Preparation method and application of hapten, immunogen, coating antigen and antibody of 5-chloro-diacetoxine

Abstract

The invention relates to a preparation method and application of a hapten, an immunogen, a coating antigen and an antibody of 5-chloro diacetoxine, the hapten of 5-chloro diacetoxine is successfully synthesized by using 5-chloro diacetoxine as an initiator through a simple chemical method, and the immunogen hapten-BSA and the coating antigen hapten-OVA are further synthesized. The synthesized 5-chloro-diacetoxantine antigen can be specifically combined with an antibody, the cross reaction of diacetoxantine and other analogues is less than 0.2%, and the 5-chloro-diacetoxantine antigen is high in titer, strong in specificity and high in affinity; meanwhile, the invention establishes an immunoassay method of the 5-chloro-diacetoxantine antibody, the 5-chloro-diacetoxantine antibody is used for detecting 5-chloro-diacetoxantine, the 5-chloro-diacetoxantine antibody has high specificity and high sensitivity, the lowest limit of detection (LOD) is 1.07 ng / mL, and the half inhibitory concentration IC50 is 27.67 ng / mL. The method can be widely applied to detection of jellies, solid beverages, enzyme beverages, tablet candies and fruit vinegar beverages, especially can be used for rapidly, qualitatively and quantitatively detecting the phenol butyl substances (5-chloro-diacetobacter) in weight-losing foods, and is simple and convenient to operate and accurate and reliable in detection result.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food detection, and relates to immunology, in particular to a preparation method and application of a 5-chloro diacetyldiphenylheptane hapten, immunogen, coating antigen and antibody. BACKGROUND

[0002] Research has found that diacetyldiphenylheptane and its analogues contained in food may have a negative impact on human health, including but not limited to nausea, vomiting, intestinal colic and diarrhea, etc. Such compounds are generally polluted into food through industrial wastewater, waste gas and other ways, thereby endangering human health.

[0003] Currently reported detection methods for diacetyldiphenylheptane and its analogues include HPLC, liquid chromatography-mass spectrometry, isotope mass spectrum, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, etc. However, most of the above methods need to be combined with complex pretreatment processes, and have problems such as low sensitivity, long separation time, interference of matrix components, etc. Although GC and GC-MS methods have high sensitivity, they need a long derivatization process. In addition, the above methods need high cost, and also need to control precise experimental conditions. Therefore, it is urgent to develop a detection method with low cost, high sensitivity and more simple operation. The immunological analysis method derived from the specific binding principle of antigen-antibody has a broad prospect, and has the characteristics of high sensitivity, low cost and good specificity. SUMMARY

[0004] The present application aims to overcome the deficiencies of the prior art, and provides a preparation method and application of a 5-chloro diacetyldiphenylheptane hapten, immunogen, coating antigen and antibody. The 5-chloro diacetyldiphenylheptane hapten is successfully synthesized by a simple chemical method using 5-chloro diacetyldiphenylheptane as a starting material, and an immunogen hapten-BSA and a coating antigen hapten-OVA are further synthesized. The antibody of 5-chloro diacetyldiphenylheptane is finally prepared, which has high titer, strong specificity and high affinity. At the same time, an immunological analysis method of the 5-chloro diacetyldiphenylheptane antibody is established, which has high specificity and high sensitivity for detecting phenylbutazone substances.

[0005] The present application solves its technical problem by adopting the following technical solution: A preparation method of a 5-chloro diacetyldiphenylheptane hapten, comprising the following method steps: (1) 0.1 mmol of 43.58 mg of 5-chloro diacetyldiphenylheptane is dissolved in 5 mL of methanol; (2) 0.2 mmol of ethyl chloroacetate and 0.6 mmol of potassium carbonate are added, and the reaction is stirred at room temperature 25℃ for 12 hours; (3) 5 mL of 2 mol / L NaOH solution is added to the reaction system, and the stirring is continued at room temperature for 3 hours; (4) After the reaction is completed, HCl is added dropwise to adjust the pH to 6-7, and the solution is allowed to stand and separate into layers, and the upper organic phase is taken and washed and purified to obtain a clear organic phase; (5) The clear organic phase is dried under reduced pressure to obtain 5-chlorobutenolide hapten.

[0006] The 5-chlorobutenolide hapten has the following structural formula: .

[0007] A method for preparing a 5-chlorobutenolide immunogen: the 5-chlorobutenolide hapten prepared above is completely dissolved in methanol, and bovine serum albumin BAS (the ratio of hapten to BSA is hapten: BSA = 1:1-1.5) is added, and the mixture is stirred at 25°C for 18 hours, and the solution is collected and dialyzed in 0.01 mol / L phosphate buffer at pH 7.4 for 72 hours to obtain an immunogen hapten-BSA.

[0008] The 5-chlorobutenolide immunogen hapten-BSA has the following structural formula: .

[0009] A method for preparing a 5-chlorobutenolide coated antigen: the 5-chlorobutenolide hapten prepared above is completely dissolved in methanol, and OVA is added, and the addition ratio is hapten: OVA = 1:1-1:1.5, and the mixture is stirred at 25°C for 18 hours, and the solution is collected and dialyzed in 0.01 mol / L phosphate buffer at pH 7.4 for 72 hours to obtain a coated antigen hapten-OVA.

[0010] The 5-chlorobutenolide coated antigen hapten-OVA has the following structural formula: .

[0011] A method for preparing a 5-chlorobutenolide antibody, the specific method steps are as follows: (1) New Zealand white rabbits are selected as experimental animals, and the above immunogen hapten-BSA is used as an antigen; (2) Immunization is performed by subcutaneous injection, with an interval of 2 weeks between each injection, and a total of 6 immunizations are performed; (3) Complete Freund's adjuvant is used for initial immunization, and incomplete Freund's adjuvant is used for subsequent immunization; (4) After immunization, blood is collected and centrifuged to separate the antiserum at 4°C; (5) The antibody is purified by Protein A-Sepharose 4B affinity chromatography to obtain polyclonal IgG antibody.

[0012] An immunoassay method of 5-chlorodioctylphenol antibody, using indirect competitive ELISA or ic-ELISA mode, comprising the following steps: (1) fixing the coating antigen hapten-OVA on a 96-well microplate; (2) adding different concentrations of the sample to be tested and mixing with the antibody for incubation; (3) adding enzyme-labeled secondary antibody for color development and then measuring the absorbance at 450 nm; (4) calculating the inhibition rate and drawing a standard curve to realize quantitative detection of 5-chlorodioctylphenol, The minimum detection limit LOD of the above method is 1.07 ng / mL, and the half-inhibitory concentration IC 50 is 27.67 ng / mL, and the cross-reactivity of the antibody to dioctylphenol and other structural analogs is less than 0.2%.

[0013] The hapten, immunogen, coating antigen and antibody described above are applied to the detection of 5-chlorodioctylphenol in food samples, including jelly, solid beverage, enzyme beverage, tablet candy and fruit vinegar beverage.

[0014] The advantages and positive effects of the present application are: 1. The method of the present application successfully synthesizes 5-chlorodioctylphenol hapten by replacing the methyl connected to the ester group of the benzene ring on both sides of the 5-chlorodioctylphenol structure with a carboxyl group through a simple chemical method, and the whole preparation process is simple and does not require complex operations. The finally synthesized 5-chlorodioctylphenol antigen can specifically bind to the antibody, and the cross-reactivity to dioctylphenol and other structural analogs is less than 0.2%. 2. The method of the present application prepares the antibody of 5-chlorodioctylphenol substance based on the immunogen hapten-BSA, which has high titer, strong specificity and high affinity, and provides core materials for establishing an immunoassay method of 5-chlorodioctylphenol. The method establishes an immunoassay method of 5-chlorodioctylphenol antibody based on the coating antigen hapten-OVA, which has high specificity and high sensitivity for detecting phenol substances, and the minimum detection limit LOD is 1.07 ng / mL, and the half-inhibitory concentration IC 50 is 27.67 ng / mL.

[0015] 3. The method of the present application can be widely applied in the detection of jelly, solid beverage, enzyme beverage, tablet candy and fruit vinegar beverage, and can especially be used for rapid qualitative and quantitative detection of phenol substances in weight loss food, which is simple in operation and accurate and reliable in detection results. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1Synthetic route of 5-chlorobutenafine hapten in the method embodiment of the present application; Figure 2 Synthetic route of 5-chlorobutenafine hapten-BSA in the embodiment of the present application; Figure 3 Synthetic route of 5-chlorobutenafine hapten-OVA in the embodiment of the present application; Figure 4 Standard curve of 5-chlorobutenafine in the experimental embodiment of the method of the present application; Figure 5 Mass spectrum verification result of hapten in the experimental embodiment of the method of the present application; Figure 6 Verification result of ultraviolet spectrophotometer in the experimental embodiment of the method of the present application; Figure 7 Actual sample spiking detection and LC-MS method verification recovery rate result in the experimental embodiment of the method of the present application. DETAILED DESCRIPTION

[0017] The present application will be further described in detail by specific embodiments, the following examples are only descriptive, not limiting, and cannot limit the protection scope of the present application.

[0018] Example 1 A preparation method of 5-chlorobutenafine hapten, as shown in Figure 1 , comprising the following method steps: (1) 0.1 mmol of 43.58 mg of 5-chlorobutenafine A was dissolved in 5 mL of methanol; (2) 0.2 mmol of ethyl chloroacetate B and 0.6 mmol of potassium carbonate were added, and the reaction was stirred at room temperature 25℃ for 12 hours; (3) 5 mL of 2 mol / L NaOH solution was added to the reaction system, and the stirring was continued at room temperature for 3 hours; (4) After the reaction was completed, HCl was added dropwise to adjust the pH to 6-7, and the upper organic phase was taken after standing and layering, and the clear organic phase was obtained after washing and purification treatment; (5) The clear organic phase was dried under reduced pressure to obtain 5-chlorobutenafine hapten C.

[0019] The structure of the 5-chlorobutenafine hapten is as follows: .

[0020] Example 2 A preparation method of 5-chlorobutenafine hapten, as shown in Figure 2The 5-chlorobisantrene hapten-BSA immunogen is prepared as follows: the 5-chlorobisantrene hapten C prepared above is dissolved in methanol, and bovine serum albumin (BSA) is added (the ratio of hapten to BSA is 1:1-1.5), and the mixture is stirred at 25°C for 18 hours. The solution is collected and dialyzed in 0.01 mol / L phosphate buffer at pH 7.4 for 72 hours to obtain the immunogen hapten-BSA, which is D.

[0021] The 5-chlorobisantrene hapten-BSA immunogen is prepared as follows: the 5-chlorobisantrene hapten C prepared above is dissolved in methanol, and bovine serum albumin (BSA) is added (the ratio of hapten to BSA is 1:1-1.5), and the mixture is stirred at 25°C for 18 hours. The solution is collected and dialyzed in 0.01 mol / L phosphate buffer at pH 7.4 for 72 hours to obtain the immunogen hapten-BSA, which is D. .

[0022] Example 3 A method for preparing a 5-chlorobisantrene coating antigen is as follows: Figure 3 The 5-chlorobisantrene hapten-BSA immunogen is prepared as follows: the 5-chlorobisantrene hapten C prepared above is dissolved in methanol, and bovine serum albumin (BSA) is added (the ratio of hapten to BSA is 1:1-1.5), and the mixture is stirred at 25°C for 18 hours. The solution is collected and dialyzed in 0.01 mol / L phosphate buffer at pH 7.4 for 72 hours to obtain the immunogen hapten-BSA, which is D.

[0023] The 5-chlorobisantrene hapten-BSA immunogen is prepared as follows: the 5-chlorobisantrene hapten C prepared above is dissolved in methanol, and bovine serum albumin (BSA) is added (the ratio of hapten to BSA is 1:1-1.5), and the mixture is stirred at 25°C for 18 hours. The solution is collected and dialyzed in 0.01 mol / L phosphate buffer at pH 7.4 for 72 hours to obtain the immunogen hapten-BSA, which is D. .

[0024] Example 4 Construction of standard curve and evaluation of method specificity 1) The 96-well plate with a coating amount of 0.01, 0.05, and 0.1 μg per well is washed 2-3 times with 0.01 M PBS buffer at pH 7.4; 2) 50 μL of the sample to be tested at different dilution gradients (0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 200, 500, and 1000 μg / L) is added to each well, followed by the addition of 50-200 μL of the signal probe dissolved in PBS, and the mixture is shaken and incubated for 15 min; 3) The reaction solution in the microwells is removed, and each well is washed 2 times with 200-250 μL of PBS (0.01 M, pH 7.4) buffer, 200 μL of the color developing solution is added, and the mixture is color developed at 37°C for 5-10 min; 4) The signal value is read using a Thermo Multifunctional Microplate Reader, and data analysis is performed according to the concentration and inhibition rate value to construct a 5-chlorobisantrene immunoassay method; 5) The standard curve of 5-chlorobisantrene is as follows: Figure 4The detection range was 1.07-27.67 ng / mL, and the detection limit of the constructed immunoassay was 1.07 ng / mL.

[0025] Example 5 Preparation of antibodies The antibodies were prepared by using New Zealand white rabbits. The New Zealand white rabbits were subcutaneously immunized with the prepared immunogen (Hapten-BSA) for 6 times with an interval of 2 weeks. In the initial injection and subsequent booster immunization, equal volumes of immunogen were emulsified with Freund's complete adjuvant and incomplete adjuvant, respectively. After bleeding the rabbits, the collected whole blood was allowed to clot, and the antiserum was separated by centrifugation at 4°C. The antiserum was purified by Protein A-Sepharose 4B affinity column. The animal experiments were performed in accordance with the "Regulations for the Administration of Experimental Animals". Protein A-Sepharose 4B column affinity chromatography was used. The antibodies were purified by specific adsorption of IgG antibodies by Protein A-Sepharose 4B, and then the purified IgG antibodies were obtained by eluting the impure proteins with phosphate buffer, adsorbing IgG antibodies on the column, and then washing the Protein A-Sepharose 4B column with glycine solution.

[0026] Example 6 Mass spectrometry verification The prepared hapten was verified by Thermos TSQ Quantum Ultra, and the results are shown in Figure 5 The mass spectrometry results show that the molecular weight of the prepared hapten is 442.85, which is very close to the mass-to-charge ratio 441.04 corresponding to its structural formula in Chemdraw software. The slight error may be due to the fact that the chlorine atom has two isotopes with atomic weights of 34.97 and 36.97, and the natural abundance is close to 3:1, so there will be two molecular ion peaks of 155.05 and 157.05 in the mass spectrum, and the intensity ratio is close to 3:1.

[0027] Example 7 Ultraviolet spectrophotometer verification The ultraviolet spectrophotometer was used to measure the absorbance spectrum of BSA, 5-chlorobisacodyl, hapten and immunogen, and the results are shown in Figure 6 BSA itself has a protein peak (tryptophan / tyrosine) at 280 nm, and occasionally has Rayleigh scattering at 310 nm. After coupling the hapten with BSA, the absorbance value in the 220-280 nm region is significantly enhanced (peak value exceeds 4000), which proves that the hapten is successfully coupled with BSA.

[0028] Example 8 Specificity evaluation results The method specificity was evaluated by using structural analogs or functional analogs of diacetyldiphenylhydramine, bisphenol sandine acetate, bisphenol TMC, 3,3-bis(4-hydroxyphenyl)-2(3H)-indolone, phenolphthalein, thymolphthalein, bisphenol fluorene, orlistat, sennoside B, α-naphthol, and the like, and the results showed that the cross-reactivity rates were all less than 0.2%, and the results are shown in Table 1.

[0029] Table 1. Cross-reactivity detection results (n=3)

[0030] Example 9 Actual sample spiked detection According to the constructed immunoassay method, the 5-chlorodihydroxyphenylpropene in five types of commercially available weight loss products including zero calorie jelly, solid beverage, enzyme beverage, tablet candy, and fruit vinegar beverage was detected by spiking, and the method was verified by LC-MS, and the recovery rate results of the two methods are shown in Figure 7 .

[0031] Although the embodiments of the present application are disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, therefore, the scope of the present application is not limited to the disclosed content of the embodiments.

Claims

1. A 5-chlorodioctylphenol hapten, characterized in that: The structural formula is as follows: 。 2. A process for the preparation of a 5-chlorodiosephenyline hapten, characterized by: The method steps include the following: (1) 0.1 mmol of 43.58 mg of 5-chlorobisantrene is dissolved in 5 mL of methanol; (2) 0.2 mmol of ethyl chloroacetate and 0.6 mmol of potassium carbonate are added, and the reaction is stirred at room temperature of 25 DEG C for 12 hours; (3) 5 mL of 2 mol / L NaOH solution is added to the reaction system, and the stirring is continued at room temperature for 3 hours; (4) After the reaction is completed, HCl is added dropwise to adjust the pH to 6-7, and the upper organic phase is taken after standing and layering, and the clear organic phase is obtained after washing and purification treatment; (5) The clear organic phase is dried under reduced pressure to obtain 5-chlorobisantrene hapten.

3. A 5-chlorodioctophyllin immunogen, characterized in that: The structural formula is as follows: 。 4. A method of preparing a 5-chlorodioctophyllin immunogen, characterized in that: The 5-chlorobisantrene hapten of claim 1 or 2 is completely dissolved in methanol, BAS is added, and the mixture is stirred at 25 DEG C for 18 hours, and the mixed solution is reacted at 4 DEG C overnight, and the solution is collected and dialyzed in 0.01 mol / L phosphate buffer for 72 hours to obtain the immunogen hapten-BSA, and the pH of the phosphate buffer is 7.

4.

5. A process for the preparation of a 5-chlorodioctophyllin immunogen according to claim 4, characterized in that: The BAS is bovine serum albumin, and the ratio of hapten to BAS is 1:1-1.5 when the BAS is added.

6. A 5-chlorocholecalciferol-coated antigen, characterized in that: The structural formula is as follows: 。 7. A method of preparing a 5-chloro double diphosphine antigen, characterized by: The 5-chlorobisantrene hapten of claim 1 or 2 is completely dissolved in methanol, OVA is added, and the addition ratio is hapten:OVA=1:1-1:1.5, and the mixture is stirred at 25 DEG C for 18 hours, and the mixed solution is reacted at 4 DEG C overnight, and the solution is collected and dialyzed in 0.01 mol / L phosphate buffer with a pH of 7.4 for 72 hours to obtain the coated antigen hapten-OVA.

8. A method of preparing 5-chlorodiosephenyline antibodies, characterized by: The method steps are as follows: (1) New Zealand white rabbits are selected as experimental animals, and the immunogen hapten-BSA of claim 3 or 4 is used as an antigen; (2) The immunization is performed by subcutaneous injection, and the interval between each injection is 2 weeks, and a total of 6 immunizations are performed; (3) Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used for subsequent immunization; (4) After the immunization is completed, blood is collected, and the antiserum is separated by centrifugation at 4 DEG C; (5) The antibody is purified by Protein A-Sepharose 4B affinity chromatography to obtain polyclonal IgG antibody.

9. An immunoassay method based on the coated antigen of claim 6 or 7 for 5-chloro bisacodyl antibodies, characterized by: An indirect competitive ELISA mode is used, including the following steps: (1) The coated antigen hapten-OVA is fixed on a 96-well microplate; (2) Different concentrations of the sample to be tested are mixed with the antibody and incubated; (3) The absorbance at 450 nm wavelength is measured after adding enzyme-labeled secondary antibody and developing color; (4) The inhibition rate is calculated, and a standard curve is drawn to realize quantitative detection of 5-chlorobisantrene, The lowest detection limit LOD of the above method is 1.07 ng / mL, and the half-inhibitory concentration IC50 is 27.67 ng / mL, and the cross-reactivity of the antibody to diethylstilbestrol and other structural analogues is less than 0.2%. 50 The lowest detection limit LOD of the above method is 1.07 ng / mL, and the half-inhibitory concentration IC50 is 27.67 ng / mL, and the cross-reactivity of the antibody to diethylstilbestrol and other structural analogues is less than 0.2%.

10. Use of the hapten, immunogen, coating antigen and antibody according to any one of claims 1 to 9 for the detection of 5-chloroacetophenoxyl in a food sample, characterized in that: The food samples include jelly, solid beverage, enzyme beverage, tablet candy, and fruit vinegar beverage.

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