Hapten, artificial antigen for simultaneously detecting phenolphthalein substances in weight loss foods and applications thereof

Abstract

The present invention discloses a hapten, an artificial antigen of phenolphthalein substances in weight loss foods and their applications. The hapten and artificial antigen of phenolphthalein substances are designed and obtained in the present invention, and an antibody capable of simultaneously recognizing phenolphthalein, bisacodyl and bispacol is prepared. The antibody has good recognition performance for phenolphthalein, bisacodyl and bispacol, and the lowest detection limits LOD for the three are 0.08 ng / mL, 0.44 ng / mL and 1.23 ng / mL respectively, and the half inhibitory concentration IC 50 are 0.40 ng / mL, 2.88 ng / mL and 12.11 ng / mL respectively, and there is no cross-reaction with functional analogues of phenolphthalein, bisacodyl and bispacol. Using the hapten, artificial antigen and antibody of phenolphthalein substances provided by the present invention can provide a technical basis for establishing a synchronous immunological detection method for phenolphthalein substances in weight loss foods, and has good application prospects.

Description

Technical Field

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

[0002] Phenbuterol, bisacetaminophen, and bispropargol are laxative chemicals in the bispropargol class. Phenbuterol has a strong irritating effect on the intestinal mucosa, promoting intestinal motility and relieving constipation. Bisacetaminophen and bispropargol are derivatives of bispropargol. After oral administration, they gradually break down in the intestines to produce bispropargol, a strong irritant to the intestinal mucosa. This product has a strong laxative effect, and long-term use can lead to dehydration, gastrointestinal discomfort, dizziness, nausea, and other symptoms, which can be life-threatening in severe cases. As people's living standards improve, obesity has become a health issue of increasing concern. The huge demand for weight loss has spawned a market for weight loss foods, with products such as weight loss coffee, weight loss tea, weight loss capsules, and weight loss enzymes emerging in large quantities.

[0003] The reported detection methods for this type of drugs in weight-loss foods are 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; Determination of 19 compounds including bisacetaminophen in foods, Food Supplement Inspection Method, BJS202209; Determination of bisacetaminophen in foods by high-performance liquid chromatography-tandem mass spectrometry, Group Standard, T / SATA 033-2-22). Although high-performance liquid chromatography-tandem mass spectrometry has high accuracy, the sample pretreatment is cumbersome, the detection time is long, the instrument is expensive, and professional operators are required. It is difficult to meet the regulatory requirements for rapid on-site detection, which limits its promotion and application in grassroots testing units. Therefore, establishing a method for quickly and accurately detecting illegally added bisphenol A substances in food is of great significance to improving my country's food safety monitoring system.

[0004] Among existing rapid detection technologies, immunoassays are widely used in drug residue detection due to their high sensitivity, specificity, rapidity, and ease of use. They offer numerous advantages over high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). However, there are currently no reports on the synthetic structure of haptens for phenolic compounds, and antibodies for their detection are also lacking. Therefore, the development of efficient methods for detecting phenolic compounds is crucial, and it is imperative to identify immunoassay raw materials, such as haptens and artificial antigens, that can generate antibodies with high specificity for phenolic compounds. Summary of the Invention

[0005] 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 phenolic substances, and to provide haptens, artificial antigens and their applications for simultaneous detection of phenolic substances in weight-loss foods.

[0006] The first object of the present invention is to provide two semi - antigens of phenolphthalein butylamine substances.

[0007] The second object of the present invention is to provide the application of the above - mentioned semi - antigens of phenolphthalein butylamine substances in the preparation of artificial antigens of phenolphthalein butylamine substances.

[0008] The third object of the present invention is to provide two artificial antigens of phenolphthalein butylamine substances.

[0009] The fourth object of the present invention is to provide the application of the above - mentioned artificial antigens of phenolphthalein butylamine substances in the preparation of antibodies against phenolphthalein butylamine substances.

[0010] The fifth object of the present invention is to provide a combination of artificial antigens of phenolphthalein butylamine substances.

[0011] The sixth object of the present invention is to provide the application of the above - mentioned combination of artificial antigens of phenolphthalein butylamine substances in the preparation of reagents for detecting phenolphthalein butylamine substances.

[0012] The seventh object of the present invention is to provide a kit for detecting phenolphthalein butylamine substances.

[0013] The eighth object of the present invention is to provide an immunoassay method for detecting phenolphthalein butylamine substances.

[0014] In order to achieve the above objects, the present invention is realized through the following solutions:

[0015] First, the present invention prepares two semi - antigens OPH - 1 and OPH - 2 for simultaneously detecting phenolphthalein butylamine substances in weight - loss foods, and then couples the two semi - antigens with bovine serum albumin (BSA) and chicken ovalbumin (OVA) respectively by the active ester method to obtain artificial antigens of phenolphthalein butylamine substances OPH - 1 - OVA, OPH - 1 - BSA, OPH - 2 - OVA and OPH - 2 - BSA. The artificial antigens of phenolphthalein butylamine substances OPH - 1 - BSA and OPH - 2 - BSA are used as immunogens to immunize animals to obtain antibodies against phenolphthalein butylamine substances. At the same time, the artificial antigens of phenolphthalein butylamine substances OPH - 1 - OVA and OPH - 2 - OVA are used as coating antigens, and the antiserum prepared with OPH - 1 - BSA and OPH - 2 - BSA as immunogens is used to select the best combination of immunogens and coating antigens by detecting the titer and inhibition rate of the antiserum.

[0016] Therefore, the present invention claims the following:

[0017] A semi - antigen OPH - 1 of phenolphthalein butylamine substance, whose structural formula is shown in formula (Ⅰ),

[0018]

[0019] The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

[0020] The hapten OPH-1 of the phenolphthalein substances is named by the systematic nomenclature as: 2-(4-(3-(4-hydroxyphenyl)-2-oxoindolin-3-yl)phenoxy)acetic acid; that is, 2-(4-(3-(4-hydroxyphenyl)-2-oxoindolin-3-yl)phenoxy)acetic acid.

[0021] A hapten OPH-2 of the phenolphthalein substances, whose structural formula is shown in formula (Ⅱ),

[0022]

[0023] The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

[0024] The hapten OPH-2 of the phenolphthalein substances is named by the systematic nomenclature as: 4-((4-(3-(4-hydroxyphenyl)-2-oxoindolin-3-yl)phenoxy)methyl)benzoic acid; that is, 4-((4-(3-(4-hydroxyphenyl)-2-oxoindolin-3-yl)phenoxy)methyl)benzoic acid.

[0025] A preparation method of the hapten OPH-1 of the phenolphthalein substances shown in the structural formula of formula (Ⅰ) includes the following steps: Dissolve phenolphthalein in methanol and react fully with ethyl bromoacetate and potassium carbonate, and fully hydrolyze the obtained reactant under alkaline conditions, and then adjust the pH to acidic to obtain it. The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

[0026] Preferably, the preparation method of the hapten OPH-1 of the phenolphthalein substances shown in the structural formula of formula (Ⅰ) includes the following steps: Dissolve 1 mol of phenolphthalein in 8 mL of methanol, then add 2 mol of ethyl bromoacetate and 6 mol of potassium carbonate, stir at room temperature (25 °C) overnight (11 h) to obtain a reactant; Use silica gel powder as the stationary phase and a mixed solution with a volume ratio of dichloromethane:methanol = 80:1 as the mobile phase, separate and purify the reactant by column chromatography, 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 OPH-1 of the phenolphthalein substances (the structural formula is shown in formula (Ⅰ)).

[0027] The preparation method of the phenolphthalein butyl ester semi - antigen OPH - 2 with the structural formula shown in formula (Ⅱ) includes the following steps: Phenolphthalein butyl ester is dissolved in methanol and fully reacted with methyl 4 - (bromomethyl) benzoate and potassium carbonate. The obtained reactant is fully hydrolyzed under alkaline conditions, and then the pH is adjusted to acidic to obtain the product. The phenolphthalein butyl ester substances are one or more of phenolphthalein butyl ester, bisacodyl butyl ester, and bis - propylphenolphthalein butyl ester.

[0028] Preferably, the preparation method of the phenolphthalein butyl ester semi - antigen OPH - 2 with the structural formula shown in formula (Ⅱ) includes the following steps: Dissolve 1 mol of phenolphthalein butyl ester in 8 mL of methanol, then add 2 mol of methyl 4 - (bromomethyl) benzoate and 6 mol of potassium carbonate, and stir at room temperature (25 °C) overnight (11 h) to obtain a reactant; Use silica gel powder as the stationary phase and a mixed solution with a volume ratio of dichloromethane: methanol = 80:1 as the mobile phase to separate and purify the reactant by column chromatography. 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, adjust the pH to 6 - 7, and the obtained precipitate is the phenolphthalein butyl ester semi - antigen OPH - 2 (with the structural formula shown in formula (Ⅱ)).

[0029] The application of the phenolphthalein butyl ester semi - antigen OPH - 1 (with the structural formula shown in formula (Ⅰ)) or the phenolphthalein butyl ester semi - antigen OPH - 2 (with the structural formula shown in formula (Ⅱ)) in the preparation of the artificial antigen of phenolphthalein butyl ester substances should also be within the protection scope of the present invention. The phenolphthalein butyl ester substances are one or more of phenolphthalein butyl ester, bisacodyl butyl ester, and bis - propylphenolphthalein butyl ester.

[0030] An artificial antigen of phenolphthalein butyl ester substances is obtained by coupling the phenolphthalein butyl ester semi - antigen OPH - 1 with the structural formula shown in formula (Ⅰ) to a carrier protein, and its structural formula is shown in formula (Ⅲ).

[0031]

[0032] Among them, P is the carrier protein, and the phenolphthalein butyl ester substances are one or more of phenolphthalein butyl ester, bisacodyl butyl ester, and bis - propylphenolphthalein butyl ester.

[0033] Preferably, the carrier protein is chicken ovalbumin or bovine serum albumin, that is, the artificial antigen of phenolphthalein butyl ester substances OPH - 1 - OVA or OPH - 1 - BSA.

[0034] More preferably, the carrier protein is bovine serum albumin, that is, the artificial antigen of phenolphthalein butyl ester substances OPH - 1 - BSA (with the structural formula shown in formula (Ⅲ), where P is bovine serum albumin).

[0035] An artificial antigen of phenolphthalein butyl ester substances is obtained by coupling the phenolphthalein butyl ester semi - antigen OPH - 2 with the structural formula shown in formula (Ⅱ) to a carrier protein, and its structural formula is shown in formula (Ⅳ).

[0036]

[0037] Among them, P is a carrier protein, and the phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

[0038] Preferably, the carrier protein is chicken ovalbumin or bovine serum albumin, that is, the artificial antigen OPH-2-OVA or OPH-2-BSA of the phenolphthalein substance.

[0039] More preferably, the carrier protein is chicken ovalbumin, that is, the artificial antigen OPH-2-OVA of the phenolphthalein substance (the structural formula is shown in formula (IV), where P is chicken ovalbumin).

[0040] A method for preparing the artificial antigen of the above-mentioned phenolphthalein substance, coupling the hapten of the above-mentioned phenolphthalein substance with a carrier protein by the active ester method, and the phenolphthalein substance is one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

[0041] Preferably, the active ester method includes the following steps:

[0042] S1. React the hapten of the phenolphthalein substance, 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;

[0043] S2. After solution A and solution B react sufficiently, dialysis is carried out to obtain the artificial antigen of the phenolphthalein substance.

[0044] More preferably, in step S1, the mass-volume ratio of the hapten of the phenolphthalein substance, N,N-dimethylformamide, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is (8 mg - 12 mg):(50 μL - 100 μL):(1 mg - 3 mg):(2 mg - 4 mg).

[0045] Further preferably, in step S1, the mass-volume ratio of the hapten of the phenolphthalein substance, N,N-dimethylformamide, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 10 mg:100 μL:2 mg:3 mg.

[0046] More preferably, in step S1, the mass-volume ratio of the carrier protein to the phosphate buffer is (8 mg - 12 mg):(0.5 mL - 1.5 mL).

[0047] Further preferably, in step S1, the mass-volume ratio of the carrier protein to the phosphate buffer is 10 mg:1 mL.

[0048] More preferably, in step S1, first dissolve the phenolphthalein-like substance 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.

[0049] Further preferably, in step S1, dissolve 10 mg of the phenolphthalein-like substance 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.

[0050] More preferably, in step S2, the volume ratio of solution A to solution B is (1 to 3):(5 to 10).

[0051] Further preferably, in step S2, the volume ratio of solution A to solution B is 1:10.

[0052] 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.

[0053] Further preferably, in step S2, add solution A dropwise to solution B, and stir at 4°C for 12 h.

[0054] More preferably, in step S2, the dialysis method is to dialyze with PBS buffer for 2 days, 4 times a day.

[0055] The artificial antigen of the phenolphthalein-like substance prepared by any of the above methods should also be within the protection scope of the present invention, and the phenolphthalein-like substance is one or more of phenolphthalein, bisacodyl, and bispicol.

[0056] The application of any of the above artificial antigens of the phenolphthalein-like substance in the preparation of an antibody against the phenolphthalein-like substance should also be within the protection scope of the present invention, and the phenolphthalein-like substance is one or more of phenolphthalein, bisacodyl, and bispicol.

[0057] The application of any of the above artificial antigens of the phenolphthalein-like substance in the detection of the phenolphthalein-like substance should also be within the protection scope of the present invention, the detection is for non-disease treatment and diagnosis purposes, and the phenolphthalein-like substance is one or more of phenolphthalein, bisacodyl, and bispicol.

[0058] Preferably, the artificial antigen of the phenolphthalein-like substance is used to identify the phenolphthalein-like substance in weight loss foods, and the phenolphthalein-like substance is one or more of phenolphthalein, bisacodyl, and bispicol.

[0059] 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.

[0060] An artificial antigen combination of phenolphthalein substances, comprising a coating antigen and an immunogen. The coating antigen is obtained by conjugating the phenolphthalein hapten OPH-1 (structural formula shown in formula (Ⅰ)) or the phenolphthalein hapten OPH-2 (structural formula shown in formula (Ⅱ)) with chicken ovalbumin; the immunogen is obtained by conjugating the phenolphthalein hapten OPH-1 (structural formula shown in formula (Ⅰ)) or the phenolphthalein hapten OPH-2 (structural formula shown in formula (Ⅱ)) with bovine serum albumin. The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispacolax.

[0061] Preferably, the coating antigen is obtained by conjugating the phenolphthalein hapten OPH-2 (structural formula shown in formula (Ⅱ)) with chicken ovalbumin, that is, the coating antigen is the artificial antigen OPH-2-OVA of phenolphthalein substances (structural formula shown in formula (Ⅳ), where P is chicken ovalbumin).

[0062] Preferably, the immunogen is obtained by conjugating the phenolphthalein hapten OPH-1 (structural formula shown in formula (Ⅰ)) with bovine serum albumin, that is, the immunogen is the artificial antigen OPH-1-BSA of phenolphthalein substances (structural formula shown in formula (Ⅲ), where P is bovine serum albumin).

[0063] More preferably, the coating antigen is obtained by conjugating the phenolphthalein hapten OPH-2 (structural formula shown in formula (Ⅱ)) with chicken ovalbumin; the immunogen is obtained by conjugating the phenolphthalein hapten OPH-1 (structural formula shown in formula (Ⅰ)) with bovine serum albumin; that is, the coating antigen is the artificial antigen OPH-2-OVA of phenolphthalein substances (structural formula shown in formula (Ⅳ), where P is chicken ovalbumin), and the immunogen is OPH-1-BSA (structural formula shown in formula (Ⅲ), where P is bovine serum albumin).

[0064] An antibody against phenolphthalein substances is prepared by immunizing an animal with the above immunogen. The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispacolax.

[0065] Preferably, the antibody against phenolphthalein substances is a monoclonal antibody. Hybridoma cells are obtained by immunizing an animal with any of the above immunogens, the obtained hybridoma cells are cultured and the cell supernatant is collected, and after identification and purification, the monoclonal antibody against phenolphthalein substances is obtained.

[0066] Preferably, the antibody against phenolphthalein substances is a polyclonal antibody. An animal is immunized with any of the above immunogens, the serum is collected, and after purification by ammonium sulfate precipitation method, the polyclonal antibody against phenolphthalein substances is obtained.

[0067] More preferably, the immunogen is obtained by conjugating the phenolphthalein semi - antigen OPH - 1 (structural formula shown in formula (Ⅰ)) with bovine serum albumin, that is, the immunogen is OPH - 1 - BSA (structural formula shown in formula (Ⅲ), where P is bovine serum albumin).

[0068] An immunoassay method for phenolphthalein substances, which uses any of the above - mentioned artificial antigen combinations of phenolphthalein substances for detection, and uses the antibody against the phenolphthalein substance as the detection antibody for detection; the immunoassay method is for non - disease treatment diagnosis purposes, and the phenolphthalein substance is one or more of phenolphthalein, bisacodyl, and bis - p - propylphenolphthalein.

[0069] Preferably, in the artificial antigen combination of the phenolphthalein substance, the coating antigen is obtained by conjugating the phenolphthalein semi - antigen OPH - 2 (structural formula shown in formula (Ⅱ)) with chicken ovalbumin; the antibody against the phenolphthalein substance is prepared by immunizing an animal with the immunogen obtained by conjugating the phenolphthalein semi - antigen OPH - 1 (structural formula shown in formula (Ⅰ)) with bovine serum albumin.

[0070] The application of the above - mentioned artificial antigen combination of phenolphthalein substances in the preparation of reagents for detecting phenolphthalein substances should also be within the protection scope of the present invention, and the phenolphthalein substance is one or more of phenolphthalein, bisacodyl, and bis - p - propylphenolphthalein.

[0071] Preferably, the reagent is used to identify phenolphthalein substances in weight - loss foods.

[0072] 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, etc.

[0073] A kit for detecting phenolphthalein substances, which contains any of the above - mentioned artificial antigen combinations of phenolphthalein substances, and the phenolphthalein substance is one or more of phenolphthalein, bisacodyl, and bis - p - propylphenolphthalein.

[0074] Preferably, the coating antigen is obtained by conjugating the phenolphthalein semi - antigen OPH - 2 (structural formula shown in formula (Ⅱ)) with chicken ovalbumin; the immunogen is obtained by conjugating the phenolphthalein semi - antigen OPH - 1 (structural formula shown in formula (Ⅰ)) with bovine serum albumin.

[0075] More preferably, the kit contains an antibody, and the antibody is obtained by immunizing an animal with the immunogen obtained by conjugating the phenolphthalein semi - antigen OPH - 1 (structural formula shown in formula (Ⅰ)) with bovine serum albumin.

[0076] Preferably, the kit further contains an enzyme - labeled plate, standards of phenolphthalein, bisacodyl, and bis - p - propylphenolphthalein, and a substrate chromogenic solution.

[0077] More preferably, the enzyme-linked immunosorbent assay (ELISA) plate is coated with a coating antigen obtained by conjugating the semi-hapten OPH-2 of the phenolphthalein substance (the structural formula is shown in formula (II)) with chicken ovalbumin.

[0078] More preferably, the substrate chromogenic solution contains urea peroxide and tetramethylbenzidine.

[0079] More preferably, the kit further comprises a termination solution, a washing solution, a blocking solution, an enzyme conjugate concentrate, and an enzyme conjugate diluent.

[0080] Further preferably, the termination solution is 1 mol / L to 3 mol / L H2SO4.

[0081] Most preferably, the termination solution is 2 mol / L H2SO4.

[0082] Further preferably, the washing solution is a 0.1 mol / L to 0.3 mol / L phosphate buffer solution containing 0.5% w / v to 1.0% w / v Tween-20 and 0.01% w / v to 0.03% w / v sodium azide, with a pH of 7.2 to 7.6.

[0083] Most preferably, the washing solution is a 0.2 mol / L phosphate buffer solution containing 0.8% w / v Tween-20 and 0.02% w / v sodium azide, with a pH of 7.4.

[0084] Further preferably, the blocking solution is a 0.1 mol / L to 0.3 mol / L phosphate buffer solution containing 1% w / v to 3% w / v casein, with a pH of 7.1 to 7.5.

[0085] [[ID=2)4]]Most preferably, the blocking solution is a 0.2 mol / L phosphate buffer solution containing 2% w / v casein, with a pH of 7.3.

[0086] Further preferably, the enzyme conjugate concentrate is a horseradish peroxidase-labeled goat anti-rabbit antibody or a goat anti-mouse antibody.

[0087] Further preferably, the enzyme conjugate diluent is a 0.1 mol / L to 0.3 mol / L phosphate buffer solution.

[0088] Most preferably, the enzyme conjugate diluent is a 0.2 mol / L phosphate buffer solution.

[0089] Preferably, the kit further comprises an immunochromatographic test strip. The immunochromatographic test strip 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 artificial antigen combination of the phenolphthalein substance, and the quality control area is coated with IgG.

[0090] Preferably, the artificial antigen of phenolic substances is obtained by coupling the hapten OPH-2 of phenolic substances (the structural formula of which is shown in formula (II)) with chicken ovalbumin.

[0091] Preferably, the IgG is goat anti-rabbit IgG or goat anti-mouse IgG.

[0092] The method for using the colloidal gold rapid detection test strip for phenolic substances is as follows:

[0093] The sample to be tested is mixed evenly with the colloidal gold-labeled antibody, and incubated at 25° C. for 5 minutes. The antibody is a polyclonal antibody or a monoclonal antibody. The sample pad 2 of the colloidal gold rapid detection test strip for phenolic substances is placed in the mixed solution for 3 minutes, and then the sample pad 2 is removed and the result is determined.

[0094] The specific determination method is:

[0095] 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 phenols, and the result is positive or weakly positive; if the test line shows red, it means that the sample to be tested does not contain phenols, and the result is negative.

[0096] Preferably, the kit is used to identify phenolic substances in weight-loss foods, and the phenolic substances are one or more of phenolic substances, diacetylbutamol and propylbutamol.

[0097] The weight-loss food involved in the present invention is a food with weight-loss function, including but not limited to compressed candies, beverages, fruit and vegetable powders, substitute teas, candied fruits, blended alcoholic beverages, fruit wines and jellies, etc. with weight-loss function.

[0098] Compared with the prior art, the present invention has the following beneficial effects:

[0099] The present invention provides a universal hapten for the simultaneous detection of phenolic substances in weight-loss foods, and prepares phenolic substances antibodies with high titer, strong specificity, and high affinity, providing core raw materials for the establishment of immunoassay methods for phenolic substances. The present invention also establishes a highly specific and sensitive immunoassay method for the detection of phenolic substances, wherein the minimum detection limit LOD for phenolic substances is 0.08 ng / mL and the half inhibitory concentration IC is 0.08 ng / mL. 50 The minimum detection limit LOD of diacetyl is 0.44 ng / mL, and the half inhibition concentration IC 50 The minimum detection limit LOD of bifenthrin was 1.23 ng / mL, and the half inhibition concentration IC 50It is 12.11 ng / mL. This method has no cross-reaction with functional analogues of phenolphthalein butyl, bisacodyl butyl and bisphenol butyl, and can rapidly conduct qualitative and quantitative detection of phenolphthalein butyl substances in weight-loss foods. The operation is simple, and the detection results are accurate and reliable. Description of the Drawings

[0100] Figure 1 It is a synthetic route diagram of haptens OPH-1 and OPH-2 of phenolphthalein butyl substances.

[0101] Figure 2 It is an ultraviolet scanning identification result diagram of artificial antigens OPH-1-OVA and OPH-1-BSA of phenolphthalein butyl substances.

[0102] Figure 3 It is an ultraviolet scanning identification result diagram of artificial antigens OPH-2-OVA and OPH-2-BSA of phenolphthalein butyl substances.

[0103] Figure 4 It is an indirect competitive ELISA standard curve of monoclonal antibodies of phenolphthalein butyl substances.

[0104] Figure 5 It is a schematic structural diagram of a colloidal gold rapid detection test strip for phenolphthalein butyl substances in Example 8 of the present application; it includes: a PVC plastic bottom plate, a sample pad, an NC membrane, a water absorption pad, a test line, and a control line.

[0105] Figure 6 It is a result determination diagram of a colloidal gold rapid detection test strip for phenolphthalein butyl substances in Example 8 of the present application; where: A is the detection result of a valid negative sample, B is the detection result of a valid positive sample, and C and D are invalid detection results. Detailed Embodiments

[0106] The present invention will be further elaborated in detail below in conjunction with the drawings in 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 all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0107] Example 1 Synthesis and Identification of Haptens of Phenolphthalein Butyl Substances

[0108] I. Synthesis and Identification of Hapten OPH-1 of Phenolphthalein Butyl Substances

[0109] 1. Experimental Method

[0110] The synthetic route of hapten OPH-1 of phenolphthalein butyl substances is as Figure 1 shown, and the specific steps are as follows:

[0111] Dissolve 1 mol of phenolphthalein butyl ether in 8 mL of methanol, then add 2 mol of ethyl bromoacetate and 6 mol of potassium carbonate, and stir at room temperature (25 °C) overnight (11 h) to obtain a reactant; use silica gel powder as the stationary phase and a mixed solution with a volume ratio of dichloromethane:methanol = 80:1 as the mobile phase, and separate and purify the reactant by column chromatography. 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, adjust the pH to 6 - 7, and the obtained precipitate is the hapten OPH-1 of phenolphthalein butyl ether substances.

[0112] Perform nuclear magnetic resonance and mass spectrometry identification on the hapten OPH-1.

[0113] 2. Experimental results

[0114] The results of the 1H nuclear magnetic resonance spectrum of the hapten OPH-1 of phenolphthalein butyl ether substances are as follows: 1 H NMR(600MHz,Methanol-d4)δ7.12(td,J=7.7,1.2Hz,1H),7.05 - 7.01(m,3H),6.94 - 6.90(m,3H),6.87(dt,J=7.8,0.8Hz,1H),6.75(d,J=8.4Hz,2H),6.59(d,J=8.3Hz,2H),4.51(s,2H).

[0115] The results of the mass spectrometry of the hapten OPH-1 of phenolphthalein butyl ether substances are as follows: MS: C 22 H 17 NO5: 375.38, ESI+[M + H]+: 376.36.

[0116] It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results can correspond to the molecular weight of OPH-1 and the 1H nuclear magnetic resonance spectrum numbers can correspond to the 1H nuclear magnetic resonance spectrum numbers on the structure of OPH-1, indicating that the hapten OPH-1 of phenolphthalein butyl ether substances has been successfully prepared, and its structural formula is shown in Formula (Ⅰ).

[0117]

[0118] The hapten OPH-1 of phenolphthalein butyl ether substances is named by the systematic nomenclature as: 2-(4-(3-(4-hydroxyphenyl)-2-oxoindolin-3-yl)phenoxy)acetic acid; that is, 2-(4-(3-(4-hydroxyphenyl)-2-oxoindolin-3-yl)phenoxy)acetic acid.

[0119] II. Synthesis and identification of the hapten OPH-2 of phenolphthalein butyl ether substances

[0120] 1. Experimental method

[0121] Dissolve 1 mol of phenolphthalein butyl ether in 8 mL of methanol, then add 2 mol of methyl 4-(bromomethyl)benzoate and 6 mol of potassium carbonate, and stir at room temperature (25 °C) overnight (11 h) to obtain the reactant; use silica gel powder as the stationary phase and a mixed solution with a volume ratio of dichloromethane:methanol = 80:1 as the mobile phase, and separate and purify the reactant by column chromatography. 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, adjust the pH to 6-7, and the resulting precipitate is the hapten OPH-2 of phenolphthalein butyl ether substances.

[0122] Perform nuclear magnetic resonance and mass spectrometry identification on the hapten OPH-2.

[0123] 2. Experimental results

[0124] The results of the 1H nuclear magnetic resonance spectrum of the hapten OPH-2 of phenolphthalein butyl ether substances are as follows: 1 H NMR(600MHz,Methanol-d4)δ7.90(d,J=7.9Hz,2H),7.39(d,J=7.8Hz,2H),7.11(td,J=7.7,1.2Hz,1H),7.05-7.01(m,3H),6.94-6.89(m,3H),6.87(dt,J=7.8,0.8Hz,1H),6.79(d,J=8.4Hz,2H),6.59(d,J=8.3Hz,2H),4.98(s,2H).

[0125] The results of the mass spectrometry of the hapten OPH-2 of phenolphthalein butyl ether substances are as follows: MS: C 28 H 21 NO5: 451.48, ESI+[M+H]+: 452.47.

[0126] It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results can correspond to the molecular weight of OPH-2 and the hydrogen spectrum numbers of the nuclear magnetic resonance can correspond to the hydrogen spectrum numbers on the structure of OPH-2, indicating that the hapten OPH-2 of phenolphthalein butyl ether substances has been successfully prepared, and its structural formula is shown in Formula (Ⅱ).

[0127]

[0128] The systematic nomenclature of the hapten OPH-2 of phenolphthalein butyl ether substances is: 4-((4-(3-(4-hydroxyphenyl)-2-oxoindolin-3-yl)phenoxy)methyl)benzoic acid; that is, 4-((4-(3-(4-hydroxyphenyl)-2-oxoindolin-3-yl)phenoxy)methyl)benzoic acid.

[0129] Example 2 Synthesis and Identification of Artificial Antigens of Phenolphthalein Butyl Ether Substances

[0130] I. Synthesis and Identification of Artificial Antigens OPH-1-OVA and OPH-1-BSA of Phenolphthalein Substances

[0131] Synthesize an artificial antigen with the general structural formula shown in Formula (Ⅲ),

[0132]

[0133] where P is bovine serum albumin (BSA) or chicken ovalbumin (OVA).

[0134] 1. Experimental Method

[0135] Couple the hapten OPH-1 of the phenolphthalein substance prepared in Example 1 (the structural formula is shown in Formula (Ⅰ)) with chicken ovalbumin (OVA) and bovine serum albumin (BSA) respectively by the active ester method.

[0136] The preparation method of OPH-1-OVA is as follows:

[0137] Dissolve 10 mg of the hapten OPH-1 of the phenolphthalein substance in Example 1 (the structural formula is shown in Formula (Ⅰ)) 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.

[0138] 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 used PBS buffer is: 2.90 g of Na2HPO4·12H2O, 8.50 g of NaCl, 0.20 g of KCl, 0.20 g of KH2PO4, and make up to 1000 mL with distilled water.

[0139] 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 artificial antigen OPH-1-OVA of the phenolphthalein substance, aliquot it into centrifuge tubes, and store it at -20 °C for use.

[0140] The preparation method of OPH-1-BSA is similar to the synthesis method of the artificial antigen OPH-1-OVA of the above phenolphthalein substance, except that BSA is used instead of OVA, and the hapten OPH-1 of the phenolphthalein substance prepared in Example 1 (the structural formula is shown in Formula (Ⅰ)) is used to synthesize the artificial antigen OPH-1-BSA of the phenolphthalein substance.

[0141] Perform ultraviolet scanning determination (200 - 400 nm) on OPH-1-OVA and OPH-1-BSA respectively.

[0142] 2. Experimental Results

[0143] The ultraviolet scanning determination results of OPH-1-OVA are as shown in Figure 2 a in the figure. Compared with the ultraviolet absorption peak of the hapten OPH-1 (structural formula as shown in formula (Ⅰ)), the ultraviolet absorption peak of the artificial antigen OPH-1-OVA of phenolphthalein substances has a significant shift, and the artificial antigen OPH-1-OVA of phenolphthalein substances simultaneously has the characteristic absorption peaks of the hapten OPH-1 (structural formula as shown in formula (Ⅰ)) and OVA, indicating that the artificial antigen OPH-1-OVA of phenolphthalein substances (structural formula as shown in formula (Ⅲ), where P is chicken ovalbumin) is successfully conjugated.

[0144] The ultraviolet scanning determination results of OPH-1-BSA are as shown in Figure 2 b in the figure. Compared with the ultraviolet absorption peak of the hapten OPH-1 (structural formula as shown in formula (Ⅰ)), the ultraviolet absorption peak of the artificial antigen OPH-1-BSA of phenolphthalein substances has a significant shift, and the artificial antigen OPH-1-BSA of phenolphthalein substances simultaneously has the characteristic absorption peaks of the hapten OPH-1 (structural formula as shown in formula (Ⅰ)) and BSA, indicating that the artificial antigen OPH-1-BSA of phenolphthalein substances (structural formula as shown in formula (Ⅲ), where P is bovine serum albumin) is successfully conjugated.

[0145] II.Synthesis and Identification of Artificial Antigens OPH-2-OVA and OPH-2-BSA of Phenolphthalein Substances

[0146] Synthesize an artificial antigen with the general structural formula as shown in formula (Ⅳ),

[0147]

[0148] where P is bovine serum albumin (BSA) or chicken ovalbumin (OVA).

[0149] 1. Experimental Method

[0150] The preparation method of OPH-2-OVA is similar to the synthesis method of the artificial antigen OPH-1-OVA of the above phenolphthalein substances, with the only difference being that the hapten OPH-2 of the phenolphthalein substance (structural formula as shown in formula (Ⅱ)) is used instead of the hapten OPH-1 of the phenolphthalein substance (structural formula as shown in formula (Ⅰ)) to prepare the artificial antigen OPH-2-OVA of the phenolphthalein substance.

[0151] The preparation method of OPH-2-BSA is similar to the synthesis method of the above-mentioned phenolic butyric acid artificial antigen OPH-2-OVA. The difference is that BSA is used instead of OVA, and the hapten OPH-2 of phenolic butyric acid prepared in Example 1 (the structural formula is shown in Formula (Ⅱ)) is used to synthesize the phenolic butyric acid artificial antigen OPH-2-BSA.

[0152] UV scanning determinations (200~400nm) were performed on OPH-2-OVA and OPH-2-BSA respectively.

[0153] 2. Experimental results

[0154] The UV scanning determination result of OPH-2-OVA is as shown in Figure 3 a in. Compared with the UV absorption peak of the hapten OPH-2 (the structural formula is shown in Formula (Ⅱ)), the UV absorption peak of the phenolic butyric acid artificial antigen OPH-2-OVA has a significant shift, and the phenolic butyric acid artificial antigen OPH-2-OVA simultaneously has the characteristic absorption peaks of the hapten OPH-2 (the structural formula is shown in Formula (Ⅱ)) and OVA, indicating that the phenolic butyric acid artificial antigen OPH-2-OVA (the structural formula is shown in Formula (Ⅳ), where P is chicken ovalbumin) is successfully conjugated.

[0155] The UV scanning determination result of OPH-2-BSA is as shown in Figure 3 b in. Compared with the UV absorption peak of the hapten OPH-2 (the structural formula is shown in Formula (Ⅱ)), the UV absorption peak of the phenolic butyric acid artificial antigen OPH-2-BSA has a significant shift, and the phenolic butyric acid artificial antigen OPH-2-BSA simultaneously has the characteristic absorption peaks of the hapten OPH-2 (the structural formula is shown in Formula (Ⅱ)) and BSA, indicating that the phenolic butyric acid artificial antigen OPH-2-BSA (the structural formula is shown in Formula (Ⅳ), where P is bovine serum albumin) is successfully conjugated.

[0156] Example 3 is used for the preparation of antibodies for simultaneously detecting phenolic butyric acid substances

[0157] 1. Preparation of polyclonal antibodies

[0158] The artificial antigens OPH-1-BSA (structural formula shown in formula (Ⅲ), where P is bovine serum albumin) and OPH-2-BSA (structural formula shown in formula (Ⅳ), where P is bovine serum albumin) of phenolphthalein substances prepared in Example 2 were used as immunogens, and emulsified uniformly with an immune adjuvant (incomplete Freund's adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunizations) at a volume ratio of 1:1 to immunize New Zealand white rabbits weighing 2.5 - 3 kg. The rabbits were immunized by multiple subcutaneous injections in the neck and back. The second immunization was carried out 4 weeks later, and subsequent booster immunizations were carried out every 3 weeks. One week after the third booster immunization, blood was collected from the marginal ear vein, and the serum titer was measured using indirect competitive ELISA.

[0159] When the titer no longer increased, the rabbits were boosted by intravenous injection into the marginal ear vein. One week later, the rabbits were bled from the heart. The collected blood was incubated at 37°C for 0.5 h, then left to stand overnight at 4°C. Then, the precipitated serum was aspirated with a pipette, and then centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was taken to obtain the antiserum. The antiserum was purified by ammonium sulfate precipitation method to obtain the polyclonal antibody, which was stored at -20°C for later use.

[0160] 2. Preparation of monoclonal antibody

[0161] The artificial antigens OPH-1-BSA (structural formula shown in formula (Ⅲ), where P is bovine serum albumin) and OPH-2-BSA (structural formula shown in formula (Ⅳ), where P is bovine serum albumin) of phenolphthalein substances prepared in Example 2 were used as immunogens, and emulsified uniformly with an immune adjuvant (complete Freund's adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunizations) at a volume ratio of 1:1 for immunizing female Balb / c mice.

[0162] The Balb / c mice were immunized by multiple subcutaneous injections in the abdomen. One week after each booster immunization, the venous blood of the mouse tail was collected to detect the serum titer. After the antibody titer no longer increased, another booster immunization was carried out. Seven days later, the spleen cells of the mice were fused with mouse myeloma cells to obtain fused cells.

[0163] 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 one week, detection, well selection, and recloning were carried out again. After 3 times of cloned culture and detection, the hybridoma cells producing monoclonal antibody were obtained. After the hybridoma cells were amplified and cultured, they were inoculated into the abdominal cavity of mice to produce ascites containing antibodies. The ascites were purified by the octanoic acid-ammonium sulfate precipitation method to obtain monoclonal antibodies, which were stored at -20°C for later use.

[0164] Combination of Immunogen and Coating Antigen in Example 4

[0165] I. Experimental Method

[0166] Using the artificial antigens OPH-1-OVA (the structural formula is shown in Formula (III), where P is chicken ovalbumin) and OPH-2-OVA (the structural formula is shown in Formula (IV), where P is chicken ovalbumin) prepared in Example 2 as coating antigens, and using the antiserum prepared in Example 3 with OPH-1-BSA (the structural formula is shown in Formula (III), where P is bovine serum albumin) and OPH-2-BSA (the structural formula is shown in Formula (IV), where P is bovine serum albumin) as immunogens, 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:

[0167] 1. Dilute the coating antigens OPH-1-OVA (the structural formula is shown in Formula (III), where P is chicken ovalbumin) and OPH-2-OVA (the structural formula is shown in Formula (IV), where P is chicken ovalbumin) to 1 μg / mL with coating buffer (0.05 M carbonate buffer solution, pH 9.6), and coat a 96-well enzyme-linked immunosorbent assay (ELISA) plate at an addition amount of 100 μL / well, incubate in a 37 °C constant temperature water bath for 12 h, discard the coating buffer, and wash 2 times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v));

[0168] 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 an oven at 37 °C for standby;

[0169] 3. Dilute the antiserum obtained from immunized female Balb / c mice to 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000 with PBST, and at the same time set a blank control well (replaced with PBST); dilute the standards of phenolphthalein, bisacodyl, and bisphenol A butyrate at 1 mg / mL 1000-fold to a concentration of 1 μg / mL with PBST to obtain the diluted solutions of phenolphthalein, bisacodyl, and bisphenol A butyrate standards;

[0170] 4. Setting of titer column: First add 50 μL of PBST to each well, then add 50 μL of antiserum at different dilution ratios to each well, and add 50 μL of PBST to the last well instead of antiserum;

[0171] 5. Setting of inhibition column: First add 50 μL of the diluted solutions of phenolphthalein, bisacodyl, and bisphenol A butyrate standards to each well, then add 50 μL of antiserum at different dilution ratios to each well, and add 50 μL of PBST to the last well instead of antiserum;

[0172] 6. Incubate at 37°C for 40 min and wash 5 times;

[0173] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000-fold with PBST), incubate at 37°C for 30 min and wash 5 times;

[0174] 8. Add the chromogenic solution, incubate at 37°C for color development for 10 min. The chromogenic solution is obtained by uniformly mixing urea peroxide and tetramethylbenzidine in a volume ratio of 1:1;

[0175] 9. Add 10% v / v H2SO4 to terminate the reaction and read the OD value at 450 nm; Calculate the titer and inhibition rate. The titer is the dilution factor of the antiserum corresponding to an OD of about 1.0, and the inhibition rate = (OD value of the titer - OD value of the inhibition) / OD value of the inhibition × 100%. 450 [[ID=URL]]The URL for this translated text is: [URL] The titer is the dilution factor of the antiserum corresponding to an OD of about 1.0, and the inhibition rate = (OD value of the titer - OD value of the inhibition) / OD value of the inhibition × 100%.

[0176] II. Experimental Results

[0177] The ELISA detection results of different combinations of immunogens and coating antigens are shown in Table 1.

[0178] Table 1 ELISA Detection Results of Immunogens and Coating Antigens

[0179]

[0180] a Indicates the inhibition rates of mouse antiserum against phenolphthalein butyrate, bisacodyl butyrate, and bispropanol butyrate respectively.

[0181] As can be seen from Table 1, the artificial antigens OPH-1-BSA (structural formula as shown in formula (III), where P is bovine serum albumin) and OPH-2-BSA (structural formula as shown in formula (IV), where P is bovine serum albumin) of phenolphthalein substances as immunogens for female Balb / c mice produced antiserum with different degrees of inhibitory effects on the target analytes phenolphthalein butyrate, bisacodyl butyrate, and bispropanol butyrate. Among them, when using the combination of immunogen OPH-1-BSA (structural formula as shown in formula (III), where P is bovine serum albumin) and coating antigen OPH-2-OVA (structural formula as shown in formula (IV), where P is chicken ovalbumin), the inhibition rates for phenolphthalein butyrate, bisacodyl butyrate, and bispropanol butyrate are the highest, and it can be used to simultaneously identify the target analytes phenolphthalein butyrate, bisacodyl butyrate, and bispropanol butyrate. Therefore, OPH-1-BSA (structural formula as shown in formula (III), where P is bovine serum albumin) is used as the best immunogen, and OPH-2-OVA (structural formula as shown in formula (IV), where P is chicken ovalbumin) is used as the best coating antigen.

[0182] Example 5 Establishment of an Indirect Competitive ELISA Detection Method for Phenolphthalein Substances

[0183] I. Experimental Method

[0184] An indirect competitive ELISA detection method for phenolphthalein substances, comprising the following steps:

[0185] 1. Use the artificial antigen OPH-2-OVA of phenolphthalein substances prepared in Example 2 (the structural formula is shown in Formula (Ⅳ), where P is chicken ovalbumin) as the coating antigen, and dilute it to 250 ng / mL with the coating solution (0.05 M carbonate buffer solution, pH 9.6); coat a 96-well enzyme-linked immunosorbent assay (ELISA) plate at an addition amount of 100 μL / well, and incubate overnight (12 h) at 37 °C;

[0186] 2. Discard the coating solution, wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), and pat dry;

[0187] 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;

[0188] 4. Discard the blocking solution, take it out after drying at 37 °C for 30 min, and store it in a self-sealing bag for later use;

[0189] 5. Dilute the monoclonal antibody prepared in Example 3 with PBST at a volume ratio of 1:4000, and dilute the standards of phenolphthalein, bisacodyl, and bispropanolol 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 respectively;

[0190] 6. Add 50 μL / well of diluted standards of phenolphthalein, bisacodyl, and bispropanolol at different concentrations (in triplicate), then add 50 μL / well of the diluted monoclonal antibody, incubate at 37 °C for 40 min, and wash five times;

[0191] 7. Add 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37 °C for 30 min, and wash five times;

[0192] 8. 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;

[0193] 9. Add 50 μL of 10% (v / v) H2SO4 solution to terminate the reaction, and read the OD value at 450 nm.

[0194] 10. ELISA standard curve drawing: Using B / B0 as the ordinate (B is the absorbance OD of phenolphthalein butyl, bisacodyl butyl, and bispropanol butyl standard products at different concentrations 450 , B0 is the absorbance OD of the blank control well 450 ), and the logarithm of the standard product concentration as the abscissa, the curve is fitted using the Logistic function to obtain the formula of the standard curve and the standard curve is prepared.

[0195] II. Experimental results

[0196] The indirect competitive ELISA standard curve of the antibody used to detect phenolphthalein butyl substances is as Figure 4 shown. The half-inhibitory concentration (IC 50 ) of phenolphthalein butyl is 0.40 ng / mL, and the lowest detection limit (LOD) is 0.08 ng / mL; the half-inhibitory concentration (IC 50 ) of bisacodyl butyl is 2.88 ng / mL, and the lowest detection limit (LOD) is 0.44 ng / mL; the half-inhibitory concentration (IC 50 ) of bispropanol butyl is 12.11 ng / mL, and the lowest detection limit (LOD) is 1.23 ng / mL; it shows that the antibody prepared by the present invention for simultaneously recognizing phenolphthalein butyl substances can meet the detection requirements, and the indirect competitive ELISA detection method for phenolphthalein butyl substances established by the present invention has high recognition ability and high sensitivity for phenolphthalein butyl substances.

[0197] Example 6 Specificity of the antibody for simultaneously detecting phenolphthalein butyl substances

[0198] I. Experimental method

[0199] Emodin, aloe-emodin, rhein, sodium picosulfate, and bisacodyl are all functional analogs of phenolphthalein butyl substances and are often added to weight loss products.

[0200] In this example, a cross-reaction experiment is used to determine the specificity of the antibody prepared by the present invention for detecting phenolphthalein butyl substances.

[0201] According to the method of Example 5, the difference is only that: the phenolphthalein butyl, bisacodyl butyl, and bispropanol butyl standard products are replaced with standard products 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 detected at the same dilution multiple to obtain the IC 50 values of each functional analog.

[0202] Calculate the cross-reaction rate (CR) according to the following formula: CR(%) = IC 50(Phendimetrazine) / IC 50 (Functional analog) × 100%, the smaller the cross-reactivity rate, the stronger the specificity.

[0203] II. Experimental Results

[0204] Table 2 Cross-reactivity results of monoclonal antibodies against phendimetrazine substances with phendimetrazine substances and their functional analogs

[0205]

[0206]

[0207] Note: NR indicates no reaction, that is, the antibody does not recognize the functional analog.

[0208] As can be seen from Table 2, the cross-reactivity rates of the monoclonal antibody used to detect phendimetrazine substances with phendimetrazine, bisacodyl acetate, and bisacodyl propionate are 100%, 13.89%, and 3.3% respectively, and the IC 50 are 0.4 ng / mL, 2.88 ng / mL, and 12.11 ng / mL respectively, and there is no cross-reactivity with the functional analogs of phendimetrazine substances such as emodin, aloe-emodin, rhein, sodium picosulfate, and bisacodyl; it shows that the antibody used to detect phendimetrazine substances has high recognition ability and strong specificity for phendimetrazine, bisacodyl acetate, and bisacodyl propionate, can effectively exclude the interference of the detection of functional analogs emodin, aloe-emodin, rhein, sodium picosulfate, and bisacodyl, and can be specifically used for the detection of phendimetrazine substances.

[0209] Example 7 An ELISA kit for simultaneously detecting phendimetrazine substances

[0210] 1. Composition

[0211] (1) Microtiter plate coated with coating antigen:

[0212] The microtiter plate is prepared by the following method:

[0213] Using the artificial antigen OPH-2-OVA of phendimetrazine substances prepared in Example 2 (the structural formula is shown in Formula (IV), where P is chicken ovalbumin) as the coating antigen, diluting it to 250 ng / mL with the coating stock solution (0.05 M carbonate buffer solution, pH 9.6), coating a 96-well microtiter plate at an addition amount of 100 μL / well, incubating overnight at 37°C in the dark; pouring out the liquid in the wells, washing 2 times with the washing solution in this kit, 30 s each time, and patting dry; then adding the blocking solution in this kit at 200 μL / well, incubating at 25°C in the dark for 2 h; pouring out the liquid in the wells and patting dry, and storing it in a vacuum-sealed manner with an aluminum film after drying;

[0214] (2) Standard substances: Ten standard substances of phenolphthalein butyl ester, bisacodyl butyl ester, and bispropanolol butyl ester with different concentrations, which are 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 respectively;

[0215] (3) Antibody: The polyclonal antibody or monoclonal antibody prepared in Example 3 for detecting phenolphthalein butyl ester substances;

[0216] (4) Enzyme conjugate working solution: Goat anti-rabbit secondary antibody labeled with horseradish peroxidase or goat anti-mouse secondary antibody labeled with horseradish peroxidase;

[0217] (5) Substrate chromogenic solution: Composed of Solution A and Solution B. Solution A is urea peroxide, and Solution B is tetramethylbenzidine;

[0218] (6) Stop solution: 2 mol / L H2SO4;

[0219] (7) Washing solution: The pH value is 7.4, containing phosphate buffer solution with 0.8% (v / v) Tween-20, 0.02% (w / w) sodium azide preservative, and 0.2 mol / L; Dilute the washing solution 20 times with water before use (that is, add 19 parts of water to 1 part of the washing solution, prepare and use immediately), and the washing solution working solution can be obtained;

[0220] (8) Diluent: 0.2 mol / L phosphate buffer solution; Dilute the diluent 20 times with water before use (that is, add 19 parts of water to 1 part of the diluent, prepare and use immediately), and the diluent working solution can be obtained;

[0221] 2. Usage

[0222] (1) Sample detection

[0223] Number the corresponding micro wells of the samples and the standard substances of this kit in sequence. Make 2 parallel wells for each sample and standard substance, add them to the enzyme-linked immunosorbent assay (ELISA) plate coated with the coating antigen, and record the positions of the standard wells and sample wells. Dilute the antibody with the diluent according to a volume ratio of 1:40 as needed (that is, add 1 part of the antibody to 40 parts of the diluent, prepare and use immediately) to obtain the antibody working solution. Dilute the enzyme conjugate concentrate with the diluent according to a volume ratio of 1:10 as needed (that is, add 1 part of the enzyme conjugate concentrate to 10 parts of the diluent, prepare and use immediately) to obtain the enzyme conjugate working solution.

[0224] Add 50 μL of the standard substance or sample to the corresponding micro well, then add 50 μL of the antibody working solution 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.

[0225] Centrifuge the liquid in the wells dry, and add 250 μL of washing solution per well. Wash thoroughly 4 - 5 times, with a 10 - second interval each time. Discard the washing solution in the wells, and pat dry with absorbent paper (any remaining bubbles after pat - drying can be punctured with a new pipette tip).

[0226] Add 100 μL of enzyme - conjugate working solution per well to the corresponding micro - wells, gently mix by shaking, cover with a cover film, and incubate in the dark at 25 °C for 30 minutes.

[0227] Centrifuge the liquid in the wells dry, and add 250 μL of washing solution per well. Wash thoroughly 4 - 5 times, with a 10 - second interval each time. Discard the washing solution in the wells, and pat dry with absorbent paper (any remaining bubbles after pat - drying can be punctured with a new pipette tip).

[0228] Add 50 μL of substrate chromogenic solution A per well, then add 50 μL of substrate chromogenic solution B per well. Gently mix by shaking, cover with a cover film, and incubate in the dark at 25 °C for 10 minutes.

[0229] Add 50 μL of stop solution per well, gently mix by shaking, set the microplate reader at 450 nm, and measure the OD value of each well.

[0230] (2) Plotting the standard curve

[0231] Draw the ELISA standard curve according to the above - mentioned optimal conditions. Use B / B0 as the ordinate (B is the absorbance OD value when adding the standard, 450 B0 is the absorbance OD value when not adding the standard), 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 draw the standard curve.

[0232] (3) Calculating the sample concentration

[0233] Substitute the measured absorbance OD value of the sample 450 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 the phenolphthalein - related substances in the test sample.

[0234] Example 8 A colloidal - gold test strip for detecting phenolphthalein - related substances

[0235] 1. Assembly of the colloidal - gold rapid - detection test strip

[0236] As shown in Figure 5 , the colloidal - gold rapid - detection test strip is composed of an NC membrane (nitrocellulose membrane), a sample pad, an absorbent pad, and a PVC plastic bottom plate stacked together.

[0237] The coated original OPH-2-OVA (structural formula shown in Formula (IV), where P is chicken ovalbumin) was sprayed onto the NC membrane at a spray volume of 0.8 μL / cm using an XYZ three-dimensional spray dot film apparatus as a 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 as a 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.

[0238] 2. Preparation of Gold-labeled Antibodies

[0239] 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:

[0240] 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.

[0241] 3. Preparation of test sample solution

[0242] Compressed candies, beverages, fruit and vegetable powders, tea substitutes, candied fruits, blended alcoholic beverages, and fruit wines: 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). Ultrasonicate and extract for 5 minutes. Cool to room temperature. Centrifuge at 4000 rpm for 5 minutes. Pipette 1.0 mL of supernatant and dilute fourfold with 0.02 M PB. Vortex thoroughly, then filter through a microporous membrane to obtain the sample solution for testing.

[0243] Jelly: 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 min, centrifuge at 4000 rpm for 5 min, aspirate 1.0 mL of supernatant, dilute fourfold with 0.02 M PB, vortex evenly, and filter through a microporous filter membrane to obtain the sample solution for testing.

[0244] 4. Detection steps

[0245] Take 120 μL of the sample solution and mix it evenly with 5 μL of the gold-labeled antibody by repeated pipetting. Incubate at room temperature for 5 min. Then insert the test strip into the sample solution and react for 3 min. Then take out the test strip and remove the sample pad, and make the result determination 3 - 5 minutes later.

[0246] 5. Result determination of the detection

[0247] As Figure 6 shown, if the sample does not contain the analyte phenolphthalein substances, the gold-labeled antibody binds to the coating antigen on the T line (test line) of the rapid test strip, making the test line show a clear red line, indicating that the test sample is negative (such as Figure 6 A in); if the sample contains the analyte phenolphthalein substances, the phenolphthalein substances bind to the gold-labeled antibody and cannot be captured by the test line on the rapid test strip, and then the test line does not show color, indicating positive (such as Figure 6 B in); similarly, the gold-labeled antibody also binds to the goat anti-mouse IgG on the C line (control line) of the cellulose membrane, making the quality control line show red. The presence or absence of the color of the control line indicates the validity or invalidity of this test strip respectively (such as Figure 6 A and B in are valid, C and D in are invalid).

[0248] 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, based on the above description and ideas, other different forms of changes or modifications can also be made. 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-like substance hapten OPH-1, characterized in that, Its structural formula is shown in Formula (I), Formula (I), The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

2. A phenolphthalein semi - antigen OPH - 2, characterized in that, Its structural formula is shown in Formula (II), Formula (II), The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

3. Use of the semi - antigen of the phenolphthalein - type substance according to claim 1 or 2 in the preparation of an artificial antigen of the phenolphthalein - type substance, characterized in that, The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

4. An artificial antigen of phenolphthalein substances, characterized in that, It is obtained by coupling the hapten OPH-1 of the phenolphthalein substance described in Claim 1 with a carrier protein, and its structural formula is shown in Formula (III), Formula (III), wherein, P is the carrier protein; The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

5. An artificial antigen of phenolphthalein substances, characterized in that, It is obtained by coupling the hapten OPH-2 of the phenolphthalein substance described in Claim 2 with a carrier protein, and its structural formula is shown in Formula (IV), Formula (IV), wherein, P is the carrier protein; The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

6. Use of the phenolphthalein-like substance artificial antigen according to claim 4 or claim 5 in the preparation of an antibody against a phenolphthalein-like substance, characterized in that, The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

7. An artificial antigen combination of phenolphthalein substances, characterized in that, It contains an immunogen and a coating antigen. The immunogen is obtained by coupling the hapten of the phenolphthalein substance described in Claim 1 or Claim 2 with bovine serum albumin, and the coating antigen is obtained by coupling the hapten of the phenolphthalein substance described in Claim 1 or Claim 2 with chicken ovalbumin. The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

8. Use of the artificial antigen combination of phenolphthalein substances according to claim 7 in the preparation of a reagent for detecting phenolphthalein substances, characterized in that, The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

9. A kit for detecting phenolphthalein substances, characterized in that, It contains the artificial antigen combination of the phenolphthalein substance described in Claim 7. The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

10. An immunoassay method for detecting phenolphthalein substances, characterized in that, Detecting with the artificial antigen combination described in Claim 7. The phenolphthalein substances are one or more of phenolphthalein, bisacodyl, and bispropylphenolphthalein.

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