N,N-dimethylformamide (DMF), add 2 mg of N-hydroxysuccinimide (NHS) and 3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and stir in the dark at 25 °C for 4 h to obtain Solution A.

[0128] Dissolve 10 mg of OVA in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain Solution B. The formulation of the used PBS buffer is: Na 2 HPO 4 ·12H 2 O 2.90 g, NaCl 8.50 g, KCl 0.20 g, KH 2 PO 4 0.20 g, and make up to 1000 mL with distilled water.

[0129] Dropwise add 100 μL of Solution A to 1 mL of Solution B, stir at 4 °C for 12 h; dialyze with PBS buffer for two days, 4 times a day, to obtain the phenolphthalein-coated antigen PT-4C-OVA, aliquot it into centrifuge tubes, and store it at -20 °C for use.

[0130] According to the above method, the difference is only that the phenolphthalein hapten PT-6C is used instead of the phenolphthalein hapten PT-4C to prepare the phenolphthalein-coated antigen PT-6C-OVA.

[0131] 2. Identification of the phenolphthalein-coated antigen

[0132] Perform ultraviolet scanning measurements (200 - 400 nm) on OVA, PT-4C, and PT-4C-OVA respectively. The measurement results are as shown in Figure 1 a in. The ultraviolet absorption peak of the phenolphthalein-coated antigen PT-4C-OVA shows a significant shift compared with the ultraviolet absorption peak of the hapten PT-4C, and the phenolphthalein-coated antigen PT-4C-OVA simultaneously has the characteristic absorption peaks of both the hapten PT-4C and OVA, indicating that the phenolphthalein-coated antigen PT-4C-OVA is successfully conjugated. Its structural formula is as shown in Formula (Ⅲ),

[0133]

[0134] wherein, P is the carrier protein OVA.

[0135] Perform ultraviolet scanning measurements (200 - 400 nm) on OVA, PT-6C, and PT-6C-OVA respectively. The measurement results are as shown in Figure 1As shown by b in [Figure], the ultraviolet absorption peak of the phenolphthalein-coated original PT-6C-OVA shows a significant shift compared to that of the hapten PT-6C, and the phenolphthalein-coated original PT-6C-OVA simultaneously has the characteristic absorption peaks of both the hapten PT-6C and OVA, indicating that the coupling of the phenolphthalein-coated original PT-6C-OVA is successful. Its structural formula is as shown in Formula (IV),

[0136]

[0137] wherein P is the carrier protein OVA.

[0138] II. Synthesis and Identification of Phenolphthalein Immunogen

[0139] 1. Synthesis of Phenolphthalein Immunogen

[0140] The preparation method is similar to the above-mentioned synthesis method of the phenolphthalein-coated original, except that BSA is used instead of OVA, and the phenolphthalein hapten PT-4C and the phenolphthalein hapten PT-6C prepared in Example 1 are used to synthesize the phenolphthalein immunogens PT-4C-BSA and PT-6C-BSA.

[0141] 2. Identification of Phenolphthalein Immunogen

[0142] Ultraviolet scanning measurements (200 - 400 nm) were respectively performed on BSA, PT-4C, and PT-4C-BSA. The measurement results are as Figure 2 shown by a in [Figure]. The ultraviolet absorption peak of the phenolphthalein immunogen PT-4C-BSA shows a significant shift compared to that of the hapten PT-4C, and the phenolphthalein immunogen PT-4C-BSA simultaneously has the characteristic absorption peaks of both the hapten PT-4C and BSA, indicating that the coupling of the phenolphthalein immunogen PT-4C-BSA is successful. Its structural formula is as shown in Formula (III),

[0143]

[0144] wherein P is the carrier protein BSA.

[0145] Ultraviolet scanning measurements (200 - 400 nm) were respectively performed on BSA, PT-6C, and PT-6C-BSA. The measurement results are as Figure 2 shown by b in [Figure]. The ultraviolet absorption peak of the phenolphthalein immunogen PT-6C-BSA shows a significant shift compared to that of the hapten PT-6C, and the phenolphthalein immunogen PT-6C-BSA simultaneously has the characteristic absorption peaks of both the hapten PT-6C and BSA, indicating that the coupling of the phenolphthalein immunogen PT-6C-BSA is successful. Its structural formula is as shown in Formula (IV),

[0146]

[0147] wherein P is the carrier protein BSA.

[0148] Preparation of Antibodies in Example 3

[0149] 1. Preparation of Polyclonal Antibodies

[0150] The phenolphthalein immunogens PT-4C-BSA and PT-6C-BSA prepared in Example 2 were respectively emulsified uniformly with an immunoadjuvant (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 immunizing animals.

[0151] New Zealand white rabbits weighing 2.5 - 3 kg were immunized by various injection methods including subcutaneous injection on the back, subcutaneous injection at various sites, intramuscular injection in the leg, and marginal ear vein injection. The second immunization was carried out 4 weeks later, and subsequent booster immunizations were carried out every 3 weeks. Blood was collected from the marginal ear vein 1 week after the third booster immunization, and the serum titer was measured by indirect competitive ELISA. When the titer no longer increased, booster immunization was carried out through the marginal ear vein. One week after the booster immunization, blood was collected from the heart, incubated in a water bath for 0.5 - 1 h, centrifuged at 4°C and 10,000 rpm for 15 min, and the supernatant was taken as the antiserum. The antiserum was purified by ammonium sulfate precipitation to obtain polyclonal antibodies, which were stored at -20°C for later use.

[0152] 2. Preparation of Monoclonal Antibodies

[0153] The phenolphthalein immunogens PT-4C-BSA and PT-6C-BSA prepared in Example 2 were respectively emulsified uniformly with an immunoadjuvant (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 immunizing animals.

[0154] BALB / c 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. After the antibody titer no longer increased, another booster immunization was carried out. Seven days later, spleen cells of the mice were fused with mouse myeloma cells to obtain fused cells.

[0155] After screening out hybridoma cells from the fused cells using HAT medium, the hybridoma cells were cultured in complete medium. The cell supernatant of the hybridoma cells was detected by ic-ELISA method. The cells in the wells with strongly positive detection results were cloned and cultured by the limiting dilution method. After 3 times of cloning and culturing and detection, hybridoma cells producing monoclonal antibodies were obtained. After amplifying the culture of the hybridoma cells, they were inoculated into the abdominal cavity of mice to produce ascites containing antibodies. The ascites was purified by octanoic acid-ammonium sulfate precipitation to obtain monoclonal antibodies, which were stored at -20°C for later use.

[0156] Determination of the Combination of Phenolphthalein Immunogen and Coating Antigen in Example 4

[0157] Using the artificial antigens PT-4C-OVA and PT-6C-OVA prepared in Example 2 as coating antigens, and using the antiserum prepared with PT-4C-BSA and PT-6C-BSA as immunogens in Example 3, the best combination of immunogen and coating antigen was selected by detecting the titer and inhibition rate of the antiserum. The specific method steps are as follows:

[0158] 1. Dilute the coating antigens PT-4C-OVA and PT-6C-OVA to 1 μg / mL respectively with coating buffer (0.05 M carbonate buffer solution, pH 9.6), coat a 96-well ELISA plate at 100 μL / well, and incubate at 37 °C for 12 h;

[0159] 2. Discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v));

[0160] 3. Add 120 μL of blocking solution (1 wt% fish skin collagen, diluted with PBST) to each well, and block at 37 °C for 3 h;

[0161] 4. Discard the blocking solution, take it out after drying at 37 °C for 30 min;

[0162] 5. Dilute the antiserum obtained by immunizing female Balb / c mice with PBST buffer into 7 gradients by serial dilution, namely 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, and set a blank control well (i.e., PBST buffer) at the same time; and dilute the phenolphthalein standard product (CAS No.: 77-09-8) to 1 μg / mL with PBST buffer to obtain the phenolphthalein standard product dilution solution;

[0163] 6. The method of adding samples in the titer row is: add 50 μL of PBST to each well, then add the 7 serially diluted antisera to the wells in sequence at 50 μL / well, and add 50 μL of PBST buffer to the last well;

[0164] The method of adding samples in the inhibition row is: add 50 μL of 1 μg / mL phenolphthalein standard product dilution solution to each well, then add the 7 serially diluted antisera to the wells in sequence at 50 μL / well, and add 50 μL of PBST buffer to the last well;

[0165] 7. Incubate at 37 °C for 40 min and wash 5 times;

[0166] 8. Add 100 μL / well of goat anti-rabbit secondary antibody-HRP (diluted 5000-fold) to the titer row and the inhibition row, incubate at 37 °C for 30 min, and wash five times;

[0167] 9. Add 100 μL / well of the hole chromogenic solution and develop color for 10 min. The chromogenic solution is obtained by uniformly mixing urea peroxide and tetramethylbenzidine in a volume ratio of 1:1.

[0168] 10. Add 50 μL / well of 2 mol / L H 2 SO 4 solution to terminate the reaction, and read the OD value at 450 nm. Calculate the titer and inhibition rate. The titer is the dilution multiple of the antiserum corresponding to an OD 450 of about 1.0. Inhibition rate = (OD value of the titer - OD value of the inhibition) / OD value of the inhibition × 100%.

[0169] The results of the titers and inhibition rates of the antisera for different combinations of immunogens and coating antigens are shown in Table 1.

[0170] Table 1 Titers and inhibition rates of sera for different combinations of immunogens and coating antigens

[0171]

[0172] As can be seen from Table 1, when the artificial antigens PT-3-BSA and PT-5-BSA of phenolphthalein are used as immunogens, the antisera produced all have a certain titer, and both are above 1:128,000; at the same time, the obtained antisera all have different degrees of inhibitory effects on the target analyte phenolphthalein, and both are above 80%. The above results indicate that the immunogens and coating antigens prepared in the present invention can not only specifically recognize the target analyte phenolphthalein, but also have high sensitivity.

[0173] Among them, the titer of 1:256,000 and the inhibition rate of 98.87% shown by the combination of the immunogen and coating antigen No. 3 are higher than the titers and inhibition rates of the other combinations of immunogens and coating antigens. Therefore, PT-6C-BSA is used as the best immunogen and PT-4C-OVA is used as the best coating antigen.

[0174] Example 5 Establishment of an indirect competitive ELISA assay for rapid identification of phenolphthalein

[0175] I. An indirect competitive ELISA method for rapid identification of phenolphthalein, comprising the following steps:

[0176] 1. Use the artificial antigen PT-4C-OVA or PT-6C-OVA prepared in Example 2 as the coating antigen, dilute it to 50 μg / L with the coating solution (0.05 M carbonate buffer solution, pH 9.6), coat a 96-well enzyme-linked immunosorbent assay (ELISA) plate at 100 μL / well, and incubate at 37°C for 12 h.

[0177] 2. Discard the coating solution, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0178] 3. Add 120 μL of blocking solution (i.e., 1 wt% fish skin collagen, diluted with PBST) to each well and block at 37 °C for 3 h;

[0179] 4. Discard the blocking solution, take it out after drying at 37 °C for 30 min;

[0180] 5. Dilute the monoclonal antibody prepared in Example 3 16,000 times with PBST buffer, and dilute the phenolphthalein standard (CAS No.: 77-09-8) with PBST buffer to 1000 μg / L, 200 μg / L, 40 μg / L, 8 μg / L, 1.6 μg / L, 0.32 μg / L, 0.064 μg / L, and 0.0128 μg / L respectively;

[0181] 6. Add the different concentrations of phenolphthalein standards obtained in the previous step to the enzyme-linked immunosorbent assay (ELISA) plate at 50 μL / well; then add the monoclonal antibody prepared in Example 3 diluted 16,000 times in the previous step to the ELISA plate at 50 μL / well; set 3 parallel replicates;

[0182] 7. Incubate at 37 °C for 40 min and wash 5 times;

[0183] 8. Add 100 μL / well of goat anti-rabbit secondary antibody-HRP (diluted 5000 times), incubate at 37 °C for 30 min, wash five times, and pat dry;

[0184] 9. Add 100 μL / well of chromogenic solution and develop color for 10 min. The chromogenic solution is obtained by mixing urea peroxide and tetramethylbenzidine evenly at a volume ratio of 1:1;

[0185] 10. Add 50 μL / well of 2 mol / L H 2 SO 4 solution to terminate the reaction and read the OD value at 450 nm.

[0186] 11. Use B / B 0 as the ordinate (B is the absorbance OD 450 when adding the standard, B 0 is the absorbance OD 450 when not adding the standard), the logarithm of the standard concentration as the abscissa, and use the Logistic function for curve fitting to obtain the formula of the standard curve and prepare the standard curve.

[0187] II. Experimental Results

[0188] A total of 4 groups of standard curves for indirect competitive ELISA of antibodies for detecting phenolphthalein were obtained. Among them, the standard curve prepared with the monoclonal antibody obtained using PT-6C-BSA as the immunogen and PT-4C-OVA as the coating antigen is as Figure 3As shown, it can be seen that the half-inhibitory concentration IC of the monoclonal antibody prepared in Example 3 for phenolphthalein 50 is 0.19 ng / mL, and the linear range for quantitative detection IC 20 ~IC 80 is 0.02 - 1.64 ng / mL, and the detection limit is 0.007 ng / mL. It shows that the antibody for detecting phenolphthalein prepared in the present invention can meet the detection requirements, and an indirect competitive ELISA detection method for phenolphthalein in weight loss foods is successfully established, and the sensitivity of this method is high.

[0189] Example 6 Specificity Evaluation of the Phenolphthalein Antibody of the Present Invention

[0190] I. Experimental Method

[0191] Phendimetrazine, bisacodyl, bispropyldine, bisoxatin, bisoxatin acetate, o-cresolphthalein, α-naphtholphthalein, thymolphthalein, and diphenyllactone are all structural analogs of phenolphthalein, and phenolphthalein, phendimetrazine, bisacodyl, bispropyldine, bisoxatin, and bisoxatin acetate are often added to weight loss products.

[0192] In this example, a cross-reaction experiment was conducted to determine the specificity of the antibody prepared in the present invention for detecting phenolphthalein.

[0193] According to the detection method of Example 5, the difference is only that: the phenolphthalein standard is replaced with phendimetrazine (CAS No.: 125-13-3), bisacodyl (CAS No.: 115-33-3), bispropyldine (manufacturer: Macklin; product number: D916002), bisoxatin (CAS No.: 17692-24-9), bisoxatin acetate (CAS No.: 14008-48-1), o-cresolphthalein (CAS No.: 596-27-0), α-naphtholphthalein (CAS No.: 596-01-0), thymolphthalein (CAS No.: 125-20-2), and diphenyllactone (CAS No.: 596-29-2), and detection was carried out at the same dilution multiple to obtain the IC 50 value of each structural analog.

[0194] The cross-reaction rate (CR) of phenolphthalein was calculated according to the following formula: CR(%) = IC 50 (phenolphthalein) / IC 50 (structural analog) × 100%, and the smaller the cross-reaction rate, the stronger the specificity.

[0195] II. Experimental Results

[0196] The cross-reaction results of phenolphthalein with its structural analogs are shown in Table 2.

[0197] Table 2 Cross-reaction Results of Phenolphthalein with Its Structural Analogs

[0198]

[0199] Note: NR means no reaction.

[0200] As can be seen from Table 2, the antibodies used to detect phenolphthalein have no cross-reaction to a variety of structural analogs (phenolphthalein, diacetylphenolphthalein, dipropenolphthalein, bisphenolphthalein, bisphenolphthalein acetate, o-cresolphthalein, α-naphtholphthalein, thymolphthalein and diphenylphthalide), indicating that the antibody prepared in Example 3 for detecting phenolphthalein has a strong specificity for phenolphthalein, can effectively eliminate the interference of its structural analogs on phenolphthalein, and can be specifically used for the detection of phenolphthalein.

[0201] Example 7 An ELISA kit for detecting phenolphthalein

[0202] 1. Composition

[0203] 1. ELISA plate coated with coating agent:

[0204] The ELISA plate is prepared by the following method:

[0205] The artificial antigen PT-4C-OVA or PT-6C-OVA prepared in Example 2 was used as a coating source, and the coating source was diluted to 50 μg / L with a coating buffer (0.05 M carbonate buffer solution, pH 9.6), and 100 μL / well was added to the ELISA plate, incubated at 37° C. in the dark overnight, the liquid in the well was removed, and the well was washed twice with the washing solution in the kit, each time for 30 seconds, and then 200 μL / well of the blocking solution of the kit was added, incubated at 25° C. in the dark for 2 hours, the liquid in the well was removed, and after drying, it was vacuum-sealed with aluminum film for storage;

[0206] 2. Standard products:

[0207] 8 different concentrations of phenolphthalein standard solutions, including 1000 ng / mL, 200 ng / mL, 40 ng / mL, 8 ng / mL, 1.6 ng / mL, 0.32 ng / mL, 0.064 ng / mL, and 0.0128 ng / mL;

[0208] 3. Antibodies:

[0209] The polyclonal antibody or monoclonal antibody prepared using the phenolphthalein immunogen PT-6C-BSA as the immunogen in Example 3;

[0210] 4. Enzyme conjugates:

[0211] Horseradish peroxidase-labeled goat anti-rabbit secondary antibody or horseradish peroxidase-labeled goat anti-mouse secondary antibody;

[0212] 5. Substrate color developing solution:

[0213] It is composed of liquid A and liquid B, liquid A is carbamide peroxide, and liquid B is tetramethylbenzidine;

[0214] 6. Stop solution:

[0215] 2 mol / L H 2 SO 4 ;

[0216] 7. Other reagents

[0217] Washing solution: with a pH value of 7.4, containing 0.8% (v / v) Tween-20, 0.02% (w / w) sodium azide preservative, and 0.2 mol / L phosphate buffer; before use, dilute the washing solution 20-fold with water (i.e., add 1 part of the washing solution to 19 parts of water, prepare and use immediately), and the working solution of the washing solution is obtained;

[0218] Blocking solution: with a pH value of 7.3, containing 2% (w / w) casein in 0.2 mol / L phosphate buffer;

[0219] Enzyme conjugate diluent: 0.2 mol / L phosphate buffer; before use, dilute the diluent 20-fold with water (i.e., add 1 part of the diluent to 19 parts of water, prepare and use immediately), and the working solution of the enzyme conjugate diluent is obtained.

[0220] II. Usage

[0221] 1. Sample detection

[0222] Number the corresponding micro-wells of the samples and the standards in this kit in sequence. Do 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells. Dilute the antibody with the working solution of the diluent at a volume ratio of 1:40 according to the required amount (i.e., add 1 part of the antibody to 40 parts of the working solution of the diluent, prepare and use immediately) to obtain the working solution of the antibody. Dilute the enzyme conjugate concentrate with the working solution of the diluent at a volume ratio of 1:10 according to the required amount (i.e., add 1 part of the enzyme conjugate concentrate to 10 parts of the working solution of the diluent, prepare and use immediately) to obtain the working solution of the enzyme conjugate.

[0223] Add 50 μL of the standard or sample to the corresponding micro-well, then add 50 μL of the working solution of the antibody to the corresponding micro-well, gently shake and mix well, cover with a cover film, and react in a dark environment at 25 °C for 40 min.

[0224] Discard the liquid in the wells by flicking, and add 250 μL / well of the working solution of the washing solution. Wash thoroughly 4 - 5 times, with an interval of 10 s each time. Discard the working solution of the washing solution in the plate wells, and pat dry with absorbent paper (the unremoved bubbles after patting dry can be pricked with an unused pipette tip).

[0225] Add 100 μL / well of the working solution of the enzyme conjugate to the corresponding micro-well, gently shake and mix well, cover with a cover film, and react in a dark environment at 25 °C for 30 min.

[0226] Centrifuge the liquid in the wells to dryness, and add 250 μL / well of the washing solution working solution. Wash thoroughly 4 - 5 times, with a 10 - second interval each time. Discard the washing solution working solution in the wells of the plate, and pat dry with absorbent paper (any unbroken bubbles remaining after pat - drying can be punctured with an unused pipette tip).

[0227] Add 50 μL / well of Substrate Chromogenic Solution A, and then add 50 μL / well of Substrate Chromogenic Solution B. Gently shake and mix well. Cover the plate with a cover film and place it in a 25 °C dark environment for reaction for 10 min.

[0228] Add 50 μL / well of the stop solution, gently shake and mix well. Set the microplate reader at 450 nm and measure the OD value of each well.

[0229] 2. Plotting the standard curve

[0230] Plot the ELISA standard curve according to the above - mentioned optimal conditions. Use B / B 0 as the ordinate (B is the absorbance OD value when adding the standard product 450 , and B 0 is the absorbance OD value when not adding the standard product 450 ), and the logarithm of the standard product concentration as the abscissa. Use the Logistic function for curve fitting to obtain the formula of the standard curve and prepare the standard curve.

[0231] 3. Calculation of the sample concentration

[0232] Substitute the measured absorbance OD 450 value of the sample into the above - mentioned calculation formula to calculate the percentage absorbance rate of the sample; substitute the percentage absorbance rate of the sample into the formula of the above - mentioned standard curve to obtain the concentration of the sample, and then multiply it by its corresponding dilution factor to obtain the actual concentration of phenolphthalein in the test sample.

[0233] Example 8 Preparation of a colloidal gold rapid test strip for rapid identification of phenolphthalein in weight - loss foods and establishment of a detection method

[0234] I. Preparation of a colloidal gold rapid test strip for rapid identification of phenolphthalein in weight - loss foods

[0235] As Figure 4 shown, the colloidal gold rapid test strip is composed of an NC membrane (nitrocellulose membrane), a sample pad, an absorbent pad, and a PVC plastic bottom plate stacked together.

[0236] The coating source (PT-4C-OVA or PT-6C-OVA) was sprayed on the NC membrane at a spray volume of 0.8 μL / cm using an XYZ three-dimensional spray dot film apparatus as test line 5; goat anti-rabbit IgG or goat anti-mouse IgG was sprayed on the NC membrane using the same method and dosage as control line 6; the test line 5 and the control line 6 were located in the middle of the NC membrane and were 6 mm apart from each other; after drying at 37°C for 12 h, the cellulose membrane was pasted on the middle part of the backing board, the sample pad overlapped the test line end of the NC membrane by 1 mm, the absorbent pad was pasted on the upper side of the cellulose and overlapped the cellulose membrane 3 by 1 mm, and the assembled test paper was cut into 3.5 mm wide test strips using a chopper.

[0237] 2. Preparation of gold-labeled antibodies

[0238] The 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:

[0239] Take 1mL of colloidal gold solution and add 0.2mol / L of K 2 CO 3 The solution was adjusted to pH 8.0, and 10 μg of polyclonal antibody or monoclonal antibody was added and incubated for 30 min; 10% wt BSA solution was then added and incubated for 30 min; the mixture was centrifuged at 10000 rpm for 20 min at 4°C, and the supernatant was discarded; the precipitate was resuspended with 200 μL 0.2 mol / L phosphate buffer solution (containing 0.5% v / v Tween-20, 0.5% wt BSA, 5% wt sucrose, 0.3% wt PVP, 0.03% v / v procline-300, pH 7.4) to obtain the gold-labeled antibody, which was stored at 4°C.

[0240] 3. Sample Pretreatment

[0241] 1. Tablet candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, blended alcoholic beverages and fruit wines

[0242] 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 min, cool to room temperature, centrifuge at 4000 rpm for 5 min, aspirate 1.0 mL of supernatant, dilute four times with 0.02 M PB, vortex evenly, and filter with a microporous filter membrane to obtain the test solution for testing.

[0243] 2. Jelly

[0244] Accurately weigh 1.0 g of the sample into a 10 mL centrifuge tube, add 5 mL of water, heat in a water bath at 80 °C until the sample dissolves, cool to room temperature, add 5 mL of methanol, extract by ultrasound for 5 min, centrifuge at 4000 rpm for 5 min, aspirate 1.0 mL of the supernatant, dilute it four-fold with 0.02 M PB, vortex evenly and filter through a microporous filter membrane to obtain the test solution for determination.

[0245] IV. Detection method

[0246] Mix 150 μL of the test solution evenly with 5 μL of the gold-labeled antibody and incubate at 25 °C for 5 min; insert the sample pad 2 of the phenolphthalein colloidal gold rapid test strip into the mixed solution, react for 3 min and then take it out, remove the sample pad 2, and make a result determination.

[0247] V. Result determination

[0248] As Figure 5 shown in A of , when both the test line 5 and the control line 6 show a clear red line, it indicates that the gold-labeled antibody binds to the coating antigen of the test line 5 and the goat anti-rabbit IgG or goat anti-mouse IgG of the control line 6 respectively, that is, the test sample does not contain the analyte phenolphthalein, and the test result is determined to be negative; as Figure 5 shown in B of , the test line 5 does not develop color, and only the control line 6 shows a clear red line, indicating that the gold-labeled antibody binds to the goat anti-rabbit IgG or goat anti-mouse of the control line 6 and the phenolphthalein in the test sample respectively, and does not bind to the coating antigen of the test line 5, that is, the test sample contains the analyte phenolphthalein, and the test result is determined to be positive; as Figure 5 shown in C and D of , when the control line 6 does not show a red line, regardless of whether the test line 5 develops color or not, the test result is determined to be invalid and needs to be retested.

[0249] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description and ideas. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A phenolphthalein hapten, characterized in that, the structural formula of the phenolphthalein hapten is shown in formula (Ⅰ), Formula (Ⅰ).

2. A phenolphthalein hapten, characterized in that, the structural formula of the phenolphthalein hapten is shown in formula (Ⅱ), Formula (Ⅱ).

3. Use of the phenolphthalein hapten according to claim 1 or 2 in the preparation of a phenolphthalein artificial antigen.

4. A phenolphthalein artificial antigen, characterized in that, it is obtained by conjugating the phenolphthalein hapten according to claim 1 with a carrier protein, and its structural formula is shown in formula (Ⅲ), Formula (Ⅲ), wherein, P is the carrier protein, and the carrier protein is chicken ovalbumin or bovine serum albumin.

5. A phenolphthalein artificial antigen, characterized in that, it is obtained by conjugating the phenolphthalein hapten according to claim 2 with a carrier protein, and its structural formula is shown in formula (Ⅳ), Formula (Ⅳ), wherein, P is the carrier protein, and the carrier protein is chicken ovalbumin or bovine serum albumin.

6. Use of the phenolphthalein artificial antigen according to claim 4 or claim 5 in the preparation of an antibody against phenolphthalein.

7. A phenolphthalein artificial antigen composition, characterized in that, it comprises a coating antigen and an immunogen. The coating antigen is obtained by conjugating the phenolphthalein hapten according to claim 1 or claim 2 with chicken ovalbumin; the immunogen is obtained by conjugating the phenolphthalein hapten according to claim 1 or claim 2 with bovine serum albumin.

8. Use of the phenolphthalein artificial antigen composition according to claim 7 in the preparation of a reagent for detecting phenolphthalein.

9. A kit for detecting phenolphthalein, characterized in that, it contains the phenolphthalein artificial antigen composition according to claim 7.

10. According to the kit of claim 9, characterized in that, it further comprises an immunochromatographic test strip. The immunochromatographic test strip comprises a bottom plate, and a sample pad, a reaction membrane and a water absorption pad are sequentially arranged on the bottom plate. 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 described in claim 9, and the quality control area is coated with IgG.

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