WO2021088209A1 - 用于地沟油检测的辣椒素半抗原、人工抗原及其制备方法与应用 - Google Patents

用于地沟油检测的辣椒素半抗原、人工抗原及其制备方法与应用 Download PDF

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WO2021088209A1
WO2021088209A1 PCT/CN2019/125894 CN2019125894W WO2021088209A1 WO 2021088209 A1 WO2021088209 A1 WO 2021088209A1 CN 2019125894 W CN2019125894 W CN 2019125894W WO 2021088209 A1 WO2021088209 A1 WO 2021088209A1
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capsaicin
hapten
formula
add
compound
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PCT/CN2019/125894
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French (fr)
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柯跃斌
沈建忠
王战辉
李金峰
蒋慧
于雪芝
江海洋
赵芳
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深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所）
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/20—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a carbon atom of an acyclic unsaturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the invention belongs to the technical field of biochemical industry, and specifically relates to capsaicin hapten and artificial antigen used for the detection of waste oil, and a preparation method and application thereof.
  • Waste oil also known as slop oil or garbage oil, is generally only used as industrial oil. Because waste oil does not meet the relevant sanitation and safety regulations from the source of raw materials, processing technology, filling and transportation, the quality of the produced oil cannot be guaranteed, and it is seriously harmful to human health. Long-term intake will cause liver, heart, and lung damage. , And even cause cancer.
  • CPC capsaicin
  • the most widely used detection methods for capsaicin are instrumental methods, including high performance liquid chromatography and chromatography-mass spectrometry.
  • the sensitivity and accuracy of the instrumental methods are relatively high, and the detection results are reliable, but professional operators and Large-scale instruments, complex sample processing procedures and high analysis costs cannot meet the requirements for rapid on-site testing.
  • the immunoassay method not only has the advantages of high sensitivity and good specificity of instrument detection technology, and is simple to operate, can meet the needs of on-site detection, and has been widely used in the detection of food contaminants.
  • the Chinese invention patent (Announcement No. CN103951577A, announcement date 2015-07-22, title of invention: a capsaicin artificial hapten, artificial antigen and preparation method thereof) discloses a method for synthesizing capsaicin artificial hapten compound, which is based on N-(4-hydroxy-3-methoxybenzyl)nonanamide is reacted with ethyl 4-bromobutyrate as raw materials to obtain the compound of formula III, and then the ester group is hydrolyzed to produce capsaicin
  • the artificial hapten compound I specifically includes the following steps: (1) Combine N-(4-hydroxy-3-methoxybenzyl)nonanamide and 4-bromobutyric acid ethyl in a molar ratio of 1:(1.2 ⁇ 1.5) The ester was dissolved in N,N-dimethylformamide to obtain the corresponding N-(4-hydroxy-3-methoxybenzyl)nonanamide solution and 4-bromobutyric acid e
  • Another example is the Chinese invention patent (publication number CN104447383A, publication date 2015-3-25, a dihydrocapsaicin artificial hapten, artificial antigen and preparation method thereof) discloses a preparation method of dihydrocapsaicin artificial hapten, Specifically, it includes the following steps: Weigh 5.86g of N-(4-hydroxy-3-methoxybenzyl)nonanamide, 2.36g of maleic anhydride and 0.49g of N,N'-dicyclohexylcarbodiimide, 25mL of dichloromethane was reacted in a reaction flask at room temperature for 0.5h, and then the above reaction solution was placed in an ice bath to control the temperature within the range of 0 ⁇ 5°C, and 0.30g of 4-dimethylaminopyridine and 4mL of dichloromethane were put together.
  • the experimental results show that the titer of antiserum obtained by immunizing animals with the dihydrocapsaicin artificial hapten can reach 4000, but the IC50 value of the half inhibitory concentration of dihydrocapsaicin is 3.06 ⁇ g/mL, the IC50 is relatively high, and the sensitivity needs to be improved. .
  • the purpose of the present invention is to provide capsaicin haptens and artificial antigens for the detection of cooking oil, and preparation methods and applications thereof.
  • the present invention provides a capsaicin hapten, the structure of which is shown in Formula I or Formula II:
  • the present invention provides a method for preparing the capsaicin hapten.
  • the preparation method includes the following steps: using natural capsaicin and 4-bromomethyl benzoic acid
  • the methyl ester is used as a raw material to perform a reflux reaction to obtain a compound of formula III, and then the reaction is heated under alkaline conditions, and finally acid is added to adjust to acidity to obtain a capsaicin hapten compound, that is, a compound of formula I.
  • the synthetic route is as follows:
  • the preparation method specifically includes the following steps:
  • Step A1 Add 1 eq of natural capsaicin and 2 eq of potassium carbonate to acetonitrile, stir well, add 1.2 eq 4-bromomethyl methyl benzoate, reflux for 24 hours, post-treatment and concentrate to remove most of acetonitrile, pour into water, ethyl acetate Ester, extraction, water washing, drying, column purification, the yield is about 70%.
  • Step A2 Dissolve the product of Step A1 in tetrahydrofuran, add the same amount of ethanol, add 2eq of sodium hydroxide aqueous solution dropwise, heat to 50°C for 1 hour, post-treatment and concentrate to remove most of the ethanol and tetrahydrofuran, cool to room temperature, add hydrochloric acid The product is adjusted to acidity, the product is separated out, collected and dried, and the yield is 90%, and the capsaicin hapten is obtained.
  • the preparation method includes the following steps: using dihydrocapsaicin and methyl 4-bromomethyl benzoate as raw materials to perform a reflux reaction to obtain a compound of formula IV, and then Heat the reaction under mild conditions, and finally add acid to adjust to acidity to obtain the capsaicin hapten compound, that is, the compound of formula II.
  • the synthetic route is as follows:
  • the preparation method specifically includes the following steps:
  • Step B1 Add 1 eq of dihydrocapsaicin and 2 eq of potassium carbonate to acetonitrile, stir well, add 1.2 eq 4-bromomethyl methyl benzoate, reflux for 24 hours, post-treatment and concentrate to remove most of the acetonitrile, pour into water, and ethyl acetate Ester, extraction, water washing, drying, column purification, the yield is about 70%.
  • Step B2 Dissolve the product of Step B1 in tetrahydrofuran, add the same amount of ethanol, and then add dropwise 2eq of sodium hydroxide aqueous solution, heat to 50°C for 1 hour, post-treatment and concentrate to remove most of the ethanol and tetrahydrofuran, cool to room temperature, add hydrochloric acid The product is adjusted to acidity, the product is separated out, collected and dried, and the yield is 90%, and the capsaicin hapten is obtained.
  • the present invention provides an artificial antigen of capsaicin, which is obtained by coupling the capsaicin hapten with a carrier protein; wherein, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, Thyroxin, a kind of human serum albumin. Preferred are bovine serum albumin BSA and keyhole limpet hemocyanin KLH.
  • the present invention provides a method for preparing the capsaicin artificial antigen, which uses the activated ester method to couple the carrier protein to the carboxyl carbon of the hapten of claim 1.
  • the molar ratio of the compound represented by formula I and the carrier protein during coupling is 8.4:1; the molar ratio of the compound represented by formula II and the carrier protein during coupling is 5.8:1.
  • the present invention provides a specific antibody prepared from the capsaicin artificial antigen, which is a polyclonal antibody or a monoclonal antibody, preferably a polyclonal antibody.
  • the polyclonal antibody can be obtained by immunizing experimental animals (such as New Zealand white rabbits) with capsaicin artificial antigen, and collecting serum for purification.
  • the present invention provides any of the following applications of the capsaicin hapten or the capsaicin artificial antigen:
  • the present invention provides a capsaicin detection reagent or kit prepared from the specific antibody.
  • the present invention provides any of the following applications of the specific antibody:
  • the present invention discloses for the first time two new capsaicin haptens, artificial antigens and preparation methods thereof.
  • By immunizing animals with the capsaicin artificial antigens specific antibodies with high titer and high sensitivity can be obtained.
  • the capsaicin hapten and the antibody prepared by the invention provide a new method for establishing a rapid, simple, inexpensive, sensitive and specific capsaicin detection method.
  • the structure of the hapten is a key factor in the preparation of high-sensitivity small molecule antibodies.
  • the two new haptens designed and synthesized by the inventors retain the characteristic structure of capsaicin to a greater extent, appropriately increase the length of the spacer, and introduce rigid spacers.
  • Benzoic acid with the synergistic effect of the three, can better expose the chemical structure of capsaicin to the immune system and improve the stimulatory properties of the immune hapten. Therefore, antibodies with low IC50 and high sensitivity are obtained, and the IC50 can reach 1.02ng/ mL, the sensitivity is about 3000 times higher than that of the antibody in the prior art, which is also an unexpected technical effect in this research and development process.
  • the capsaicin antibody (polyantibody) is prepared by using the conjugate of the hapten and the carrier protein provided by the present invention, the preparation process is simple and economical, the detection sensitivity of the antibody to capsaicin can reach 1.02ng/mL, and the practical value is high.
  • the invention has good application prospects in the detection of waste oil.
  • Figure 1 is a flow chart of the preparation of the capsaicin hapten represented by formula I in Example 1 of the present invention.
  • Figure 2 is a flow chart of the preparation of the capsaicin hapten represented by formula II in Example 1 of the present invention.
  • Figure 3 is a mass spectrum of the capsaicin hapten represented by formula I in Example 1 of the present invention.
  • Figure 4 is a mass spectrum of the capsaicin hapten represented by formula II in Example 1 of the present invention.
  • Fig. 5 is a 1 H-NMR chart of the capsaicin hapten represented by formula I in Example 1 of the present invention.
  • Fig. 6 is a 1 H-NMR chart of the capsaicin hapten represented by formula II in Example 1 of the present invention.
  • Figure 7 is a MALDI-TOF-MS diagram of BSA in Example 2 of the present invention.
  • Figure 8 is a MALDI-TOF-MS diagram of CPC-BSA in Example 2 of the present invention.
  • Figure 9 is a MALDI-TOF-MS diagram of CPC2-BSA in Example 2 of the present invention.
  • Fig. 10 is a standard curve diagram of the detection of capsaicin using polyclonal antibodies in Example 4 of the present invention.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • the PBS buffers used in the examples are all pH 7.4, 0.01M PBS buffers.
  • the carbonate buffers used in the examples are all sodium carbonate buffers with pH 9.6 and 0.05 mol/L.
  • NHS is an abbreviation for N-hydroxysuccinimide.
  • EDC is an abbreviation for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
  • DMF is an abbreviation for N,N-dimethylformamide.
  • NHS, EDC, bovine serum albumin (Albumin from bovine serum, BSA), Keyhole Limpet Hemocyanin (KLH), Freund's complete adjuvant and Freund's incomplete adjuvant were all purchased from Sigma.
  • the stationary phase used in column chromatography is 200-300 mesh silica gel.
  • the capsaicin hapten represented by formula I is prepared by refluxing natural capsaicin and methyl 4-bromomethyl benzoate as raw materials to obtain the compound of formula III, and then heating and reacting under alkaline conditions, and finally adding The acid is adjusted to acidity to obtain the capsaicin hapten compound, that is, the compound of formula I.
  • the synthetic route is shown in Figure 1. The specific synthetic steps are as follows:
  • Step A1 Weigh 3.05g of natural capsaicin and 2.76g of potassium carbonate into acetonitrile, stir well, add 2.75g methyl 4-bromomethyl benzoate, reflux for 24h, post-treatment concentrated to remove most of acetonitrile, pour into water , Ethyl acetate, extraction, water washing, drying, column purification, the yield is about 70%.
  • Step A2 Dissolve the product of Step A1 in tetrahydrofuran, add the same amount of ethanol, then add dropwise 0.02 mol sodium hydroxide aqueous solution, heat to 50 degrees for 1 hour, post-treatment and concentrate to remove most of the ethanol and tetrahydrofuran, cool to room temperature, add The hydrochloric acid is adjusted to acidity (PH value is 4-6), the product is precipitated, and the capsaicin hapten 4-(2-methoxy-4-((8-methyl-6-nonenamide)methyl)phenoxy is obtained Base) benzoic acid, the molecular formula is C 26 H 33 NO 5 , collected and dried, the yield is 90%.
  • the capsaicin hapten represented by formula II is prepared by refluxing dihydrocapsaicin and methyl 4-bromomethyl benzoate as raw materials to obtain the compound of formula IV, and then heating and reacting under alkaline conditions, and finally Add acid to adjust to acidity to obtain the capsaicin hapten compound, namely the compound of formula II.
  • the synthetic route is shown in Figure 2. The specific synthetic steps are as follows:
  • Step B1 Weigh 3.07g of dihydrocapsaicin and 2.76g of potassium carbonate into acetonitrile, stir well, add 2.75g methyl 4-bromomethyl benzoate, reflux for 24h, post-processing concentration to remove most of the acetonitrile, pour In water, ethyl acetate, extraction, water washing, drying, column purification, the yield is about 70%.
  • Step B2 Dissolve the product of Step B1 in tetrahydrofuran, add the same amount of ethanol, then add dropwise 0.02mol sodium hydroxide aqueous solution, heat to 50°C for 1 hour, post-treatment and concentrate to remove most of the ethanol and tetrahydrofuran, cool to room temperature, and add The hydrochloric acid is adjusted to acidity, the product is precipitated, and the capsaicin hapten 4-(2-methoxy-4-((8-methylnonanamide)methyl)phenoxymethyl)benzoic acid is obtained.
  • the molecular formula is C 26 H 35 NO 5 , collected and dried, with a yield of 90%.
  • the immunogen carrier protein mainly uses KLH
  • the coating original carrier protein mainly uses BSA
  • the coupling method used is active ester. law.
  • MALDI-TOF-MS Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry
  • Binding ratio ⁇ M(conjugate)-M(protein) ⁇ /M(hapten), where M is the molecular weight
  • the molecular weight of the hapten of formula I is 439.6, the molecular weight of BSA is 64671.3 and the molecular weight of the conjugate is 68378.7 from the highest peak of mass spectrometry, and the binding ratio of BSA to hapten is calculated to be 8.4, that is, one BSA molecule in CPC1-BSA On average, 8.4 haptens were coupled.
  • the molecular weight of the hapten of formula II is 441.6, and the molecular weight of the conjugate analyzed from the highest peak of mass spectrometry is 67240.6.
  • the binding ratio of BSA to hapten is calculated to be 5.8, that is, 5.8 conjugates on one BSA molecule in CPC2-BSA. Hapten.
  • KLH was used instead of BSA, and the others were the same as step 1 of step 1 of Example 2 (that is, 1. preparation of capsaicin coating agent).
  • the synthetic capsaicin immunogen of the compound represented by formula I is referred to as CPC1-KLH.
  • the synthetic capsaicin immunogen of the compound represented by formula II is referred to as CPC2-KLH.
  • the CPC1-KLH and CPC2-KLH solutions prepared in Example 2 were respectively immunized with 2 groups of female New Zealand white rabbits aged 3-4 months and weighing 1.5-2.0 kg, 2 in each group.
  • Each immunogen was diluted with normal saline to 1 mg/mL, and emulsified with the same amount of Freund's adjuvant.
  • Freund's complete adjuvant was used for the first immunization, multiple intradermal injections on the back of the neck, and the immunization dose was 1 mg/mouse.
  • the booster immunization was carried out. The immunization was performed once every 4 weeks for a total of 3 times.
  • the adjuvant was changed to Freund's incomplete adjuvant, and the immunization dose was unchanged, and it was changed to multiple subcutaneous injections on the back of the neck.
  • One week after the fourth immunization a large amount of blood was collected by heart blood collection. After blood collection, the blood was allowed to stand at 37°C for 2 hours, then at 4°C overnight, and then centrifuged at 3000 rpm for 20 minutes, and the supernatant was collected, which was the antiserum, and stored in aliquots at -20°C.
  • the indirect ELISA method is used to detect the titer of antiserum. The specific steps are as follows:
  • Example 2 The antigen in Example 2 was diluted with 0.05 M, pH 9.6 carbonate buffer from 10 ⁇ g/mL, 100 ⁇ L/well, and reacted at 37° C. for 2 hours.
  • Blocking After patted dry, add 200 ⁇ L/well blocking solution and react at 37°C for 2h. Dry it for later use after washing.
  • Termination and determination Add 100 ⁇ L of termination solution to each well to terminate the reaction, and then measure the OD 450 value of each well with a microplate reader.
  • Coating Dilute the original coating buffer by 5000 times, 100 ⁇ L/well, and react at 37°C for 2 hours.
  • Preparing capsaicin CPC standard solution Dissolve CPC standard product in DMF to prepare a 5mg/mL mother solution, and then dilute it to the required concentration with 0.01mol/L, pH7.4 PBS solution before adding the sample.
  • concentration of CPC is 0.1 ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL, 24.3 ng/mL).
  • Termination and determination Add 100 ⁇ L of stop solution to each well to terminate the reaction, and then measure the OD 450 value of each well with a microplate reader.
  • the level of potency is not directly related to the level of IC50.
  • the titer and IC50 are not positively correlated.
  • the difference in antibody titer has no effect on the detection of small molecules.
  • Sensitivity is the most important property parameter.
  • the antibody has no cross-reactivity with chemical residual pollutants such as fenvalerate and deltamethrin, and biotoxin pollutants such as aflatoxin, indicating that the antibody can specifically react with capsaicin.
  • the capsaicin haptens and artificial antigens mentioned above can prepare capsaicin-specific antibodies with high affinity and higher sensitivity (IC50 value as low as 1.02ng/mL).
  • the sensitivity of the antibody obtained by the present invention provides about 3000 times, which is an unexpected technical effect of the present invention. The possible reasons for this technical effect are as follows:
  • the hapten synthesis in the prior art uses N-(4-hydroxy-3-methoxybenzyl)nonanamide as a raw material, which is insufficient in retaining the characteristic structure of capsaicin; furthermore, The spacer arm is only 4 carbons in length, and cannot fully expose the target recognition site when coupled with the carrier protein.
  • the spacer arm is linear butyric acid, and the linear spacer arm has a certain degree of flexibility, which is not necessarily good when used as an antigenic determinant to stimulate the body to produce antibodies.
  • the present invention directly uses capsaicin and dihydrocapsaicin as raw materials, which can retain the characteristic structure of capsaicin to a greater extent; the spacer arm of the present invention is slightly longer and can be more fully exposed when coupled with carrier protein Target recognition site:
  • the inventors first tried to use benzoic acid in the process of hapten synthesis, which has a rigid structure and may achieve better immune effects in the process of immune stimulation.
  • the experimental results show that the immune hapten designed and synthesized by the inventors can stimulate the animal body to produce a stronger immune response and obtain more sensitive antibodies.
  • the structure of the hapten is a key factor in the preparation of high-sensitivity small-molecule antibodies.
  • the synthetic raw materials, synthesis method, and synthesis path of the hapten of the present invention are different from those of the prior art, and the structure of the hapten obtained by preparation is also different. Lay the foundation for the preparation of more sensitive antibodies.
  • the rational design of haptens is the key to the preparation of small molecule antibodies.
  • the present invention uses natural capsaicin and dihydrocapsaicin as starting materials to design and synthesize two haptens with different structures but with the same spacer.
  • the synergistic effect of the three can better expose the chemical structure of capsaicin to the immune system and improve the stimulatory nature of the immune hapten. This is an antibody with low IC50 and high sensitivity.

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Abstract

本发明涉及用于地沟油检测的辣椒素半抗原和人工抗原及其制备方法与应用。所述辣椒素半抗原的结构如式I或式Ⅱ所示；所述辣椒素人工抗原是由式I或式Ⅱ所示半抗原与载体蛋白偶联得到。利用所述辣椒素人工抗原免疫动物，可得到效价高，灵敏度高的特异性抗体。本发明提供的辣椒素半抗原及其制备的抗体，为建立快速、简便、价廉、灵敏、特异的辣椒素检测方法提供了新手段。

Description

用于地沟油检测的辣椒素半抗原、人工抗原及其制备方法与应用 技术领域
本发明属于生物化工技术领域，具体地说，涉及用于地沟油检测的辣椒素半抗原和人工抗原及其制备方法与应用。
背景技术
地沟油，又称潲水油、垃圾油，一般只作为工业用油。因地沟油从原料来源、加工工艺及灌装运输过程等均不符合有关卫生安全规定，生产出的油脂质量无法保证，对人体健康有严重危害，长期摄入将会导致肝、心、肺损害，甚至引发癌症。
据文献统计，通过监测辣椒素类物质判别地沟油的准确率可以达到80％以上。而在组成辣椒素类物质的多种辣椒碱类化合物中，最主要的是辣椒素(capsaicin，CPC)，在天然辣椒素中占比高达69％。因此，食用油中辣椒素的检测对鉴别地沟油具有重要意义。
辣椒素检测方法中目前应用较多的是仪器方法，包括高效液相色谱法以及色谱-质谱联用技术，仪器方法灵敏度及准确度相对较高，且检测结果可靠，但是需要专业的操作人员以及大型仪器，样品处理过程复杂，分析成本高，无法满足现场快速检测的要求。而免疫分析方法既具有仪器检测技术灵敏度高、特异性好的优点，且操作简便、能够满足现场检测需求，已经被广泛应用到食品污染物的检测中。
如中国发明专利(公告号CN103951577A，公告日2015-07-22，发明名称一种辣椒素人工半抗原、人工抗原及其制备方法)公开了一种辣椒素人工半抗原化合物的合成方法，是以N-(4-羟基-3-甲氧基苄基)壬酰胺与4-溴代丁酸乙酯为原料经成醚反应，得到式III化合物，然后将其中的酯基团水解制得辣椒素人工半抗原化合物I，具体包括以下步骤：(1)将摩尔比为1∶(1.2～1.5)的N-(4-羟基-3-甲氧基苄基)壬酰胺和4-溴丁酸乙酯分别溶解于N，N-二甲基甲酰胺中，得到相应的N-(4-羟 基-3-甲氧基苄基)壬酰胺溶液和4-溴丁酸乙酯溶液，然后将4-溴丁酸乙酯溶液置于恒压漏斗，缓慢滴加至N-(4-羟基-3-甲氧基苄基)壬酰胺溶液中，待滴加完毕，在105～110℃条件下反应5-7h，后处理得到中间产物III；(2)将中间产物III在碱性条件下水解，后处理得到辣椒素人工抗原。实验结果表明，用该辣椒素人工半抗原化合物抗原免疫动物得到的抗血清效价可达128000，但对辣椒素的半抑制浓度IC50值为3.34μg/mL，IC50偏高，灵敏度有待提高。
又如中国发明专利(公开号CN104447383A，公开日2015-3-25，一种二氢辣椒素人工半抗原、人工抗原及其制备方法)公开了一种二氢辣椒素人工半抗原的制备方法，具体包括以下步骤：称取N-(4-羟基-3-甲氧基苄基)壬酰胺5.86g、顺丁烯二酸酐2.36g和N，N’-二环己基碳二亚胺0.49g，25mL二氯甲烷于反应瓶中室温反应0.5h，然后将上述反应液置于冰浴中使其温度控制在0～5℃范围内，取4-二甲氨基吡啶0.30g和4mL二氯甲烷置于恒压滴液漏斗中充分混匀，缓慢向反应瓶中滴加，0.5h滴加完毕，待滴加完毕，保持室温25℃条件继续搅拌6小时，将反应液过滤，减压蒸馏除去溶剂得淡黄色的油状物粗产品，然后将该粗产品通过硅胶柱层析，柱层析试剂∶石油醚和乙酸乙酯体积比为1∶1的混合溶液，脱色得到二氢辣椒素半抗原(E)-4-[2-甲氧基-4-(壬酰胺甲基)苯氧基]-4-氧-2-丁烯酸，分子式为C21NO6H29。实验结果表明，用该二氢辣椒素人工半抗原免疫动物得到的抗血清效价可达4000，但是对二氢辣椒素的半抑制浓度IC50值为3.06μg/mL，IC50偏高，灵敏度有待提高。
因此有必要再设计免疫半抗原分子，制备出一种辣椒素检测抗体对于地沟油的快速检测且具有高灵敏度特性尤为重要。
发明概述
技术问题
问题的解决方案
技术解决方案
本发明的目的是提供用于地沟油检测的辣椒素半抗原和人工抗原及其制备方法与应用。
为了实现本发明目的，第一方面，本发明提供辣椒素半抗原，其结构如式I或 式II所示：
Figure PCTCN2019125894-appb-000001
第二方面，本发明提供所述辣椒素半抗原的制备方法，当所述辣椒素半抗原为式I所示化合物时，制备方法包括如下步骤：以天然辣椒素与4-溴甲基苯甲酸甲酯为原料进行回流反应，得到式III化合物，然后在碱性条件下加热反应，最后加酸调至酸性，得到辣椒素半抗原化合物，即式I化合物，合成路线如下：
Figure PCTCN2019125894-appb-000002
当所述辣椒素半抗原为式I所示化合物时，制备方法具体包括如下步骤：
步骤A1：将1eq天然辣椒素和2eq碳酸钾加入乙腈中，搅拌均匀，加入1.2eq 4- 溴甲基苯甲酸甲酯，回流反应24h，后处理浓缩除去大部分乙腈，倒入水中，乙酸乙酯，提取，水洗，干燥，过柱纯化，收率约70％。
步骤A2：将步骤A1产物溶于四氢呋喃中，加入等量的乙醇，再滴加2eq氢氧化钠水溶液，加热至50度反应1h，后处理浓缩除去大部分乙醇和四氢呋喃，冷却至室温，加盐酸调至酸性，产品析出，收集干燥，收率90％，即得辣椒素半抗原。
当所述辣椒素半抗原为式II所示化合物时，制备方法包括如下步骤：以二氢辣椒素与4-溴甲基苯甲酸甲酯为原料进行回流反应，得到式IV化合物，然后在碱性条件下加热反应，最后加酸调至酸性，得到辣椒素半抗原化合物，即式II化合物，合成路线如下：
Figure PCTCN2019125894-appb-000003
当所述辣椒素半抗原为式II所示化合物时，制备方法具体包括如下步骤：
步骤B1：1eq二氢辣椒素和2eq碳酸钾加入乙腈中，搅拌均匀，加入1.2eq 4-溴甲基苯甲酸甲酯，回流反应24h，后处理浓缩除去大部分乙腈，倒入水中，乙酸乙酯，提取，水洗，干燥，过柱纯化，收率约70％。
步骤B2：将步骤B1产物溶于四氢呋喃中，加入等量的乙醇，再滴加2eq氢氧化钠水溶液，加热至50℃反应1h，后处理浓缩除去大部分乙醇和四氢呋喃，冷却至室温，加盐酸调至酸性，产品析出，收集干燥，收率90％，即得辣椒素半抗原。
第三方面，本发明提供辣椒素人工抗原，其由上述辣椒素半抗原与载体蛋白偶联后得到；其中，所述载体蛋白选自牛血清白蛋白、卵清蛋白、钥孔血蓝蛋白 、甲状腺蛋白、人血清白蛋白的一种。优选牛血清白蛋白BSA、钥孔血蓝蛋白KLH。
第四方面，本发明提供所述辣椒素人工抗原的制备方法，采用活化酯法将载体蛋白偶联于权利要求1所述半抗原的羧基碳上。
优选地，式I所示化合物与载体蛋白的偶联时摩尔比为8.4∶1；式II所示化合物与载体蛋白的偶联时摩尔比为5.8∶1。
第五方面，本发明提供由所述辣椒素人工抗原制备的特异性抗体，其为多克隆抗体或单克隆抗体，优选多克隆抗体。所述多克隆抗体可通过辣椒素人工抗原免疫实验动物(如新西兰大白兔)，收集血清纯化获得。
第六方面，本发明提供所述辣椒素半抗原或所述辣椒素人工抗原的以下任一应用：
①制备抗辣椒素特异性抗体中的应用；
②检测抗辣椒素特异性抗体中的应用。
第七方面，本发明提供由所述特异性抗体制备的辣椒素检测试剂或试剂盒。
第八方面，本发明提供所述特异性抗体的以下任一应用：
(1)在检测辣椒素中的应用；
(2)在制备辣椒素的免疫层析试纸条中的应用；
(3)在制备辣椒素的胶体金检测试纸条中的应用。
发明的有益效果
有益效果
本发明首次公开了两种新的辣椒素半抗原、人工抗原及其制备方法，用所述辣椒素人工抗原免疫动物，可得到效价高，灵敏度高的特异性抗体。本发明提供的辣椒素半抗原及其制备的抗体，为建立快速、简便、价廉、灵敏、特异的辣椒素检测方法提供了新手段。
半抗原的结构在高灵敏度小分子抗体制备过程中是关键因素，本发明人设计合成的2种新型半抗原通过更大程度的保留辣椒素的特征结构、适当增加间隔臂长度、引入刚性间隔臂苯甲酸，三者协同作用，可以更好的将辣椒素化学结构暴露于免疫系统，提高了免疫半抗原的刺激原性，因此获得了IC50低、灵敏度高 的抗体，其IC50可达1.02ng/mL，灵敏度相比现有技术中的抗体提高了约3000倍，这也是本次研发过程中意料不到的技术效果。
利用本发明提供的半抗原与载体蛋白的偶联物制备辣椒素抗体(多抗)，制备过程简单、经济，抗体对辣椒素的检测灵敏度可达1.02ng/mL、实用价值高。本发明在地沟油检测中具有良好的应用前景。
对附图的简要说明
附图说明
图1为本发明实施例1中式I所示辣椒素半抗原制备的流程图。
图2为本发明实施例1中式II所示辣椒素半抗原制备的流程图。
图3为本发明实施例1中式I所示辣椒素半抗原的质谱图。
图4为本发明实施例1中式II所示辣椒素半抗原的质谱图。
图5为本发明实施例1中式I所示辣椒素半抗原的 1H-NMR图。
图6为本发明实施例1中式II所示辣椒素半抗原的 1H-NMR图。
图7为本发明实施例2中BSA的MALDI-TOF-MS图。
图8为本发明实施例2中CPC-BSA的MALDI-TOF-MS图。
图9为本发明实施例2中CPC②-BSA的MALDI-TOF-MS图。
图10为本发明实施例4中利用多克隆抗体检测辣椒素的标准曲线图。
发明实施例
本发明的实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。实施例中所用的PBS缓冲液均为pH7.4、0.01M的PBS缓冲液。实施例中所用的碳酸盐缓冲液均为pH9.6、0.05mol/L的碳酸钠缓冲液。
NHS为N-羟基琥珀酰亚胺的缩写。EDC为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的缩写。DMF为N，N-二甲基甲酰胺的缩写。NHS、EDC、牛血清白蛋白(Albumin from bovine serum，BSA)、钥孔血蓝蛋白(Keyhole Limpet  Hemocyanin，KLH)以及弗氏完全佐剂、弗氏不完全佐剂均购自Sigma公司。柱层析所用固定相均为200-300目的硅胶。
实施例1 辣椒素半抗原的制备和表征
一、辣椒素半抗原的制备
1、式I所示辣椒素半抗原的制备，是以天然辣椒素与4-溴甲基苯甲酸甲酯为原料进行回流反应，得到式III化合物，然后在碱性条件下加热反应，最后加酸调至酸性，得到辣椒素半抗原化合物，即式I化合物，合成路线图如图1所示，具体合成步骤如下：
步骤A1：称取3.05g天然辣椒素和2.76g碳酸钾加入乙腈中，搅拌均匀，加入2.75g 4-溴甲基苯甲酸甲酯，回流反应24h，后处理浓缩除去大部分乙腈，倒入水中，乙酸乙酯，提取，水洗，干燥，过柱纯化，收率约70％。
步骤A2：将步骤A1产物溶于四氢呋喃中，加入等量的乙醇，再滴加0.02mol氢氧化钠水溶液，加热至50度反应1h，后处理浓缩除去大部分乙醇和四氢呋喃，冷却至室温，加盐酸调至酸性(PH值为4-6)，产品析出，得到辣椒素半抗原4-(2-甲氧基-4-((8-甲基-6-壬烯酰胺)甲基)苯氧基)苯甲酸，分子式为C 26H 33NO 5，收集干燥，收率90％。
2、式II所示辣椒素半抗原的制备，是以二氢辣椒素与4-溴甲基苯甲酸甲酯为原料进行回流反应，得到式IV化合物，然后在碱性条件下加热反应，最后加酸调至酸性，得到辣椒素半抗原化合物，即式II化合物，合成路线图如图2所示，具体合成步骤如下：
步骤B1：称取3.07g二氢辣椒素和2.76g碳酸钾加入乙腈中，搅拌均匀，加入2.75g 4-溴甲基苯甲酸甲酯，回流反应24h，后处理浓缩除去大部分乙腈，倒入水中，乙酸乙酯，提取，水洗，干燥，过柱纯化，收率约70％。
步骤B2：将步骤B1产物溶于四氢呋喃中，加入等量的乙醇，再滴加0.02mol氢氧化钠水溶液，加热至50℃反应1h，后处理浓缩除去大部分乙醇和四氢呋喃，冷却至室温，加盐酸调至酸性，产品析出，得到辣椒素半抗原4-(2-甲氧基-4-( (8-甲基壬酰胺)甲基)苯氧甲基)苯甲酸，分子式为C 26H 35NO 5，收集干燥，收率90％。
二、辣椒素半抗原的表征
1、质谱鉴定
步骤一制备的式I所示辣椒素半抗原的质谱鉴定结果：MS m/z[M+H] +理论值：439.55；实测值：439.9，与目标产物的分子量相吻合，质谱见图3。
步骤一制备的式II所示辣椒素半抗原的质谱鉴定结果：MS m/z[M+H] +理论值：441.57；实测值：441.9，与目标产物的分子量相吻合，质谱见图4。
2、核磁共振鉴定
步骤一制备的式I所示辣椒素半抗原的核磁鉴定结果：
1HNMR(300MHz，DMSO-d6)δ(ppm)12.99(br，s，1H，COOH)，8.29(t，1H，NH)，7.97-7.94(d，2H，CHar)，7.65-7.62(d，2H，CHar)，6.94(s，1H，Char)，6.92-6.71(dd，2H，Char)，5.35-5.32(m，2H，CH＝CH)，5.14(s，2H，CH2)，4.19-4.17(d，2H，CH2)，3.78(s，3H，CH3)，2.28-2.22(m，1H，CH)，2.14-2.09(t，2H，CH2)，1.94-1.93(q，2H，CH2)1.54-1.49(m，2H，CH2)，1.31-1.29(m，2H，CH2)，0.93-0.83(dd，6H，CH3，CH3)。核磁数据表明上述方法合成的化合物即为目标产物，磁共振鉴定结果见图5。
步骤一制备的式II所示辣椒素半抗原的核磁鉴定结果：
1HNMR(300MHz，DMSO-d6)δ(ppm)12.99(br，s，1H，COOH)，8.29(t，1H，NH)，7.97-7.94(d，2H，CHar)，7.56-7.52(d，2H，CHar)，6.94(s，1H，Char)，6.92-6.71(dd，2H，Char)，5.14(s，2H，CH2)，4.19-4.17(d，2H，CH2)，3.78(s，3H，CH3)，2.13-2.08(t，2H，CH2)，1.53-1.49(m，3H，CH2，CH)，1.1.23-1.11(m，8H，CH2，CH2，CH2，CH2)，0.85-0.83(dd，6H，CH3，CH3)。核磁数据表明上述方法合成的化合物即为目标产物，磁共振鉴定结果见图6。
实施例2 辣椒素人工抗原的制备和表征
所述免疫原与包被原的制备方法中，其区别在于载体蛋白的使用类型，所述免疫原载体蛋白主要采用KLH，所述包被原载体蛋白主要采用BSA，所用偶联方法为活泼酯法。
一、辣椒素包被原的合成和鉴定
1、辣椒素包被原的制备
(1)将43.3mg实施例1制备得到的I所示化合物溶于500μL DMF中，加入15mg NHS和30mg EDC，室温下搅拌过夜，得到溶液I。
(2)将60mg BSA加入10mL PBS缓冲液中，充分溶解，即为溶液II。
(3)将溶液I缓慢滴加至溶液II中至溶液出现浑浊，共加入溶液I约50μL，4℃下缓慢搅拌24h后装入透析袋，在生理盐水中4℃透析48h(中间换水3次)，得到辣椒素包被原溶液，-20℃保存。式I所示化合物合成的辣椒素包被原简称CPC①-BSA。
(4)取44.0mg实施例1制备得到的式II所示化合物，使用上述三步同样的方法制备式II所示化合物合成的辣椒素包被原，简称CPC②-BSA。
2、辣椒素包被原的鉴定
用基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry，MALDI-TOF-MS)法分别测定CPC①-BSA溶液和CPC②-BSA溶液中BSA与半抗原的结合比。结果见图7、图8、图9。
结合比＝{M(偶联物)-M(蛋白质)}/M(半抗原)，M为分子量；
式I半抗原的分子量为439.6，由质谱最高峰值分析BSA的分子量为64671.3，偶联物的分子量为68378.7，经计算得出BSA与半抗原的结合比为8.4，即CPC①-BSA中一个BSA分子上平均偶联8.4个半抗原。
式II半抗原的分子量为441.6，由质谱最高峰值分析偶联物的分子量为67240.6，经计算得出BSA与半抗原的结合比为5.8，即CPC②-BSA中一个BSA分子上平均偶联5.8个半抗原。
二、辣椒素免疫原的合成
1、辣椒素免疫原的制备
用KLH代替BSA，其它同实施例2的步骤一的步骤1(即1、辣椒素包被原的制备)。
式I所示化合物合成辣椒素免疫原简称CPC①-KLH。
式II所示化合物合成辣椒素免疫原简称CPC②-KLH。
实施例3 辣椒素抗血清的制备
将实施例2制备的CPC①-KLH，CPC②-KLH溶液分别免疫2组3-4月龄，体重1.5-2.0kg的雌性新西兰大白兔，每组2只。将各免疫原用生理盐水稀释至1mg/mL，与等量弗氏佐剂乳化。首免采用弗氏完全佐剂，颈背部皮内多点注射，免疫剂量为1mg/只。4周后进行加强免疫，每隔4周加免1次，共加免3次，佐剂改为弗氏不完全佐剂，免疫剂量不变，改为颈背部皮下多点注射。第4次免疫1周后，用心脏采血的方法大量采血。取血后，将血液37℃静置2h，然后4℃静置过夜，然后3000rpm离心20min，收集上清液，即为抗血清，-20℃分装保存。
实施例4 辣椒素抗血清的测定
一、采用间接ELISA方法检测抗血清效价，具体操作步骤如下：
1)包被：将实施例2中的抗原用0.05M、pH9.6碳酸盐缓冲液从10μg/mL开始进行倍比稀释，100μL/孔，37℃反应2h。
2)洗涤：将板内溶液倾去，甩干，并用洗涤液洗涤3次，每次3min。
3)封闭：拍干后，加入200μL/孔封闭液，37℃反应2h。洗涤后烘干备用。
4)加样：将抗血清从1∶1000开始进行倍比稀释，并加入到各种稀释度的包被孔中，100μL/孔，37℃反应1h；充分洗涤后，加入1∶3000稀释的HRP-羊抗兔IgG，100μL/孔，37℃反应1h。
5)显色：将酶标板取出，充分洗涤后，每孔加入100μL的TMB显色液，37℃避光反应15min。
6)终止和测定：每孔加入100μL终止液以终止反应，然后用酶标仪测定各孔的OD 450值。
7)结果判读：以OD 450值≥阴性对照孔的2.1倍(即P/N≥2.1)所对应的血清最高稀释倍数即为血清的ELISA效价。
二、最低检测限、半数抑制以及特异性的检测
具体操作步骤如下：
1)用上述的间接ELISA方法确定以CPC②-BSA作为包被原，以CPC①-KLH免疫兔子获得的血清作为抗体，以OD 450值在1.5左右时所对应的抗原和抗体浓度为最适工作浓度。
2)包被：将包被原用包被缓冲液稀释5000倍，100μL/孔，37℃反应2h。
3)洗涤和封闭：方法操作同上述间接ELISA法。
4)配辣椒素CPC标准溶液：将CPC标准品溶于DMF中配制成5mg/mL的母液，然后在加样前，再用0.01mol/L，pH7.4的PBS溶液倍比稀释成需要浓度(CPC的浓度分别为0.1ng/mL、0.3ng/mL、0.9ng/mL、2.7ng/mL、8.1ng/mL、24.3ng/mL)。
5)加样：每孔加入50μL倍比稀释的CPC各浓度标准品，然后再加入50μL/孔最适稀释倍数的抗血清，37℃反应1h。充分洗涤后，加入1∶5000稀释的HRP-羊抗兔IgG，100μL/孔，37℃反应1h。
6)显色反应：将酶标板取出，充分洗涤后，每孔加入100μL的TMB显色液，37℃避光反应15min。
7)终止和测定：每孔加入100μL终止液以终止反应，然后用酶标仪测定各孔的OD 450值。
8)数据处理：以CPC各浓度的对数为横坐标，以CPC各浓度对应的OD值为纵坐标，使用Origin 8.5软件，按照四参数对数拟合绘制标准曲线，如图10所示，通过计算IC 50值(半数抑制浓度)判定抗血清对CPC是否具有高亲和力。
结果显示，四免后，兔子抗血清效价可达3000，检测限为0.05ng/mL，IC 50值为1.02ng/mL，线性检测范围为0.16-6.63ng/mL。
需要说明的是，效价的高低与IC50高低没有直接关联。效价与IC50不是呈正相关的关系，对于小分子抗体而言抗体效价的差异对小分子的检测无影响，灵敏度才是最重要的性质参数。
经常规交叉反应测定，该抗体与氰戊菊酯、溴氰菊酯等化学残留污染物及黄曲霉毒素等生物毒素污染物均无交叉反应，表明抗体可以特异性与辣椒素反应。
综上，上述辣椒素半抗原、人工抗原是可以制备出亲和力高，灵敏度更高(IC50值低至1.02ng/mL)的辣椒素特异性抗体。而且，与现有技术中公开的辣椒素半抗原、抗原及其制备方法相比，本发明获得的抗体的灵敏度提供了约3000倍，这是本发明未曾预料到的技术效果。获得此技术效果，可能存在的原因如下：
(1)现有技术中的半抗原合成是以N-(4-羟基-3-甲氧基苄基)壬酰胺为原料，此原料在保留辣椒素的特征结构上有所不足；再者，间隔臂只有4个碳的长度，与载体蛋白偶联时能无法充分暴露目标识别位点。除此之外，其间隔臂为线性的丁酸，线性的间隔臂具有一定的柔性，在作为抗原决定簇刺激机体产生抗体时效果不一定好。
(2)本发明是直接采用辣椒素和二氢辣椒素为原料，可更大程度的保留了辣椒素的特征结构；本发明的间隔臂稍长一些，与载体蛋白偶联时能更充分暴露目标识别位点；本发明人在半抗原合成的过程中首次尝试使用苯甲酸，其具有刚性结构，在免疫刺激的过程中可能会取得较好的免疫效果。最终通过实验结果表明，本发明人设计合成的免疫半抗原可以刺激动物机体产生更强烈的免疫反应，获得更灵敏的抗体。
(3)半抗原的结构在高灵敏度小分子抗体制备过程中是关键因素，本发明半抗原的合成原料、合成方法、合成路径均与现有技术的不同，制备获得的半抗原结构也是不同，为更高灵敏度抗体的制备奠定基础。半抗原的合理设计是制备小分子抗体的关键，本发明以天然辣椒素和二氢辣椒素为起始原料，设计合成了2种不同结构但具有相同间隔臂的半抗原，通过更大程度的保留辣椒素的特征结构、适当增加间隔臂长度、引入刚性间隔臂苯甲酸，三者协同作用可以更好的将辣椒素化学结构暴露于免疫系统，提高了免疫半抗原的刺激原性，因此获得了IC50低、灵敏度高的抗体。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (10)

  1. 用于地沟油检测的辣椒素半抗原，其特征在于，其结构如式I或式II所示：
    Figure PCTCN2019125894-appb-100001
  2. 权利要求1所述辣椒素半抗原的制备方法，其特征在于，当所述辣椒素半抗原为式I所示化合物时，制备方法包括如下步骤：以天然辣椒素与4-溴甲基苯甲酸甲酯为原料进行回流反应，得到式III化合物，然后在碱性条件下加热反应，最后加酸调至酸性，得到辣椒素半抗原化合物，即式I化合物，合成路线如下：
    Figure PCTCN2019125894-appb-100002
  3. 根据权利要求2所述辣椒素半抗原的制备方法，其特征在于，包括 以下步骤：
    步骤A1：将天然辣椒素和碳酸钾加入乙腈中，搅拌均匀，加入4-溴甲基苯甲酸甲酯，回流反应，后处理浓缩除去大部分乙腈，倒入水中，乙酸乙酯，提取，水洗，干燥，过柱纯化。
    步骤A2：将步骤A1产物溶于四氢呋喃中，加入等量的乙醇，再滴加氢氧化钠水溶液，加热反应，后处理浓缩除去大部分乙醇和四氢呋喃，冷却至室温，加盐酸调至酸性，产品析出，收集干燥，即得辣椒素半抗原。
  4. 权利要求1所述辣椒素半抗原的制备方法，其特征在于，当所述辣椒素半抗原为式II所示化合物时，制备方法包括如下步骤：以二氢辣椒素与4-溴甲基苯甲酸甲酯为原料进行回流反应，得到式IV化合物，然后在碱性条件下加热反应，最后加酸调至酸性，得到辣椒素半抗原化合物，即式II化合物，合成路线如下：
    Figure PCTCN2019125894-appb-100003
  5. 根据权利要求4所述辣椒素半抗原的制备方法，其特征在于，包括以下步骤：
    步骤B1：将二氢辣椒素和碳酸钾加入乙腈中，搅拌均匀，加入4-溴甲基苯甲酸甲酯，回流反应，后处理浓缩除去大部分乙腈，倒入水中，乙酸乙酯，提取，水洗，干燥，过柱纯化。
    步骤B2：将步骤B1产物溶于四氢呋喃中，加入等量的乙醇，再滴加氢氧化钠水溶液，加热反应，后处理浓缩除去大部分乙醇和四氢呋喃，冷却至室温，加盐酸调至酸性，产品析出，收集干燥， 即得辣椒素半抗原。
  6. 辣椒素人工抗原，其特征在于，由权利要求1所述辣椒素半抗原与载体蛋白偶联后得到；
    其中，所述载体蛋白选自牛血清白蛋白、卵清蛋白、钥孔血蓝蛋白、甲状腺蛋白、人血清白蛋白的任一种。
  7. 权利要求6所述的辣椒素人工抗原的制备方法，其特征在于，采用活化酯法将载体蛋白偶联于权利要求1所述辣椒素半抗原的羧基碳上。
  8. 辣椒素特异性抗体，其特征在于，它是由权利要求6所述辣椒素人工抗原制备的特异性抗体。
  9. 由权利要求8所述辣椒素特异性抗体制备的辣椒素检测试剂或试剂盒。
  10. 权利要求8所述辣椒素特异性抗体的以下任一应用：
    (1)在检测辣椒素中的应用；
    (2)在制备辣椒素的免疫层析试纸条中的应用；
    (3)在制备辣椒素的胶体金检测试纸条中的应用。
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