WO2023102750A1 - Fenfluramine hapten, and preparation method therefor and use thereof, fenfluramine hapten artificial antigen, and fenfluramine hapten antibody - Google Patents

Fenfluramine hapten, and preparation method therefor and use thereof, fenfluramine hapten artificial antigen, and fenfluramine hapten antibody Download PDF

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WO2023102750A1
WO2023102750A1 PCT/CN2021/136186 CN2021136186W WO2023102750A1 WO 2023102750 A1 WO2023102750 A1 WO 2023102750A1 CN 2021136186 W CN2021136186 W CN 2021136186W WO 2023102750 A1 WO2023102750 A1 WO 2023102750A1
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fenfluramine
hapten
artificial antigen
antibody
carrier protein
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PCT/CN2021/136186
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French (fr)
Chinese (zh)
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雷红涛
潘康亮
全琦琪
方亚琳
关甜
王锦
沈兴
李向梅
徐振林
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华南农业大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
    • C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/14—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of carbon skeletons containing rings
    • 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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention relates to the technical field of food detection, and more specifically relates to a fenfluramine hapten, an artificial antigen, an antibody and a preparation method and application thereof.
  • Fenfluramine is a central inhibitory agent at therapeutic doses and has a sedative effect. It promotes the release of 5-HT from nerve endings, inhibits 5-HT uptake and excites 5-HT receptors, and stimulates the hypothalamic satiety center, thereby causing loss of appetite. It also increases glucose utilization and lowers blood sugar. In large doses (dextrorotary) fenfluramine has a central excitatory effect. Common side effects and central effects include dry mouth, diarrhea, drowsiness, headache, dizziness, gastrointestinal disturbances, insomnia, changes in blood pressure (mostly hypotension), restlessness, palpitations, and sweating.
  • the methods for analyzing appetite suppressants such as fenfluramine are mainly based on various chromatographic techniques, including HPLC (APCL-MS), LCMS/MS (Li Yin, et al. LC-MS/MS detects 4 kinds of weight loss health care products Research on illegal drugs [J]. New technology and new technology, 2017 (01): 66-69.), and GC/MS, etc.
  • HPLC APCL-MS
  • LCMS/MS Li Yin, et al. LC-MS/MS detects 4 kinds of weight loss health care products Research on illegal drugs [J].
  • New technology and new technology, 2017 (01): 66-69. New technology and new technology, 2017 (01): 66-69.
  • GC/MS etc.
  • these methods have the characteristics of high detection efficiency, high accuracy, and strong anti-interference ability; however, the equipment required for detection is expensive, the cost is high, the sample pretreatment is complicated, and professional operation is required, which is not suitable for large-scale sample sites. Testing requirements
  • the prior art discloses a rapid detection method for the illegal addition of fenfluramine in health food.
  • the method is to use reagent A to construct an acidic reaction environment, then add reagent B and reagent C, and use nitrite ions in the acidic medium to A diazotization reaction occurs with p-nitroaniline, and then an appropriate amount of alkaline reagent C is added to neutralize the excess acid to adjust a suitable pH environment, and then in this environment, the diazo-p-nitroaniline can be combined with the target substance fenfluridine Coupled with light to generate a red azo compound, judge whether the illegal drug fenfluramine is illegally added according to whether the red color appears, this method uses chemical reaction combined with color development to judge whether it contains fenfluramine, and cannot accurately detect the content of fenfluramine
  • the addition amount, and the chemical reaction reagents will also have an impact on the environment, which does not meet the requirements for on-site testing of large batches of samples.
  • the technical problem to be solved by the present invention is to overcome the defects and deficiencies of the fenfluramine detection method in the prior art, and provide a fenfluramine hapten, artificial antigen, antibody and its preparation method and application.
  • the object of the present invention is to provide two fenfluramine haptens.
  • the purpose of the present invention is also to provide the application of fenfluramine hapten in the preparation of fenfluramine artificial antigen.
  • the object of the present invention is also to provide artificial antigen of fenfluramine.
  • the purpose of the present invention is also to provide the application of the fenfluramine hapten and/or artificial antigen in the preparation of fenfluramine artificial antibody.
  • the purpose of the present invention is also to provide a fenfluramine antibody.
  • the object of the present invention is also to provide a kit for detecting fenfluramine.
  • the object of the present invention is also to provide an immunoassay method for detecting fenfluramine.
  • a kind of fenfluramine hapten is hapten FB or hapten FN, and the structural formula of described hapten FB is as shown in formula (I),
  • the hapten FB is named by systematic nomenclature: 4-((1-(3-(trifluoromethyl)phenyl)propan-2-yl)amino)butyric acid;
  • the hapten FN is named by systematic nomenclature: 6-((3-(trifluoromethyl)phenethyl)amino)hexanoic acid.
  • the preparation method of the hapten FB of the present invention comprises the following steps:
  • m-trifluoromethylpropiophenone and 4-aminobutyric acid methyl ester dissolve m-trifluoromethylpropiophenone and 4-aminobutyric acid methyl ester in methanol, and react overnight at 60°C; add potassium borohydride after cooling to room temperature, and heat to 60°C for 2 hours after the bubbles disappear; separate For purification, dissolve the separated and purified reactant in methanol, then stir with aqueous sodium hydroxide solution for 3-5 hours at room temperature, and adjust the pH to 6-7 after the reaction to obtain the hapten FB.
  • the molar ratio of m-trifluoromethylpropiophenone to methyl 4-aminobutyrate is 1:2-5.
  • the molar ratio of m-trifluoromethylpropiophenone to methyl 4-aminobutyrate is 1:4.
  • the molar ratio of the separated and purified reactants to methanol is 1-1.5:2-5.
  • the molar ratio of the separated and purified reactant to methanol is 1:3.
  • the molar ratio of m-trifluoromethylpropiophenone to potassium borohydride is 1-2:1-3.
  • the molar ratio of m-trifluoromethylpropiophenone to potassium borohydride is 1:1.
  • the preparation method of the hapten FN of the present invention comprises the following steps:
  • 2-(3-trifluoromethylphenyl)ethylamine and 6-bromohexanoic acid methyl ester are dissolved in acetonitrile, potassium carbonate and sodium iodide are added and heated to reflux for 12h, separated and purified, and the separated and purified
  • the reactant was dissolved in methanol, and then stirred with aqueous sodium hydroxide solution at room temperature for 3-5 hours. After the reaction, the pH was adjusted to 6-7 to obtain the hapten FN.
  • the molar ratio of 2-(3-trifluoromethylphenyl)ethylamine to methyl 6-bromohexanoate is 1:3-6.
  • the molar ratio of 2-(3-trifluoromethylphenyl)ethylamine to methyl 6-bromohexanoate is 1:5.
  • the molar ratio of 2-(3-trifluoromethylphenyl)ethylamine, sodium iodide and potassium carbonate is 1-1.5:0.1-1:3-8.
  • the molar ratio of 2-(3-trifluoromethylphenyl)ethylamine, sodium iodide and potassium carbonate is 1:0.1:6.
  • the molar ratio of the separated and purified reactants to methanol is 1-2:2-5.
  • the molar ratio of the separated and purified reactant to methanol is 1:3.
  • hapten FB and/or hapten FN in the preparation of fenfluramine artificial antigen is also within the protection scope of the present invention.
  • a fenfluramine artificial antigen obtained by coupling the hapten FB or the hapten FN to a carrier protein the structural formula of the artificial antigen FB obtained by coupling the hapten FB to the carrier protein is shown in formula (III), Wherein, P is carrier protein,
  • the carrier protein (P) is bovine serum albumin (Bovine serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), lactoferrin (Lactoferrin, LF) or chicken ovalbumin ( ovalbumin, OVA) any one or more.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • KLH keyhole limpet hemocyanin
  • lactoferrin lactoferrin
  • ovalbumin ovalbumin, OVA
  • the preparation method of the artificial antigen FB or the artificial antigen FN of the present invention uses the hapten FB or the hapten FN to couple the carrier protein by the active ester method.
  • the preparation method of the artificial antigen FB comprises the following steps:
  • step (3) Slowly add the hapten FB activation solution in step (1) dropwise to the carrier protein solution in step (2), and react at 4°C for 12 hours;
  • step (3) (4) dialyze the reaction solution obtained in step (3) with PBS buffer solution to obtain the artificial antigen FB.
  • the mass ratio of the hapten FB, NHS and EDC in step (1) is 1:1-2:1.5-2.5.
  • the mass ratio of the hapten FB, NHS and EDC in step (1) is 1:1.5:1.9.
  • the mass volume ratio of the carrier protein to PBS buffer in step (2) is 8 mg:1 mL.
  • the mass ratio of the hapten FB in step (1) to the carrier protein in step (2) is 1-2:1-4.
  • the mass ratio of the hapten FB in step (1) to the carrier protein in step (2) is 1:3.
  • the preparation method of the artificial antigen FN is the same as that of the artificial antigen FB.
  • a fenfluramine artificial antigen combination includes an immunogen and a coating source, the immunogen is obtained by coupling the hapten FB to a carrier protein, that is, the artificial antigen FB; the coating source is the fenfluramine artificial antigen.
  • the coating is obtained by coupling the hapten FN to a carrier protein, that is, the artificial antigen FN.
  • the immunogen is obtained by coupling the hapten FB to the carrier protein lactoferrin (LF), i.e. the artificial antigen FB-LF; the coating is obtained by coupling the hapten FN to the carrier protein chicken ovalbumin (OVA) obtained, that is, the artificial antigen FN-OVA.
  • LF carrier protein lactoferrin
  • OVA carrier protein chicken ovalbumin
  • the application of the artificial antigen combination in preparing fenfluramine antibody and/or detecting fenfluramine is also within the protection scope of the present invention.
  • a fenfluramine antibody is prepared by immunizing animals with the artificial antigen FB obtained by coupling the hapten FB to a carrier protein.
  • the fenfluramine antibody is prepared by immunizing animals with the artificial antigen FB-LF obtained by coupling the hapten FB to the carrier protein lactoferrin (LF).
  • FB-LF carrier protein lactoferrin
  • the fenfluramine antibody is a monoclonal antibody or a polyclonal antibody.
  • a method for preparing polyclonal antibody against fenfluramine is prepared by immunizing experimental animals with artificial antigen FB obtained by coupling carrier protein with said hapten FB.
  • the preparation method of fenfluramine polyclonal antibody comprises the following steps:
  • the artificial antigen FB is the artificial antigen FB-LF obtained by coupling the hapten FB to the carrier protein lactoferrin (LF).
  • LF carrier protein lactoferrin
  • fenfluramine antibody in detecting fenfluramine and/or preparing a kit for detecting fenfluramine is also within the protection scope of the present invention.
  • a kit for detecting fenfluramine comprising the fenfluramine artificial antigen and the fenfluramine antibody.
  • the kit includes the artificial antigen FN obtained by coupling the hapten FN to a carrier protein and the antibody prepared by immunizing animals with the artificial antigen FB obtained by coupling the hapten FB to a carrier protein.
  • the artificial antigen FN is the artificial antigen FN-OVA obtained by coupling the hapten FN to the carrier protein chicken ovalbumin (OVA), and the artificial antigen FB is the hapten FB coupled to the carrier protein milk Antibody prepared by immunizing animals with artificial antigen FB-LF obtained from ferritin (LF).
  • OVA carrier protein chicken ovalbumin
  • LF ferritin
  • the kit also includes one or more of an ELISA plate, a fenfluramine standard, an enzyme conjugate, a chromogenic solution, a stop solution or a washing solution.
  • the kit also includes the fenfluramine artificial antigen-coated ELISA plate, fenfluramine standard, enzyme conjugate, color developing solution, stop solution and concentrated washing solution.
  • the immunoassay method is a method for non-diagnostic and therapeutic purposes.
  • the artificial antigen FB obtained by coupling the hapten FN to a carrier protein is used as an antigen.
  • the artificial antigen FN-OVA coupled to the carrier protein chicken ovalbumin (OVA) is used as an antigen
  • the artificial antigen FB-LF coupled to the carrier protein lactoferrin (LF) is used as an immunogen
  • Antibodies prepared from immunized animals are used as detection antibodies for detection.
  • the immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensing, immunocolloidal gold, and the like.
  • the present invention has the following beneficial effects:
  • the present invention provides two kinds of fenfluramine haptens, hapten FB and hapten FN, use hapten FB and hapten FN coupling carrier protein to obtain artificial antigen FB-LF and artificial antigen FN-OVA;
  • the hapten FB has a high degree of overlap with the skeleton structure of the test substance fenfluramine, which effectively improves the immunogenicity of the fenfluramine artificial antigen FB-LF, and the structure of the artificial antigen FB-LF and the hapten FB is quite different , forming a larger steric hindrance, further improving the affinity of the antibody;
  • the fenfluramine artificial antigen FB-LF was used as an immunogen to immunize New Zealand white rabbits, and the antibodies were purified by octanoic acid-ammonium sulfate method. The resulting antibody has high titer, strong specificity, and high affinity.
  • the minimum detection limit LOD of the antibody to fenfluramine is 0.63ng/mL, the half-inhibitory concentration IC50 is 10.22ng/mL, and the quantitative detection range is 1.76 ⁇ 59.13ng/mL, with high detection sensitivity and wide linear range; the cross-reactivity rate to fenfluramine is 100%, and to fenfluramine analogues phentermine, chlorphentermine, benzomorphine, amphetamine There was no cross-reactivity with predone.
  • the antibody of the present invention has the characteristics of simplicity, rapidity, strong specificity, wide linear range, and high sensitivity, and has good application prospects and broad development space in the rapid and effective detection of fenfluramine. Utilizing the fenfluramine hapten, artificial antigen and antibody of the present invention can realize the purpose of fast and accurate detection of fenfluramine.
  • Fig. 1 is a synthetic route diagram of the hapten FB of Example 1 of the present invention.
  • Fig. 2 is a synthetic route diagram of the hapten FN of Example 1 of the present invention.
  • Fig. 3 is an ultraviolet scanning diagram of the haptens FB, LF, and FB-LF of Example 2 of the present application.
  • Fig. 4 is an ultraviolet scanning diagram of the haptens FN, OVA, and FN-OVA of Example 2 of the present application.
  • Fig. 5 is the antibody indirect competition ELISA standard curve of fenfluramine in Example 5 of the present application.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • hapten FB Separate and purify the reactant, then dissolve the separated and purified reactant in methanol, the molar ratio of the separated and purified reactant to methanol is 1:3, add 1mol/L sodium hydroxide aqueous solution and stir at room temperature for 3-5h, after the reaction Use hydrochloric acid with a concentration of 1mol/L to adjust the pH to 6-7 to obtain the hapten FB.
  • the synthetic route diagram of hapten FB is shown in Fig. 1 .
  • the mass spectrometry results of the hapten FB are: MS: C 15 H 20 F 3 NO 2 : 289.13, ESI-[MH] ⁇ : 288.1.
  • the hapten FB is named by systematic nomenclature: 4-((1-(3-(trifluoromethyl)phenyl)propan-2-yl)amino)butanoic acid.
  • the mass spectrum results of the hapten FN are: MSC 13 H 16 F 3 NO 2 : 303.14, ESI-[MH] ⁇ : 302.1.
  • the hapten FN is named by systematic nomenclature: 6-((3-(trifluoromethyl)phenethyl)amino)hexanoic acid.
  • the hapten FB or hapten FN prepared in Example 1 was coupled to lactoferrin (LF) or chicken ovalbumin (OVA) by the active ester method.
  • PBS buffer solution Na 2 HPO 4 ⁇ 12H 2 O 2.90g, NaCl 8.50g, KCl 0.20g, KH 2 PO 4 0.20g, add distilled water to 1000mL.
  • the preparation of the artificial antigen FN is the same as that of the artificial antigen FB, the only difference is that the carrier protein is chicken ovalbumin.
  • UV scanning was carried out on LF, hapten FB and the artificial antigen FB-LF synthesized above.
  • the UV scanning results are shown in Figure 3.
  • LF, hapten FB, and FB-LF were identified by ultraviolet (200-350nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of FB-LF was significantly different from that of the carrier protein LF.
  • FB has a characteristic peak at 240nm and 300nm respectively, and after coupling LF, the absorption peaks of FB-LF are obviously higher than LF at 240nm and 300nm, and the curve relative to the hapten FB has a significant shift.
  • the characteristic peak of the drug that appears in the coupling product is contributed by the protein-bound drug molecule, so the reaction product is the carrier protein LF and The complex of the hapten FB indicates that the FB-LF coupling is successful.
  • UV scanning was performed on OVA, the hapten FN and the artificial antigen FN-OVA synthesized above.
  • the UV scanning results are shown in Figure 4.
  • hapten FN and FN-OVA were identified by ultraviolet (200-350nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of FN-OVA was significantly different from that of the carrier protein OVA, and the hapten FN has a characteristic peak at 240nm and 260nm, and after coupling with OVA, the absorption peaks of FN-OVA are significantly higher than OVA at 240nm and 260nm, and the curve relative to the hapten FN has a significant shift.
  • the characteristic peak of the drug that appears in the coupling product is contributed by the protein-bound drug molecule, so it shows that the reaction product is the carrier protein OVA and The complex of hapten FN indicates that the coupling of FN-OVA is successful.
  • the FB-LF prepared in Example 2 was used as the immunogen and immune adjuvant (incomplete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for subsequent booster immunizations) in a volume ratio of 1:1 to emulsify evenly, Immunization of New Zealand white rabbits.
  • the New Zealand white rabbits weigh 2.5-3 kg, and are injected subcutaneously at multiple points on the neck and back, and are immunized for the second time after 4 weeks, and boosted immunization once every 3 weeks thereafter.
  • blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA.
  • the ear vein is used to boost the immunization.
  • blood was collected from the heart, and the method of obtaining serum from the collected blood was as follows: incubate at 37°C for 0.5-1 hour, then let it stand overnight at 4°C, then suck the precipitated serum with a straw, and then incubate at 4°C for 3000- Centrifuge at 5000rpm for 10min, and take the supernatant.
  • the antiserum was purified by ammonium sulfate precipitation to polyclonal antibodies, and stored at -20°C for future use.
  • the present invention also prepared artificial antigen FB-BSA with bovine serum albumin (BSA) as carrier protein and artificial antigen FB-BSA with chicken ovalbumin (OVA) as carrier protein according to the preparation method of FB-LF of embodiment 2. OVA, and all were successfully coupled.
  • BSA bovine serum albumin
  • OVA chicken ovalbumin
  • the FB-BSA prepared and the FB-LF prepared in Example 2 were used as immunogens respectively, and the fenfluramine antibody prepared by immunizing New Zealand white rabbits according to the method of Example 3 was used for coating source screening, so that the prepared FB-OVA Using the FN-OVA prepared in Example 2 as the coating source, the titer and inhibition rate of the antiserum obtained from immunizing New Zealand white rabbits were detected by ELISA.
  • Titer column first add 50 ⁇ L of PBST to each well, then add the antibody obtained by doubling dilution to each well in turn at 50 ⁇ L per well, and replace the last well with 50 ⁇ L of PBST without adding antibody;
  • Inhibition column first add 50 ⁇ L of drug to each well, then add 50 ⁇ L of the antibody obtained by doubling dilution into each well in turn, and replace the last well with 50 ⁇ L of PBST without adding antibody; incubate at 37°C for 40 minutes, wash 5 times , make a decision;
  • the titer is the dilution of antiserum corresponding to an OD 450 of about 1.0.
  • Inhibition rate (OD value of titer - OD value of inhibition) / OD value of inhibition * 100%
  • Table 1 shows the titers and inhibition rates of the antisera of the four groups of immunogens and coating agents.
  • Table 1 The potency and inhibition rate of the antiserum of 4 groups of immunogens and coating original combinations
  • fenfluramine artificial antigens are used as the antisera produced by immunized New Zealand white rabbits to have certain titers, and the obtained antisera have different degrees of inhibition to the target analyte fenfluramine Effect.
  • the antiserum titer 1:128000 and inhibition rate 88.36% shown in the combination of the immunogen and the coating original structure of No. 3 are the best combination; under this combination, the fenfluramine antibody can not only specifically recognize the target analysis fenfluramine, and the sensitivity of the antibody is good; the titer and inhibition rate of the antiserum are higher than the combination of the immunogen and the coating source of No. good combination. That is, FB-LF was used as the immunogen, and FN-OVA was used as the coating source.
  • a kind of indirect competition ELISA method that detects fenfluramine comprises the following steps:
  • Example 2 Use the artificial antigen FN-OVA prepared in Example 2 as the coating source, dilute it to 62.5ng/mL with the coating solution, coat the 96-well microtiter plate, add 100 ⁇ L to each well, and incubate overnight at 37°C (12h) ;
  • Example 4 Dilute the polyclonal antibody prepared in Example 3 with the PBST of Example 4 1:4000 times, and dilute the fenfluramine drug to be tested to 1000ng/mL, 250ng/mL, 62.5ng/mL, 15.63ng/mL mL, 3.9ng/mL, 0.98ng/mL, 0.244ng/mL, 0.06ng/mL, 0.015ng/mL;
  • the antibody indirect competition ELISA standard curve that is used to detect fenfluramine drug is as shown in Figure 5, as can be seen from Figure 5, is used to detect the antibody half-inhibitory concentration (IC 50 ) of fenfluramine drug to be 10.22ng/mL, quantitative detection The range is 1.76-59.13ng/mL, and the lower limit of detection (LOD) is 0.63ng/mL; it shows that the antibody prepared by the present invention for detecting fenfluramine has high sensitivity and can meet the detection requirements.
  • IC 50 antibody half-inhibitory concentration
  • LOD lower limit of detection
  • Embodiment 6 is used to detect the specificity evaluation of the antibody of fenfluramine
  • the specificity of the antibody used to detect fenfluramine is determined by the cross-reaction experiment of the fenfluramine antibody and the fenfluramine drug and its analogues.
  • the specificity of the antibody is expressed by the cross-reaction rate (CR), and the cross-reaction The smaller the rate, the stronger the specificity.
  • Fenfluramine and its analogs phentermine (PHE), chlorphentermine (CPT), phenmetrazine (PHM) and anfepramone (AFP) were used as times respectively Ratio dilution, adopt indirect competition ELISA method to measure, and step is with embodiment 5, obtains the IC of each analog 50 value, calculates fenfluramine cross-reactivity rate (CR) according to the following formula
  • the cross-reactivity rate of the antibody used to detect fenfluramine to fenfluramine is 100%, and the IC50 is 10.22ng/mL, and to fenfluramine analogues phentermine, chlorphentermine , benzomorphine, and amfeprarone have no crossover; it shows that the antibody used to detect fenfluramine has high recognition ability and strong specificity for fenfluramine, and can effectively exclude fenfluramine analogue benzene
  • the interference of butylamine, chlorphentermine, benzomorphine, and amfepramone on the detection of fenfluramine can be specially used for the detection of fenfluramine.
  • a kit for detecting fenfluramine comprising the following parts:
  • the coating buffer is used to dilute the coating source to 31.25 ⁇ g/ L, add 100 ⁇ L to each well, incubate overnight at 37°C in the dark, pour off the liquid in the well, wash twice with washing solution for 30 seconds each time, pat dry, then add 200 ⁇ L of blocking solution to each well, and incubate at 25°C in the dark for 2 hours , Pour off the liquid in the hole and pat dry, after drying, seal it with an aluminum film and store it in a vacuum; the coating buffer is a carbonate buffer with a pH value of 9.6 and 0.05mol/L, and the blocking solution is a pH value of 7.1 to 7.5, containing 1%-3% casein by mass ratio, 0.1-0.3mol/L phosphate buffer;
  • Fenfluramine standard solution 8 concentration gradients, respectively 1000 ⁇ g/L, 200 ⁇ g/L, 40 ⁇ g/L, 8 ⁇ g/L, 1.6 ⁇ g/L, 0.32 ⁇ g/L, 0.064 ⁇ g/L, 0.0128 ⁇ g/L;
  • Enzyme conjugate the fenfluramine polyclonal antibody prepared in Example 3 labeled with horseradish peroxidase;
  • Substrate chromogenic solution composed of A liquid and B liquid, A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;
  • the stop solution is 2mol/L H 2 SO 4 ;
  • the washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01 ⁇ to 0.03 ⁇ sodium azide preservative, and 0.1 to 0.3mol/L phosphate buffer, and the percentage is weight volume percentage.
  • the percent absorbance of a standard or sample is equal to the average of the absorbance values of the standard or sample (double wells) divided by the average of the absorbance of the first standard (0 ⁇ g/L), and multiplied by 100%. Take the percent absorbance of the standard substance as the ordinate, and take the logarithm of the concentration of fenfluramine standard substance ( ⁇ g/L) as the abscissa to draw a standard curve. Substitute the percent absorbance of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and multiply it by the corresponding dilution factor to obtain the actual concentration of fenfluramine in the sample.

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Abstract

Provided in the present invention are a fenfluramine hapten, and a preparation method therefor and the use thereof, a fenfluramine artificial antigen, and a fenfluramine antibody. According to the present invention, two haptens, namely hapten FB and hapten FN, are prepared, artificial antigen FB-LF is obtained by means of coupling hapten FB to carrier protein LF, a specific antibody for detecting fenfluramine is further prepared, and artificial antigen FN-OVA is used as a coatingen. The antibody has a good sensitivity and specificity to fenfluramine, with a half-maximal inhibitory concentration of 10.22 ng/mL, a lowest detection limit of 0.63 ng/mL and a quantitative detection range of 1.76-59.13 ng/mL, has no cross reaction to fenfluramine analogs, and can specifically detect fenfluramine. Provided in the present invention is an immunoassay method for fenfluramine, which achieves the purpose of rapidly and accurately detecting fenfluramine.

Description

一种芬氟拉明半抗原、人工抗原、抗体及其制备方法和应用A kind of fenfluramine hapten, artificial antigen, antibody and its preparation method and application 技术领域technical field
本发明涉及食品检测技术领域，更具体地，涉及一种芬氟拉明半抗原、人工抗原、抗体及其制备方法和应用。The invention relates to the technical field of food detection, and more specifically relates to a fenfluramine hapten, an artificial antigen, an antibody and a preparation method and application thereof.
背景技术Background technique
芬氟拉明(fenfluramine)在治疗剂量下是抑制中枢的，具有镇静作用。它促进神经末梢释放5-羟色胺，抑制5-羟色胺在摄取和激动5-羟色胺受体，兴奋下丘脑饱食中枢，从而引起食欲减退。它还能增加葡萄糖的利用，降低血糖。在大剂量下(右旋)芬氟拉明具有兴奋中枢作用。常见的副作用及其中枢效应有口干、腹泻、昏睡、头痛、眩晕、肠胃功能紊乱、失眠、血压的改变(大多为低血压)、不安、心悸、出汗等。单独且长时间使用芬氟拉明减肥时会出现心脏瓣膜损伤的情况。为此，1997年9月FDA发出声明敦促生产商停止芬氟拉明与右旋的市场销售。随后这两种药物相继退出全球市场。我国也已禁止在减肥类保健品中使用芬氟拉明，但仍允许其作为治疗单纯性肥胖症的处方药使用。Fenfluramine (fenfluramine) is a central inhibitory agent at therapeutic doses and has a sedative effect. It promotes the release of 5-HT from nerve endings, inhibits 5-HT uptake and excites 5-HT receptors, and stimulates the hypothalamic satiety center, thereby causing loss of appetite. It also increases glucose utilization and lowers blood sugar. In large doses (dextrorotary) fenfluramine has a central excitatory effect. Common side effects and central effects include dry mouth, diarrhea, drowsiness, headache, dizziness, gastrointestinal disturbances, insomnia, changes in blood pressure (mostly hypotension), restlessness, palpitations, and sweating. Heart valve damage has been reported when fenfluramine is used alone and for prolonged periods of time for weight loss. For this reason, in September 1997, the FDA issued a statement urging manufacturers to stop the marketing of fenfluramine and dextromethorphan. Both drugs were subsequently withdrawn from the global market. my country has also banned the use of fenfluramine in weight-loss health products, but it is still allowed to be used as a prescription drug for the treatment of simple obesity.
目前，分析芬氟拉明等食欲抑制剂的方法以各种色谱技术为主，包括HPLC(APCL-MS)、LCMS/MS(李荫,等.LC-MS/MS检测减肥保健品中4种违禁药物研究[J].新技术新工艺,2017(01):66-69.)、及GC/MS等。然而，这些方法虽然具有检测效率高、准确度高、抗干扰能力强等特点；但是，检测所需的仪器设备昂贵、成本高、样品前处理繁杂、需专业人士操作，不符合大批量样品现场检测要求。At present, the methods for analyzing appetite suppressants such as fenfluramine are mainly based on various chromatographic techniques, including HPLC (APCL-MS), LCMS/MS (Li Yin, et al. LC-MS/MS detects 4 kinds of weight loss health care products Research on illegal drugs [J]. New technology and new technology, 2017 (01): 66-69.), and GC/MS, etc. However, although these methods have the characteristics of high detection efficiency, high accuracy, and strong anti-interference ability; however, the equipment required for detection is expensive, the cost is high, the sample pretreatment is complicated, and professional operation is required, which is not suitable for large-scale sample sites. Testing requirements.
现有技术公开了一种保健食品中非法添加违禁药物芬氟拉明的快速检测方法，该方法是利用试剂A构造酸性反应环境，然后加入试剂B及试剂C，利用亚硝酸离子在酸性介质中与对硝基苯胺发生重氮化反应，再加入适量的碱性试剂C中和过量的酸，调节适宜的pH环境，然后在该环境中使重氮对硝基苯胺可与目标物质芬氟拉明偶联，生成红色偶氮化合物，根据是否出现红色判断是否非法添加了违禁药物芬氟拉明，该方法利用化学反应结合显色判断是否含有芬氟拉明，不能准确检测芬氟拉明的添加量，且化学反应的试剂也会对环境造成影响，不符合大批量样品现场检测要求。现有技术中也还未见有关于芬氟拉明半抗原、人工 抗原、抗体的相关报道。The prior art discloses a rapid detection method for the illegal addition of fenfluramine in health food. The method is to use reagent A to construct an acidic reaction environment, then add reagent B and reagent C, and use nitrite ions in the acidic medium to A diazotization reaction occurs with p-nitroaniline, and then an appropriate amount of alkaline reagent C is added to neutralize the excess acid to adjust a suitable pH environment, and then in this environment, the diazo-p-nitroaniline can be combined with the target substance fenfluridine Coupled with light to generate a red azo compound, judge whether the illegal drug fenfluramine is illegally added according to whether the red color appears, this method uses chemical reaction combined with color development to judge whether it contains fenfluramine, and cannot accurately detect the content of fenfluramine The addition amount, and the chemical reaction reagents will also have an impact on the environment, which does not meet the requirements for on-site testing of large batches of samples. In the prior art, there are no related reports about fenfluramine hapten, artificial antigen, antibody.
发明内容Contents of the invention
本发明要解决的技术问题是克服现有技术中芬氟拉明检测方法的缺陷和不足，提供一种芬氟拉明半抗原、人工抗原、抗体及其制备方法和应用。The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the fenfluramine detection method in the prior art, and provide a fenfluramine hapten, artificial antigen, antibody and its preparation method and application.
本发明的目的在于提供两种芬氟拉明半抗原。The object of the present invention is to provide two fenfluramine haptens.
本发明的目的还在于提供芬氟拉明半抗原在制备芬氟拉明人工抗原中的应用。The purpose of the present invention is also to provide the application of fenfluramine hapten in the preparation of fenfluramine artificial antigen.
本发明的目的还在于提供芬氟拉明人工抗原。The object of the present invention is also to provide artificial antigen of fenfluramine.
本发明的目的还在于提供所述芬氟拉明半抗原和/或人工抗原在制备芬氟拉明人工抗体中的应用。The purpose of the present invention is also to provide the application of the fenfluramine hapten and/or artificial antigen in the preparation of fenfluramine artificial antibody.
本发明的目的还在于提供一种芬氟拉明抗体。The purpose of the present invention is also to provide a fenfluramine antibody.
本发明的目的还在于提供一种检测芬氟拉明的试剂盒。The object of the present invention is also to provide a kit for detecting fenfluramine.
本发明的目的还在于提供一种检测芬氟拉明的免疫分析方法。The object of the present invention is also to provide an immunoassay method for detecting fenfluramine.
本发明上述目的通过以下技术方案实现：The above object of the present invention is achieved through the following technical solutions:
一种芬氟拉明半抗原，为半抗原FB或半抗原FN，所述半抗原FB的结构式如式(I)所示，A kind of fenfluramine hapten is hapten FB or hapten FN, and the structural formula of described hapten FB is as shown in formula (I),
Figure PCTCN2021136186-appb-000001
Figure PCTCN2021136186-appb-000001
所述半抗原FB采用系统命名法命名为：4-((1-(3-(三氟甲基)苯基)丙-2-基)氨基)丁酸；The hapten FB is named by systematic nomenclature: 4-((1-(3-(trifluoromethyl)phenyl)propan-2-yl)amino)butyric acid;
所述半抗原FN的结构式如式(II)所示，The structural formula of the hapten FN is shown in formula (II),
Figure PCTCN2021136186-appb-000002
Figure PCTCN2021136186-appb-000002
所述半抗原FN采用系统命名法命名为：6-((3-(三氟甲基)苯乙基)氨基)己酸。The hapten FN is named by systematic nomenclature: 6-((3-(trifluoromethyl)phenethyl)amino)hexanoic acid.
本发明所述半抗原FB的制备方法，包括如下步骤：The preparation method of the hapten FB of the present invention comprises the following steps:
将间三氟甲基苯丙酮与4-氨基丁酸甲酯溶于甲醇中，充分反应后；冷却至室温，然后硼氢化钾，充分反应；分离纯化，将分离纯化后的反应物溶解于甲醇，然后充分水解，调节pH为6～7，即得半抗原FB。Dissolve m-trifluoromethylpropiophenone and 4-aminobutyric acid methyl ester in methanol, after fully reacting; cool to room temperature, then potassium borohydride, fully react; separate and purify, dissolve the reactant after separation and purification in methanol , and then fully hydrolyzed to adjust the pH to 6-7 to obtain the hapten FB.
优选地，将间三氟甲基苯丙酮与4-氨基丁酸甲酯溶于甲醇中，60℃反应过夜；冷却至室温后加入硼氢化钾，待气泡消失后加热至60℃反应2h；分离纯化，将分离纯化后的反应物溶解于甲醇，然后与氢氧化钠水溶液在室温下搅拌3～5h，反应结束后调节pH为6～7，即得半抗原FB。Preferably, dissolve m-trifluoromethylpropiophenone and 4-aminobutyric acid methyl ester in methanol, and react overnight at 60°C; add potassium borohydride after cooling to room temperature, and heat to 60°C for 2 hours after the bubbles disappear; separate For purification, dissolve the separated and purified reactant in methanol, then stir with aqueous sodium hydroxide solution for 3-5 hours at room temperature, and adjust the pH to 6-7 after the reaction to obtain the hapten FB.
优选地，所述间三氟甲基苯丙酮与4-氨基丁酸甲酯的摩尔比为1:2～5。Preferably, the molar ratio of m-trifluoromethylpropiophenone to methyl 4-aminobutyrate is 1:2-5.
进一步优选地，所述间三氟甲基苯丙酮与4-氨基丁酸甲酯的摩尔比为1:4。Further preferably, the molar ratio of m-trifluoromethylpropiophenone to methyl 4-aminobutyrate is 1:4.
优选地，所述分离纯化后的反应物与甲醇的摩尔比为1～1.5:2～5。Preferably, the molar ratio of the separated and purified reactants to methanol is 1-1.5:2-5.
进一步优选地，所述分离纯化后的反应物与甲醇的摩尔比为1:3。Further preferably, the molar ratio of the separated and purified reactant to methanol is 1:3.
优选地，所述间三氟甲基苯丙酮与硼氢化钾的摩尔比为1～2:1～3。Preferably, the molar ratio of m-trifluoromethylpropiophenone to potassium borohydride is 1-2:1-3.
进一步优选地，所述间三氟甲基苯丙酮与硼氢化钾的摩尔比为1:1。Further preferably, the molar ratio of m-trifluoromethylpropiophenone to potassium borohydride is 1:1.
本发明所述半抗原FN的制备方法，包括如下步骤：The preparation method of the hapten FN of the present invention comprises the following steps:
将2-(3-三氟甲基苯基)乙胺与6-溴己酸甲酯溶于乙腈中，加入碳酸钾和碘化钠充分反应，分离纯化，将分离纯化后的反应物溶解于甲醇，然后充分水解，调节pH为6～7，即得半抗原FN。Dissolve 2-(3-trifluoromethylphenyl)ethylamine and methyl 6-bromohexanoate in acetonitrile, add potassium carbonate and sodium iodide to fully react, separate and purify, and dissolve the reactant after separation and purification in methanol, and then fully hydrolyzed to adjust the pH to 6-7 to obtain the hapten FN.
优选地，将2-(3-三氟甲基苯基)乙胺与6-溴己酸甲酯溶于乙腈中，加入碳酸钾和碘化钠加热回流12h，分离纯化，将分离纯化后的反应物溶解于甲醇，然后与氢氧化钠水溶液在室温下搅拌3～5h，反应结束后调节pH为6～7，即得半抗原FN。Preferably, 2-(3-trifluoromethylphenyl)ethylamine and 6-bromohexanoic acid methyl ester are dissolved in acetonitrile, potassium carbonate and sodium iodide are added and heated to reflux for 12h, separated and purified, and the separated and purified The reactant was dissolved in methanol, and then stirred with aqueous sodium hydroxide solution at room temperature for 3-5 hours. After the reaction, the pH was adjusted to 6-7 to obtain the hapten FN.
优选地，所述2-(3-三氟甲基苯基)乙胺与6-溴己酸甲酯的摩尔比为1:3～6。Preferably, the molar ratio of 2-(3-trifluoromethylphenyl)ethylamine to methyl 6-bromohexanoate is 1:3-6.
进一步优选地，所述2-(3-三氟甲基苯基)乙胺与6-溴己酸甲酯的摩尔比为1:5。Further preferably, the molar ratio of 2-(3-trifluoromethylphenyl)ethylamine to methyl 6-bromohexanoate is 1:5.
优选地，所述2-(3-三氟甲基苯基)乙胺、碘化钠与碳酸钾的摩尔比为1～1.5:0.1～1:3～8。Preferably, the molar ratio of 2-(3-trifluoromethylphenyl)ethylamine, sodium iodide and potassium carbonate is 1-1.5:0.1-1:3-8.
进一步优选地，所述2-(3-三氟甲基苯基)乙胺、碘化钠与碳酸钾的摩尔比为1:0.1:6。Further preferably, the molar ratio of 2-(3-trifluoromethylphenyl)ethylamine, sodium iodide and potassium carbonate is 1:0.1:6.
优选地，所述分离纯化后的反应物与甲醇的摩尔比为1～2:2～5。Preferably, the molar ratio of the separated and purified reactants to methanol is 1-2:2-5.
进一步优选地，所述分离纯化后的反应物与甲醇的摩尔比为1:3。Further preferably, the molar ratio of the separated and purified reactant to methanol is 1:3.
所述半抗原FB和/或半抗原FN在制备芬氟拉明人工抗原中的应用也在本发明的保护范围之内。The application of the hapten FB and/or hapten FN in the preparation of fenfluramine artificial antigen is also within the protection scope of the present invention.
一种芬氟拉明人工抗原，由所述半抗原FB或半抗原FN偶联载体蛋白得到；由所述半抗原FB偶联载体蛋白得到的人工抗原FB的结构式如式(III)所示，其中，P为载体蛋白，A fenfluramine artificial antigen obtained by coupling the hapten FB or the hapten FN to a carrier protein; the structural formula of the artificial antigen FB obtained by coupling the hapten FB to the carrier protein is shown in formula (III), Wherein, P is carrier protein,
Figure PCTCN2021136186-appb-000003
Figure PCTCN2021136186-appb-000003
由所述半抗原FN偶联载体蛋白得到的人工抗原FN的结构式如式(IV)所示，其中，P为载体蛋白，The structural formula of the artificial antigen FN obtained by coupling the hapten FN to the carrier protein is shown in formula (IV), wherein, P is the carrier protein,
Figure PCTCN2021136186-appb-000004
Figure PCTCN2021136186-appb-000004
优选地，所述载体蛋白(P)为牛血清白蛋白(Bovine serum albumin，BSA)、钥孔血蓝蛋白(Keyhole limpet hemocyanin，KLH)、乳铁蛋白(Lactoferrin，LF)或者鸡卵清白蛋白(ovalbumin，OVA)任意一种或几种。Preferably, the carrier protein (P) is bovine serum albumin (Bovine serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), lactoferrin (Lactoferrin, LF) or chicken ovalbumin ( ovalbumin, OVA) any one or more.
本发明所述人工抗原FB或人工抗原FN的制备方法，利用所述半抗原FB或半抗原FN，通过活泼酯法偶联载体蛋白。The preparation method of the artificial antigen FB or the artificial antigen FN of the present invention uses the hapten FB or the hapten FN to couple the carrier protein by the active ester method.
作为上述方法的一个具体实施方式，所述人工抗原FB的制备方法，包括如下步骤：As a specific embodiment of the above method, the preparation method of the artificial antigen FB comprises the following steps:
(1)将所述半抗原FB与N-羟基丁二酰亚胺(NHS)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶解于N,N-二甲基甲酰胺(DMF)中，室温下避光搅拌2～4h，得到半抗原FB活化液；(1) Dissolve the hapten FB, N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in In N,N-dimethylformamide (DMF), stir at room temperature in the dark for 2-4 hours to obtain the hapten FB activation solution;
(2)将载体蛋白加入到PBS缓冲液中；(2) adding the carrier protein to the PBS buffer;
(3)将步骤(1)的半抗原FB活化液缓慢逐滴加入步骤(2)的载体蛋白溶液中，4℃反应12h；(3) Slowly add the hapten FB activation solution in step (1) dropwise to the carrier protein solution in step (2), and react at 4°C for 12 hours;
(4)用PBS缓冲液透析步骤(3)所得反应液，即得人工抗原FB。(4) dialyze the reaction solution obtained in step (3) with PBS buffer solution to obtain the artificial antigen FB.
优选地，步骤(1)中所述半抗原FB、NHS与EDC的质量比为1:1～2:1.5～2.5。Preferably, the mass ratio of the hapten FB, NHS and EDC in step (1) is 1:1-2:1.5-2.5.
更优选地，步骤(1)中所述半抗原FB、NHS与EDC的质量比为1:1.5:1.9。More preferably, the mass ratio of the hapten FB, NHS and EDC in step (1) is 1:1.5:1.9.
优选地，步骤(2)中所述载体蛋白与PBS缓冲液的质量体积比为8mg:1mL。Preferably, the mass volume ratio of the carrier protein to PBS buffer in step (2) is 8 mg:1 mL.
优选地，步骤(1)中所述半抗原FB与步骤(2)中所述载体蛋白的质量比为1～2:1～4。Preferably, the mass ratio of the hapten FB in step (1) to the carrier protein in step (2) is 1-2:1-4.
更优选地，步骤(1)中所述半抗原FB与步骤(2)中所述载体蛋白的质量比为1:3。More preferably, the mass ratio of the hapten FB in step (1) to the carrier protein in step (2) is 1:3.
所述人工抗原FN的制备方法同人工抗原FB。The preparation method of the artificial antigen FN is the same as that of the artificial antigen FB.
所述芬氟拉明人工抗原在制备芬氟拉明抗体中的应用也在本发明的保护范围之内。The application of the fenfluramine artificial antigen in the preparation of fenfluramine antibody is also within the protection scope of the present invention.
一种芬氟拉明人工抗原组合，包括免疫原和包被原，免疫原由所述半抗原FB偶联载体蛋白得到的，即人工抗原FB；包被原为所述芬氟拉明人工抗原。A fenfluramine artificial antigen combination includes an immunogen and a coating source, the immunogen is obtained by coupling the hapten FB to a carrier protein, that is, the artificial antigen FB; the coating source is the fenfluramine artificial antigen.
优选地，所述包被原由所述半抗原FN偶联载体蛋白得到的，即人工抗原FN。Preferably, the coating is obtained by coupling the hapten FN to a carrier protein, that is, the artificial antigen FN.
进一步优选地，所述免疫原由所述半抗原FB偶联载体蛋白乳铁蛋白(LF)得到，即人工抗原FB-LF；所述包被原由所述半抗原FN偶联载体蛋白鸡卵清白蛋白(OVA)得到的，即人工抗原FN-OVA。Further preferably, the immunogen is obtained by coupling the hapten FB to the carrier protein lactoferrin (LF), i.e. the artificial antigen FB-LF; the coating is obtained by coupling the hapten FN to the carrier protein chicken ovalbumin (OVA) obtained, that is, the artificial antigen FN-OVA.
所述人工抗原组合在制备芬氟拉明抗体和/或检测芬氟拉明中的应用也在本发明的保护范围之内。The application of the artificial antigen combination in preparing fenfluramine antibody and/or detecting fenfluramine is also within the protection scope of the present invention.
一种芬氟拉明抗体，利用所述半抗原FB偶联载体蛋白得到的人工抗原FB免疫动物制备得到。A fenfluramine antibody is prepared by immunizing animals with the artificial antigen FB obtained by coupling the hapten FB to a carrier protein.
优选地，所述芬氟拉明抗体是利用所述半抗原FB偶联载体蛋白乳铁蛋白(LF)得到的人工抗原FB-LF免疫动物制备得到。Preferably, the fenfluramine antibody is prepared by immunizing animals with the artificial antigen FB-LF obtained by coupling the hapten FB to the carrier protein lactoferrin (LF).
优选地，所述芬氟拉明抗体为单克隆抗体或多克隆抗体。Preferably, the fenfluramine antibody is a monoclonal antibody or a polyclonal antibody.
一种芬氟拉明多克隆抗体的制备方法，利用所述半抗原FB偶联载体蛋白得到的人工抗原FB免疫实验动物制备得到。A method for preparing polyclonal antibody against fenfluramine is prepared by immunizing experimental animals with artificial antigen FB obtained by coupling carrier protein with said hapten FB.
作为上述方法的一个具体实施方式，芬氟拉明多克隆抗体的制备方法包括如下步骤：As a specific embodiment of the above method, the preparation method of fenfluramine polyclonal antibody comprises the following steps:
(1)利用所述人工抗原FB配合免疫佐剂免疫实验动物；(1) using the artificial antigen FB to cooperate with an immune adjuvant to immunize experimental animals;
(2)首次免疫时，人工抗原FB与等体积的完全弗氏佐剂乳化后免疫新西兰大白兔；(2) For the first immunization, New Zealand white rabbits were immunized after the artificial antigen FB was emulsified with an equal volume of complete Freund's adjuvant;
(3)4周后第二次免疫，以后每间隔3周加强免疫一次。(3) The second immunization after 4 weeks, and booster immunization every 3 weeks thereafter.
(4)加强免疫四次后，心脏取血分离获得血清即得多克隆抗体。(4) After four times of booster immunization, the blood was collected from the heart to separate the polyclonal antibody.
优选地，所述人工抗原FB是所述半抗原FB偶联载体蛋白乳铁蛋白(LF)得到的人工抗原FB-LF。Preferably, the artificial antigen FB is the artificial antigen FB-LF obtained by coupling the hapten FB to the carrier protein lactoferrin (LF).
所述芬氟拉明抗体在检测芬氟拉明和/或制备检测芬氟拉明试剂盒中的应用也在本发明的保护范围之内。The application of the fenfluramine antibody in detecting fenfluramine and/or preparing a kit for detecting fenfluramine is also within the protection scope of the present invention.
一种检测芬氟拉明的试剂盒，包括所述芬氟拉明人工抗原和所述芬氟拉明抗体。A kit for detecting fenfluramine, comprising the fenfluramine artificial antigen and the fenfluramine antibody.
优选地，所述试剂盒包括所述半抗原FN偶联载体蛋白得到的人工抗原FN和所述半抗原FB偶联载体蛋白得到的人工抗原FB免疫动物制备得到的抗体。Preferably, the kit includes the artificial antigen FN obtained by coupling the hapten FN to a carrier protein and the antibody prepared by immunizing animals with the artificial antigen FB obtained by coupling the hapten FB to a carrier protein.
进一步优选地，所述人工抗原FN是所述半抗原FN偶联载体蛋白鸡卵清白蛋白(OVA)得到的人工抗原FN-OVA，所述人工抗原FB是所述半抗原FB偶联载体蛋白乳铁蛋白(LF)得到的人工抗原FB-LF免疫动物制备得到的抗体。Further preferably, the artificial antigen FN is the artificial antigen FN-OVA obtained by coupling the hapten FN to the carrier protein chicken ovalbumin (OVA), and the artificial antigen FB is the hapten FB coupled to the carrier protein milk Antibody prepared by immunizing animals with artificial antigen FB-LF obtained from ferritin (LF).
优选地，所述试剂盒还包括酶标板、芬氟拉明标准品、酶结合物、显色液、终止液或洗涤液中一种或多种。Preferably, the kit also includes one or more of an ELISA plate, a fenfluramine standard, an enzyme conjugate, a chromogenic solution, a stop solution or a washing solution.
进一步优选地，所述试剂盒还包括所述芬氟拉明人工抗原包被的酶标板、芬氟拉明标准品、酶结合物、显色液、终止液和浓缩洗涤液。Further preferably, the kit also includes the fenfluramine artificial antigen-coated ELISA plate, fenfluramine standard, enzyme conjugate, color developing solution, stop solution and concentrated washing solution.
一种检测芬氟拉明的免疫分析方法，以所述芬氟拉明人工抗原为抗原，以所述半抗原FB偶联载体蛋白得到的人工抗原FB免疫动物制备得到的抗体为检测抗体进行检测；所述免疫分析方法为非诊断治疗目的方法。An immunoassay method for detecting fenfluramine, using the fenfluramine artificial antigen as an antigen, and using the antibody prepared by immunizing animals with the artificial antigen FB obtained by coupling the hapten FB with carrier protein as a detection antibody for detection ; The immunoassay method is a method for non-diagnostic and therapeutic purposes.
优选地，所述免疫分析方法，以所述半抗原FN偶联载体蛋白得到的人工抗原FB为抗原。Preferably, in the immunoassay method, the artificial antigen FB obtained by coupling the hapten FN to a carrier protein is used as an antigen.
优选地，所述免疫分析方法，以偶联载体蛋白鸡卵清白蛋白(OVA)的人工抗原FN-OVA为抗原，以偶联载体蛋白乳铁蛋白(LF)的人工抗原FB-LF为免疫原免疫动物制备得到的抗体为检测抗体进行检测。Preferably, in the immunoassay method, the artificial antigen FN-OVA coupled to the carrier protein chicken ovalbumin (OVA) is used as an antigen, and the artificial antigen FB-LF coupled to the carrier protein lactoferrin (LF) is used as an immunogen Antibodies prepared from immunized animals are used as detection antibodies for detection.
所述免疫分析方法包括但不局限于酶免疫分析、免疫层析、免疫传感、免疫胶体金等。The immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensing, immunocolloidal gold, and the like.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
(1)本发明提供了两种芬氟拉明半抗原，半抗原FB和半抗原FN，应用半抗原FB和半抗原FN偶联载体蛋白得到人工抗原FB-LF和人工抗原FN-OVA；以半抗原FB与待测物芬氟拉明的骨架结构重叠度高，有效的提高了芬氟拉明人工抗原FB-LF的免疫原性，且人工抗原FB-LF与半抗原FB结构差异较大，形成了较大空间位阻，进一步提高抗体亲和力；(1) The present invention provides two kinds of fenfluramine haptens, hapten FB and hapten FN, use hapten FB and hapten FN coupling carrier protein to obtain artificial antigen FB-LF and artificial antigen FN-OVA; The hapten FB has a high degree of overlap with the skeleton structure of the test substance fenfluramine, which effectively improves the immunogenicity of the fenfluramine artificial antigen FB-LF, and the structure of the artificial antigen FB-LF and the hapten FB is quite different , forming a larger steric hindrance, further improving the affinity of the antibody;
(2)利用所述芬氟拉明人工抗原FB-LF作为免疫原免疫新西兰大白兔，采用辛酸-硫酸铵法纯化后为抗体。所得到的抗体效价高、特异性强、亲和力高，利用所述抗体对芬氟拉明的最低检测限LOD为0.63ng/mL，半抑制浓度IC 50为10.22ng/mL，定量检测范围为1.76～59.13ng/mL，检测灵敏度高，线性范围广；对芬氟拉明的交叉反应率为100％，对芬氟拉明类似物苯丁胺、氯苯丁胺、苯甲吗啡、安非泼拉酮的均无交叉反应。 (2) The fenfluramine artificial antigen FB-LF was used as an immunogen to immunize New Zealand white rabbits, and the antibodies were purified by octanoic acid-ammonium sulfate method. The resulting antibody has high titer, strong specificity, and high affinity. The minimum detection limit LOD of the antibody to fenfluramine is 0.63ng/mL, the half-inhibitory concentration IC50 is 10.22ng/mL, and the quantitative detection range is 1.76～59.13ng/mL, with high detection sensitivity and wide linear range; the cross-reactivity rate to fenfluramine is 100%, and to fenfluramine analogues phentermine, chlorphentermine, benzomorphine, amphetamine There was no cross-reactivity with predone.
(3)本发明的抗体具有简便快速、特异性强、线性范围广，灵敏度高的特点，在芬氟拉明的快速有效检测中具有良好的应用前景和广阔的发展空间。利用本发明的芬氟拉明半抗原、人工抗原、抗体可实现快速准确检测芬氟拉明的目的。(3) The antibody of the present invention has the characteristics of simplicity, rapidity, strong specificity, wide linear range, and high sensitivity, and has good application prospects and broad development space in the rapid and effective detection of fenfluramine. Utilizing the fenfluramine hapten, artificial antigen and antibody of the present invention can realize the purpose of fast and accurate detection of fenfluramine.
附图说明Description of drawings
图1为本发明实施例1半抗原FB的合成路线图。Fig. 1 is a synthetic route diagram of the hapten FB of Example 1 of the present invention.
图2为本发明实施例1半抗原FN的合成路线图。Fig. 2 is a synthetic route diagram of the hapten FN of Example 1 of the present invention.
图3为本申请实施例2半抗原FB、LF、FB-LF紫外扫描图。Fig. 3 is an ultraviolet scanning diagram of the haptens FB, LF, and FB-LF of Example 2 of the present application.
图4为本申请实施例2半抗原FN、OVA、FN-OVA紫外扫描图。Fig. 4 is an ultraviolet scanning diagram of the haptens FN, OVA, and FN-OVA of Example 2 of the present application.
图5为本申请实施例5芬氟拉明的抗体间接竞争ELISA标准曲线。Fig. 5 is the antibody indirect competition ELISA standard curve of fenfluramine in Example 5 of the present application.
具体实施方式Detailed ways
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
除非特别说明，以下实施例所用试剂和材料均为市购。Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
实施例1 芬氟拉明半抗原的合成与鉴定Example 1 Synthesis and identification of fenfluramine hapten
1.芬氟拉明半抗原FB的合成与鉴定1. Synthesis and Identification of Fenfluramine Hapten FB
1.1芬氟拉明半抗原FB的合成1.1 Synthesis of fenfluramine hapten FB
以甲醇作为溶剂，将间三氟甲基苯丙酮(1moL)与4-氨基丁酸甲酯(4moL)常温搅拌至固体溶解，60℃反应过夜。第二天待反应冷却至室温后，投入硼氢化钾(1moL)，待气泡消失后加热至60℃反应2h。分离纯化反应物，然后将分离纯化的反应物溶解于甲醇，分离纯化的反应物与甲醇摩尔比为1:3，加入1mol/L的氢氧化钠水溶液在室温下搅拌3～5h，反应结束后用浓度为1mol/L的盐酸调节pH为6～7，即得半抗原FB。半抗原FB的合成路线图如图1所示。Using methanol as a solvent, m-trifluoromethylpropiophenone (1 moL) and methyl 4-aminobutyrate (4 moL) were stirred at room temperature until the solid was dissolved, and reacted overnight at 60°C. The next day, after the reaction was cooled to room temperature, potassium borohydride (1 moL) was added, and heated to 60° C. for 2 h after the bubbles disappeared. Separate and purify the reactant, then dissolve the separated and purified reactant in methanol, the molar ratio of the separated and purified reactant to methanol is 1:3, add 1mol/L sodium hydroxide aqueous solution and stir at room temperature for 3-5h, after the reaction Use hydrochloric acid with a concentration of 1mol/L to adjust the pH to 6-7 to obtain the hapten FB. The synthetic route diagram of hapten FB is shown in Fig. 1 .
1.2芬氟拉明半抗原FB的鉴定1.2 Identification of fenfluramine hapten FB
半抗原FB的核磁鉴定结果： 1H NMR(600MHz,Methanol-d4)δ7.53(s,1H),7.51–7.35(m,3H),3.19–3.12(m,3H),3.01(dd,J＝10.0,6.4Hz,2H),2.94(dd,J＝8.9,6.7Hz,2H),2.15(t,J＝7.3Hz,2H),1.42(d,J＝6.5Hz,4H),1.11(s,J＝7.0Hz,3H). NMR identification results of hapten FB: 1 H NMR (600MHz, Methanol-d4) δ7.53(s,1H),7.51–7.35(m,3H),3.19–3.12(m,3H),3.01(dd,J =10.0,6.4Hz,2H),2.94(dd,J=8.9,6.7Hz,2H),2.15(t,J=7.3Hz,2H),1.42(d,J=6.5Hz,4H),1.11(s ,J＝7.0Hz,3H).
半抗原FB的质谱结果为：MS：C 15H 20F 3NO 2：289.13，ESI-[M-H] -：288.1。 The mass spectrometry results of the hapten FB are: MS: C 15 H 20 F 3 NO 2 : 289.13, ESI-[MH] − : 288.1.
半抗原FB的结构式如式(I)所示：The structural formula of hapten FB is shown in formula (I):
Figure PCTCN2021136186-appb-000005
Figure PCTCN2021136186-appb-000005
半抗原FB采用系统命名法命名为：4-((1-(3-(三氟甲基)苯基)丙-2-基)氨基)丁酸。The hapten FB is named by systematic nomenclature: 4-((1-(3-(trifluoromethyl)phenyl)propan-2-yl)amino)butanoic acid.
2.芬氟拉明半抗原FN的合成与鉴定2. Synthesis and identification of fenfluramine hapten FN
2.1芬氟拉明半抗原FN的合成2.1 Synthesis of fenfluramine hapten FN
以乙腈作为溶剂，将2-(3-三氟甲基苯基)乙胺(1moL)与6-溴己酸甲酯(5moL)，加入碳酸钾(6moL)和碘化钠(0.1moL)加热回流12h，分离纯化反应物。然后将分离纯化的反应物溶解于甲醇，分离纯化的反应物与甲醇的摩尔比为1:3。再加入1mol/L的氢氧化钠水溶液在室温下搅拌3～5h，反应结束后用浓度为1mol/L的盐酸调节pH为6～7，即得半抗原FN。半抗原FN的合成路线图如图2所示。Using acetonitrile as a solvent, add 2-(3-trifluoromethylphenyl)ethylamine (1moL) and 6-bromohexanoic acid methyl ester (5moL), add potassium carbonate (6moL) and sodium iodide (0.1moL) and heat Reflux for 12h, separate and purify the reactants. Then the separated and purified reactant was dissolved in methanol, and the molar ratio of the separated and purified reactant to methanol was 1:3. Then add 1 mol/L sodium hydroxide aqueous solution and stir at room temperature for 3-5 hours. After the reaction, adjust the pH to 6-7 with 1 mol/L hydrochloric acid to obtain the hapten FN. The synthetic route map of the hapten FN is shown in Figure 2.
2.2芬氟拉明半抗原FN的鉴定2.2 Identification of fenfluramine hapten FN
半抗原FN的核磁鉴定结果： 1H NMR(600MHz,Chloroform-d)δ7.41(dd,J＝5.1,3.4Hz,1H),7.38(s,2H),7.35(dd,J＝5.0,1.3Hz,2H),3.46(dd,J＝8.2,6.8Hz,2H),3.19(t,J＝7.0Hz,2H),2.89–2.80(m,2H),2.27(t,J＝8.1Hz,2H),1.94–1.85(m,3H). NMR identification results of the hapten FN: 1 H NMR (600MHz, Chloroform-d) δ7.41(dd, J=5.1,3.4Hz,1H),7.38(s,2H),7.35(dd,J=5.0,1.3 Hz, 2H), 3.46(dd, J=8.2, 6.8Hz, 2H), 3.19(t, J=7.0Hz, 2H), 2.89–2.80(m, 2H), 2.27(t, J=8.1Hz, 2H ),1.94–1.85(m,3H).
半抗原FN的质谱结果为：MSC 13H 16F 3NO 2：303.14，ESI-[M-H] -：302.1。 The mass spectrum results of the hapten FN are: MSC 13 H 16 F 3 NO 2 : 303.14, ESI-[MH] − : 302.1.
半抗原FN的结构式如式(II)所示：The structural formula of the hapten FN is shown in formula (II):
Figure PCTCN2021136186-appb-000006
Figure PCTCN2021136186-appb-000006
半抗原FN采用系统命名法命名为：6-((3-(三氟甲基)苯乙基)氨基)己酸。The hapten FN is named by systematic nomenclature: 6-((3-(trifluoromethyl)phenethyl)amino)hexanoic acid.
实施例2 芬氟拉明人工抗原的合成和鉴定Example 2 Synthesis and Identification of Fenfluramine Artificial Antigen
1.芬氟拉明人工抗原的合成1. Synthesis of Fenfluramine Artificial Antigen
将实施例1制备的半抗原FB或半抗原FN，通过活泼酯法偶联乳铁蛋白(LF)或鸡卵清蛋白(OVA)。The hapten FB or hapten FN prepared in Example 1 was coupled to lactoferrin (LF) or chicken ovalbumin (OVA) by the active ester method.
称取1moL实施例1制备的半抗原FB，与1.4moL NHS和1.6moL EDC溶解于50～200μL DMF中，室温下避光搅拌2～4h，得到半抗原活化液；将10mg LF加入到1mL的PBS缓冲液(0.01mol/L，pH＝7.4)中；将半抗原活化液缓慢逐滴加入LF的PBS缓冲溶液中，4℃反应12h；然后用PBS缓冲液透析3天，每天3次，透析结束后即得人工抗原FB，分装于离心管中，于-20℃保存，以供使用。Weigh 1moL of the hapten FB prepared in Example 1, dissolve in 50-200μL DMF with 1.4moL NHS and 1.6moL EDC, and stir at room temperature for 2-4 hours in the dark to obtain the hapten activation solution; add 10mg LF to 1mL In PBS buffer (0.01mol/L, pH=7.4); slowly add the hapten activation solution dropwise into the PBS buffer solution of LF, react at 4°C for 12h; then dialyze with PBS buffer for 3 days, 3 times a day, dialyze After the end, the artificial antigen FB was obtained, which was divided into centrifuge tubes and stored at -20°C for use.
其中，PBS缓冲液的配方：Na 2HPO 4·12H 2O 2.90g，NaCl 8.50g，KCl 0.20g，KH 2PO 4 0.20g，加蒸馏水定容至1000mL。 Among them, the formula of PBS buffer solution: Na 2 HPO 4 ·12H 2 O 2.90g, NaCl 8.50g, KCl 0.20g, KH 2 PO 4 0.20g, add distilled water to 1000mL.
人工抗原FN的制备同人工抗原FB，区别仅在于载体蛋白为鸡卵清蛋白。The preparation of the artificial antigen FN is the same as that of the artificial antigen FB, the only difference is that the carrier protein is chicken ovalbumin.
2.芬氟拉明人工抗原的鉴定2. Identification of fenfluramine artificial antigen
对LF、半抗原FB和上述合成的人工抗原FB-LF，进行紫外扫描。紫外扫描结果如图3。Ultraviolet scanning was carried out on LF, hapten FB and the artificial antigen FB-LF synthesized above. The UV scanning results are shown in Figure 3.
LF、半抗原FB、FB-LF分别进行紫外(200～350nm)扫描鉴定，并通过比较偶联前后的各物质的最高吸光值，发现FB-LF的吸收曲线与载体蛋白LF明显 不同，半抗原FB在240nm和300nm处各有一个特征峰，而偶联LF后，FB-LF的吸收峰在240nm和300nm处明显比LF高，且相对半抗原FB的曲线发生显著位移。由于在偶联反应的透析过程已经将未反应的药物等小分子成分全部透析去除，因此偶联产物出现的药物特征峰是由蛋白结合的药物分子贡献的，故说明反应产物是载体蛋白LF与半抗原FB的复合物，说明FB-LF偶联成功。LF, hapten FB, and FB-LF were identified by ultraviolet (200-350nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of FB-LF was significantly different from that of the carrier protein LF. FB has a characteristic peak at 240nm and 300nm respectively, and after coupling LF, the absorption peaks of FB-LF are obviously higher than LF at 240nm and 300nm, and the curve relative to the hapten FB has a significant shift. Since the unreacted drug and other small molecule components have been dialyzed and removed during the dialysis process of the coupling reaction, the characteristic peak of the drug that appears in the coupling product is contributed by the protein-bound drug molecule, so the reaction product is the carrier protein LF and The complex of the hapten FB indicates that the FB-LF coupling is successful.
对OVA、半抗原FN和上述合成的人工抗原FN-OVA，进行紫外扫描。紫外扫描结果如图4。Ultraviolet scanning was performed on OVA, the hapten FN and the artificial antigen FN-OVA synthesized above. The UV scanning results are shown in Figure 4.
OVA、半抗原FN和FN-OVA分别进行紫外(200～350nm)扫描鉴定，并通过比较偶联前后的各物质的最高吸光值，发现FN-OVA的吸收曲线与载体蛋白OVA明显不同，半抗原FN在240nm和260nm处各有一个特征峰，而偶联OVA后，FN-OVA的吸收峰在240nm和260nm处明显比OVA高，且相对半抗原FN的曲线发生显著位移。由于在偶联反应的透析过程已经将未反应的药物等小分子成分全部透析去除，因此偶联产物出现的药物特征峰是由蛋白结合的药物分子贡献的，故说明反应产物是载体蛋白OVA与半抗原FN的复合物，说明FN-OVA偶联成功。OVA, hapten FN and FN-OVA were identified by ultraviolet (200-350nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of FN-OVA was significantly different from that of the carrier protein OVA, and the hapten FN has a characteristic peak at 240nm and 260nm, and after coupling with OVA, the absorption peaks of FN-OVA are significantly higher than OVA at 240nm and 260nm, and the curve relative to the hapten FN has a significant shift. Since the unreacted drug and other small molecule components have been dialyzed and removed during the dialysis process of the coupling reaction, the characteristic peak of the drug that appears in the coupling product is contributed by the protein-bound drug molecule, so it shows that the reaction product is the carrier protein OVA and The complex of hapten FN indicates that the coupling of FN-OVA is successful.
实施例3 抗体的制备Example 3 Preparation of Antibody
将实施例2制备的FB-LF作为免疫原与免疫佐剂(第一次免疫用不完全弗氏佐剂，以后加强免疫均用弗氏不完全佐剂)按体积比1:1乳化均匀，免疫新西兰大白兔。所述新西兰大白兔体重为2.5～3kg，采用颈部和背部皮下多点注射，4周后第二次免疫，以后每间隔3周加强免疫一次。第三次加强免疫后1周耳缘静脉取血，并利用间接竞争ELISA测定血清效价。当效价不再上升时，采用耳缘静脉加强免疫。一周后心脏采血，收集到的血获得血清的方式为：在37℃下温浴0.5～1h，然后在4℃下静置过夜，再用吸管吸取析出来的血清，接着在4℃下，3000～5000rpm离心10min，取上清。抗血清采用硫酸铵沉淀法纯化的到多克隆抗体，于-20℃冻存备用。The FB-LF prepared in Example 2 was used as the immunogen and immune adjuvant (incomplete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for subsequent booster immunizations) in a volume ratio of 1:1 to emulsify evenly, Immunization of New Zealand white rabbits. The New Zealand white rabbits weigh 2.5-3 kg, and are injected subcutaneously at multiple points on the neck and back, and are immunized for the second time after 4 weeks, and boosted immunization once every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, the ear vein is used to boost the immunization. One week later, blood was collected from the heart, and the method of obtaining serum from the collected blood was as follows: incubate at 37°C for 0.5-1 hour, then let it stand overnight at 4°C, then suck the precipitated serum with a straw, and then incubate at 4°C for 3000- Centrifuge at 5000rpm for 10min, and take the supernatant. The antiserum was purified by ammonium sulfate precipitation to polyclonal antibodies, and stored at -20°C for future use.
实施例4 芬氟拉明免疫原和包被原组合优化Example 4 Combination optimization of fenfluramine immunogen and coating agent
本发明还按照实施例2的FB-LF的制备方法制备了以牛血清白蛋白(BSA)为载体蛋白的人工抗原FB-BSA和以鸡卵清蛋白(OVA)作为载体蛋白的人工抗原FB-OVA，且均偶联成功。The present invention also prepared artificial antigen FB-BSA with bovine serum albumin (BSA) as carrier protein and artificial antigen FB-BSA with chicken ovalbumin (OVA) as carrier protein according to the preparation method of FB-LF of embodiment 2. OVA, and all were successfully coupled.
分别将制备的FB-BSA和实施例2制备的FB-LF作为免疫原，按照实施例3 的方法免疫新西兰大白兔制得的芬氟拉明抗体进行包被原筛选，以制备的FB-OVA和实施例2制备的FN-OVA作为包被原，经ELISA检测免疫新西兰大白兔所获得抗血清的效价和抑制率。The FB-BSA prepared and the FB-LF prepared in Example 2 were used as immunogens respectively, and the fenfluramine antibody prepared by immunizing New Zealand white rabbits according to the method of Example 3 was used for coating source screening, so that the prepared FB-OVA Using the FN-OVA prepared in Example 2 as the coating source, the titer and inhibition rate of the antiserum obtained from immunizing New Zealand white rabbits were detected by ELISA.
具体操作步骤如下：The specific operation steps are as follows:
(1)将芬氟拉明人工抗原FB-OVA、FN-OVA分别用包被液(0.05M碳酸盐缓冲溶液，pH 9.6)稀释至250ng/mL的浓度，包被96孔酶标板，每孔加入100μL，37℃恒温水浴箱温育过夜，弃包被液，用PBST(0.01M PBS，0.06％Tween-20(v/v))洗涤2次；(1) Dilute fenfluramine artificial antigens FB-OVA and FN-OVA to a concentration of 250ng/mL with coating solution (0.05M carbonate buffer solution, pH 9.6) respectively, and coat a 96-well microtiter plate, Add 100 μL to each well, incubate overnight in a constant temperature water bath at 37°C, discard the coating solution, and wash twice with PBST (0.01M PBS, 0.06% Tween-20 (v/v));
(2)每孔加入120μL封闭液(1wt％的鱼胶蛋白)，37℃封闭3h，弃去封闭液，拍板，在干燥箱37℃烘干备用；(2) Add 120 μL of blocking solution (1wt% fish gelatin) to each well, block at 37°C for 3 hours, discard the blocking solution, clap, and dry in a drying oven at 37°C for later use;
(3)将芬氟拉明抗体用PBST稀释为1:2000、1:4000、1:8000、1:16000、1:32000、1:64000、1:128000，同时设置空白对照孔(用PBST代替)；用PBST将1mg/mL芬氟拉明药物稀释1000倍，为1μg/mL；(3) Dilute the fenfluramine antibody with PBST to 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and set blank control wells (replace with PBST ); the 1mg/mL fenfluramine drug was diluted 1000 times with PBST to be 1 μg/mL;
效价列：先每孔加50μL的PBST，然后将倍比稀释得到抗体按每孔50μL依次加入孔内，最后一个孔不加抗体，用50μL PBST代替；Titer column: first add 50 μL of PBST to each well, then add the antibody obtained by doubling dilution to each well in turn at 50 μL per well, and replace the last well with 50 μL of PBST without adding antibody;
抑制列：先每孔加50μL的药物，然后将倍比稀释得到抗体按每孔50μL依次加入孔内，最后一个孔不加抗体，用50μL PBST代替；在37℃下温育40min，洗涤5次，拍板；Inhibition column: first add 50 μL of drug to each well, then add 50 μL of the antibody obtained by doubling dilution into each well in turn, and replace the last well with 50 μL of PBST without adding antibody; incubate at 37°C for 40 minutes, wash 5 times , make a decision;
(4)加入羊抗兔二抗Ig-HRP(5000倍稀释)，37℃温育30min，洗涤5次，拍板；(4) Add goat anti-rabbit secondary antibody Ig-HRP (5000-fold dilution), incubate at 37°C for 30min, wash 5 times, and take plate;
(5)加入显色液，在37℃下温育显色10min；(5) Add color developing solution and incubate at 37°C for 10 minutes;
(6)加入10％H 2SO 4终止反应，并在450nm处读取OD值； (6) Add 10% H 2 SO 4 to terminate the reaction, and read the OD value at 450nm;
效价是OD 450为1.0左右所对应的抗血清稀释倍数。 The titer is the dilution of antiserum corresponding to an OD 450 of about 1.0.
抑制率＝(效价的OD值-抑制的OD值)/抑制的OD值*100％Inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition * 100%
4组免疫原和包被原组合的抗血清的效价和抑制率如表1所示。Table 1 shows the titers and inhibition rates of the antisera of the four groups of immunogens and coating agents.
表1 4组免疫原和包被原组合的抗血清的效价和抑制率Table 1 The potency and inhibition rate of the antiserum of 4 groups of immunogens and coating original combinations
Figure PCTCN2021136186-appb-000007
Figure PCTCN2021136186-appb-000007
Figure PCTCN2021136186-appb-000008
Figure PCTCN2021136186-appb-000008
从表1中可以看出，不同的芬氟拉明人工抗原作为免疫的新西兰大白兔产生的抗血清均有一定的效价，所得抗血清对目标分析物芬氟拉明均有不同程度的抑制效果。其中，编号3的免疫原和包被原结构组合所示的抗血清效价1:128000和抑制率88.36％为最佳组合；在该组合下，芬氟拉明抗体不仅能特异性识别目标分析物芬氟拉明，而且抗体灵敏度好；抗血清效价和抑制率均高于编号1、2、4的免疫原和包被原组合，故编号3的免疫原和包被原结构组合为最佳组合。即将FB-LF作为免疫原、FN-OVA作为包被原。As can be seen from Table 1, different fenfluramine artificial antigens are used as the antisera produced by immunized New Zealand white rabbits to have certain titers, and the obtained antisera have different degrees of inhibition to the target analyte fenfluramine Effect. Among them, the antiserum titer 1:128000 and inhibition rate 88.36% shown in the combination of the immunogen and the coating original structure of No. 3 are the best combination; under this combination, the fenfluramine antibody can not only specifically recognize the target analysis fenfluramine, and the sensitivity of the antibody is good; the titer and inhibition rate of the antiserum are higher than the combination of the immunogen and the coating source of No. good combination. That is, FB-LF was used as the immunogen, and FN-OVA was used as the coating source.
实施例5 芬氟拉明的间接竞争ELISA检测方法的建立The establishment of the indirect competition ELISA detection method of embodiment 5 fenfluramine
1、实验方法1. Experimental method
一种检测芬氟拉明的间接竞争ELISA方法，包括以下步骤：A kind of indirect competition ELISA method that detects fenfluramine, comprises the following steps:
(1)将实施例2制备的人工抗原FN-OVA作为包被原，用包被液稀释至62.5ng/mL，包被96孔酶标板，每孔加入100μL，37℃孵育过夜(12h)；(1) Use the artificial antigen FN-OVA prepared in Example 2 as the coating source, dilute it to 62.5ng/mL with the coating solution, coat the 96-well microtiter plate, add 100 μL to each well, and incubate overnight at 37°C (12h) ;
(2)弃去包被液，洗涤两次，拍干；(2) Discard the coating solution, wash twice, and pat dry;
(3)每孔加入120μL封闭液(即质量比1％鱼皮胶原蛋白)，37℃封闭3h；(3) Add 120 μL of blocking solution (i.e., 1% fish skin collagen by mass ratio) to each well, and block at 37°C for 3 hours;
(4)弃去封闭液，拍板，37℃烘干30min后取出，用自封袋装好备用；(4) Discard the blocking solution, clap, dry at 37°C for 30 minutes, take it out, and put it in a ziplock bag for later use;
(5)用实施例4的PBST 1：4000倍稀释实施例3制备的多克隆抗体，并将待检测芬氟拉明药物稀释至1000ng/mL、250ng/mL、62.5ng/mL、15.63ng/mL、3.9ng/mL、0.98ng/mL、0.244ng/mL、0.06ng/mL、0.015ng/mL；(5) Dilute the polyclonal antibody prepared in Example 3 with the PBST of Example 4 1:4000 times, and dilute the fenfluramine drug to be tested to 1000ng/mL, 250ng/mL, 62.5ng/mL, 15.63ng/mL mL, 3.9ng/mL, 0.98ng/mL, 0.244ng/mL, 0.06ng/mL, 0.015ng/mL;
(6)每行加入50μL待检测芬氟拉明药物稀释液(三组平行)，再加入50μL/孔实施例3制备的多克隆抗体稀释液，37℃孵育40min，洗涤五次，拍干；(6) Add 50 μL of the fenfluramine drug dilution to be tested in each row (three parallel groups), then add 50 μL/well of the polyclonal antibody dilution prepared in Example 3, incubate at 37° C. for 40 min, wash five times, and pat dry;
(7)加入羊抗兔二抗-HRP(5000倍稀释)100μL/孔，37℃孵育30min，洗涤五次，拍干；(7) Add goat anti-rabbit secondary antibody-HRP (5000-fold dilution) 100 μL/well, incubate at 37°C for 30 minutes, wash five times, and pat dry;
(8)加入显色液，每孔100μL，显色10min；(8) Add color developing solution, 100 μL per well, develop color for 10 min;
(9)加入50μL 10％的H 2SO 4溶液终止反应，并在450nm处读取OD值 (9) Add 50 μL of 10% H 2 SO 4 solution to terminate the reaction, and read the OD value at 450 nm
2、实验结果2. Experimental results
用于检测芬氟拉明药物的抗体间接竞争ELISA标准曲线如图5所示，从图5可知用于检测芬氟拉明药物的抗体半抑制浓度(IC 50)为10.22ng/mL，定量检测范围为1.76～59.13ng/mL，最低检测限(LOD)为0.63ng/mL；说明本发明制备得到的用于检测芬氟拉明的抗体灵敏度高，可以满足检测要求。 The antibody indirect competition ELISA standard curve that is used to detect fenfluramine drug is as shown in Figure 5, as can be seen from Figure 5, is used to detect the antibody half-inhibitory concentration (IC 50 ) of fenfluramine drug to be 10.22ng/mL, quantitative detection The range is 1.76-59.13ng/mL, and the lower limit of detection (LOD) is 0.63ng/mL; it shows that the antibody prepared by the present invention for detecting fenfluramine has high sensitivity and can meet the detection requirements.
实施例6 用于检测芬氟拉明的抗体的特异性评价Embodiment 6 is used to detect the specificity evaluation of the antibody of fenfluramine
1、实验方法1. Experimental method
通过芬氟拉明抗体与芬氟拉明药物及其类似物进行交叉反应实验来确定用于检测芬氟拉明的抗体的特异性，抗体的特异性用交叉反应率(CR)表示，交叉反应率越小，特异性越强。将芬氟拉明及其类似物苯丁胺(phentermine，PHE)、氯苯丁胺(chlorphentermine，CPT)、苯甲吗啡(phenmetrazine，PHM)和安非泼拉酮(anfepramone，AFP)分别作倍比稀释，采用间接竞争ELISA法进行测定，步骤同实施例5，得到各类似物的IC 50值，按照以下公式计算芬氟拉明交叉反应率(CR) The specificity of the antibody used to detect fenfluramine is determined by the cross-reaction experiment of the fenfluramine antibody and the fenfluramine drug and its analogues. The specificity of the antibody is expressed by the cross-reaction rate (CR), and the cross-reaction The smaller the rate, the stronger the specificity. Fenfluramine and its analogs phentermine (PHE), chlorphentermine (CPT), phenmetrazine (PHM) and anfepramone (AFP) were used as times respectively Ratio dilution, adopt indirect competition ELISA method to measure, and step is with embodiment 5, obtains the IC of each analog 50 value, calculates fenfluramine cross-reactivity rate (CR) according to the following formula
CR(％)＝IC 50(芬氟拉明)/IC 50(类似物)×100％ CR (%)=IC 50 (fenfluramine)/IC 50 (analogue)×100%
2、实验结果2. Experimental results
实施例3制备的芬氟拉明多克隆抗体与芬氟拉明药物及其类似物的交叉反应结果如表2所示，The cross-reaction results of the fenfluramine polyclonal antibody prepared in Example 3 and fenfluramine drugs and analogs thereof are shown in Table 2,
表2芬氟拉明多克隆抗体与芬氟拉明及其类似物的交叉反应结果Table 2 Cross-reaction results of fenfluramine polyclonal antibody with fenfluramine and its analogues
Figure PCTCN2021136186-appb-000009
Figure PCTCN2021136186-appb-000009
注：NR表示无反应Note: NR means no response
从表2可知，用于检测芬氟拉明的抗体对芬氟拉明的交叉反应率为100％，IC 50为10.22ng/mL，对芬氟拉明类似物苯丁胺、氯苯丁胺、苯甲吗啡、安非泼拉酮的均无交叉；说明用于检测芬氟拉明的抗体对芬氟拉明的识别能力高、特异性强，可以有效地排除芬氟拉明类似物苯丁胺、氯苯丁胺、苯甲吗啡、安非泼拉酮对芬氟拉明检测的干扰，能够专门用于对芬氟拉明的检测。 It can be seen from Table 2 that the cross-reactivity rate of the antibody used to detect fenfluramine to fenfluramine is 100%, and the IC50 is 10.22ng/mL, and to fenfluramine analogues phentermine, chlorphentermine , benzomorphine, and amfeprarone have no crossover; it shows that the antibody used to detect fenfluramine has high recognition ability and strong specificity for fenfluramine, and can effectively exclude fenfluramine analogue benzene The interference of butylamine, chlorphentermine, benzomorphine, and amfepramone on the detection of fenfluramine can be specially used for the detection of fenfluramine.
实施例7 检测芬氟拉明的试剂盒的开发Example 7 Development of a kit for detecting fenfluramine
1.试剂盒的组成1. Composition of the kit
检测芬氟拉明的试剂盒，包含下述各部分：A kit for detecting fenfluramine, comprising the following parts:
(1)包被有包被原的酶标板的制备：将实施例2制备的芬氟拉明人工抗原 FN-OVA作为包被原，用包被缓冲液将包被原稀释成31.25μg/L，每孔加入100μL，37℃避光孵育过夜，倾去孔中液体，用洗涤液洗涤2次，每次30s，拍干，然后在每孔中加入200μL封闭液，25℃避光孵育2h，倾去孔内液体拍干，干燥后用铝膜真空密封保存；包被缓冲液为pH值为9.6，0.05mol/L的碳酸盐缓冲液，封闭液为pH值为7.1～7.5，含有质量比1％～3％酪蛋白、0.1～0.3mol/L的磷酸盐缓冲液；(1) Preparation of an enzyme-titer plate coated with a coating source: the fenfluramine artificial antigen FN-OVA prepared in Example 2 is used as a coating source, and the coating buffer is used to dilute the coating source to 31.25 μg/ L, add 100 μL to each well, incubate overnight at 37°C in the dark, pour off the liquid in the well, wash twice with washing solution for 30 seconds each time, pat dry, then add 200 μL of blocking solution to each well, and incubate at 25°C in the dark for 2 hours , Pour off the liquid in the hole and pat dry, after drying, seal it with an aluminum film and store it in a vacuum; the coating buffer is a carbonate buffer with a pH value of 9.6 and 0.05mol/L, and the blocking solution is a pH value of 7.1 to 7.5, containing 1%-3% casein by mass ratio, 0.1-0.3mol/L phosphate buffer;
(2)芬氟拉明标准品溶液：8个浓度梯度，分别为1000μg/L，200μg/L，40μg/L，8μg/L，1.6μg/L，0.32μg/L，0.064μg/L，0.0128μg/L；(2) Fenfluramine standard solution: 8 concentration gradients, respectively 1000μg/L, 200μg/L, 40μg/L, 8μg/L, 1.6μg/L, 0.32μg/L, 0.064μg/L, 0.0128 μg/L;
(3)实施例3制备的芬氟拉明多克隆抗体；(3) the fenfluramine polyclonal antibody prepared in embodiment 3;
(4)酶结合物：辣根过氧化物酶标记的实施例3制备的芬氟拉明多克隆抗体；(4) Enzyme conjugate: the fenfluramine polyclonal antibody prepared in Example 3 labeled with horseradish peroxidase;
(5)底物显色液：由A液和B液组成，A液为过氧化脲，B液为四甲基联苯胺；(5) Substrate chromogenic solution: composed of A liquid and B liquid, A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;
(6)终止液为2mol/L H 2SO 4； (6) The stop solution is 2mol/L H 2 SO 4 ;
(7)洗涤液为pH值为7.4，含有0.5％～1.0％吐温-20、0.01‰～0.03‰叠氮化钠防腐剂、0.1～0.3mol/L的磷酸盐缓冲液，所述百分比为重量体积百分比。(7) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide preservative, and 0.1 to 0.3mol/L phosphate buffer, and the percentage is weight volume percentage.
2.样品检测2. Sample testing
将样品和标准品对应微孔按序编号，每个样品和标准品做2孔平行，并记录标准孔和样品孔所在的位置。根据需要量将酶结合物浓缩液用酶结合物稀释液按1:10体积比进行稀释(即一份酶结合物浓缩液加入10份酶结合物稀释液，现配现用)。加入标准品/样品50μL到对应的微孔中，然后加入酶结合物工作液50μL，轻轻震荡混匀，用盖板膜盖板后置25℃避光环境中反应30min。将孔内液体甩干，加入洗涤工作液250μL/孔；充分洗涤4～5次，每次间隔10s，泼掉板孔内洗涤液，用吸水纸拍干(拍干后未被清楚的气泡可食用未使用过的枪头戳破)。加入底物显色液A液50μL/孔，再加入底物显色液B液50μL/孔，轻轻振荡混匀，用盖板膜盖板后置25℃避光环境中反应10min；加入终止液50μL/孔，轻轻振荡混匀，设定酶标仪与450nm处，测定每孔OD值。Number the corresponding microwells of the samples and standards in sequence, make 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells. Dilute the enzyme conjugate concentrated solution with the enzyme conjugate diluent according to the required amount at a volume ratio of 1:10 (that is, one part of the enzyme conjugate concentrated solution is added to 10 parts of the enzyme conjugate diluent, ready for immediate use). Add 50 μL of the standard/sample to the corresponding microwell, then add 50 μL of the enzyme conjugate working solution, shake and mix gently, cover the plate with a cover film and place it in a light-proof environment at 25°C for 30 minutes. Dry the liquid in the wells, add 250 μL/well of washing working solution; fully wash 4 to 5 times, with an interval of 10 seconds each time, pour off the washing liquid in the wells, and pat dry with absorbent paper (bubbles that are not cleared after pat dry can be Eat unused pipette tip). Add 50 μL/well of substrate chromogenic solution A, then add 50 μL/well of substrate chromogenic solution B, shake and mix gently, cover the plate with a cover film and place it in a light-proof environment at 25°C for 10 minutes; the addition is terminated 50 μL/well, shake gently to mix, set the microplate reader to 450nm, and measure the OD value of each well.
3.检测结果分析3. Analysis of test results
标准品或样本的百分吸光率等于标准品或样本的吸光度值的平均值(双孔)除以第一个标准品(0μg/L)的吸光度值的平均值，再乘以100％。以标准品百分吸光 率为纵坐标，以芬氟拉明标准品浓度(μg/L)的对数为横坐标，绘制标准曲线图。将样本的百分吸光率代入标准曲线中，从标准曲线上读出样本所对应的浓度，乘以其对应的稀释倍数即为样本中芬氟拉明的实际浓度。The percent absorbance of a standard or sample is equal to the average of the absorbance values of the standard or sample (double wells) divided by the average of the absorbance of the first standard (0 μg/L), and multiplied by 100%. Take the percent absorbance of the standard substance as the ordinate, and take the logarithm of the concentration of fenfluramine standard substance (μg/L) as the abscissa to draw a standard curve. Substitute the percent absorbance of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and multiply it by the corresponding dilution factor to obtain the actual concentration of fenfluramine in the sample.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

  1. 一种芬氟拉明半抗原，其特征在于，所述半抗原的结构式如式(I)所示，A kind of fenfluramine hapten, is characterized in that, the structural formula of described hapten is as shown in formula (I),
    Figure PCTCN2021136186-appb-100001
    Figure PCTCN2021136186-appb-100001
  2. 一种芬氟拉明半抗原，其特征在于，所述半抗原的结构式如式(II)所示，A fenfluramine hapten, characterized in that, the structural formula of the hapten is as shown in formula (II),
    Figure PCTCN2021136186-appb-100002
    Figure PCTCN2021136186-appb-100002
  3. 权利要求1所述芬氟拉明半抗原的制备方法，其特征在于，将间三氟甲基苯丙酮与4-氨基丁酸甲酯溶于甲醇中，充分反应后；冷却至室温，然后硼氢化钾，充分反应；分离纯化，将分离纯化后的反应物溶解于甲醇，然后充分水解，调节pH为6～7，即得。The preparation method of fenfluramine hapten described in claim 1 is characterized in that, m-trifluoromethyl propiophenone and 4-aminobutyric acid methyl ester are dissolved in methanol, after fully reacting; Cool to room temperature, then boron Potassium hydride, full reaction; separation and purification, dissolving the separated and purified reactant in methanol, then fully hydrolyzing, adjusting the pH to 6-7, to obtain.
  4. 权利要求2所述芬氟拉明半抗原的制备方法，其特征在于，将2-(3-三氟甲基苯基)乙胺与6-溴己酸甲酯溶于乙腈中，加入碳酸钾和碘化钠充分反应，分离纯化，将分离纯化后的反应物溶解于甲醇，然后充分水解，调节pH为6～7，即得。The preparation method of fenfluramine hapten described in claim 2 is characterized in that, 2-(3-trifluoromethylphenyl) ethylamine and 6-bromohexanoic acid methyl ester are dissolved in acetonitrile, add potassium carbonate Fully react with sodium iodide, separate and purify, dissolve the separated and purified reactant in methanol, then fully hydrolyze, adjust the pH to 6-7, and obtain the product.
  5. 权利要求1或2所述芬氟拉明半抗原在制备芬氟拉明人工抗原中的应用。The application of the fenfluramine hapten described in claim 1 or 2 in the preparation of fenfluramine artificial antigen.
  6. 一种芬氟拉明人工抗原，其特征在于，由权利要求1或2所述芬氟拉明半抗原偶联载体蛋白得到。A fenfluramine artificial antigen is characterized in that it is obtained by coupling the fenfluramine hapten described in claim 1 or 2 to a carrier protein.
  7. 一种芬氟拉明人工抗原组合，其特征在于，包括免疫原和包被原，所述免疫原由权利要求1所述半抗原偶联载体蛋白得到的；所述包被原为权利要求6所述芬氟拉明人工抗原。A fenfluramine artificial antigen combination, characterized in that it comprises an immunogen and a coating, the immunogen is obtained by coupling the carrier protein with the hapten according to claim 1; the coating is the original of claim 6 Fenfluramine artificial antigen.
  8. 一种芬氟拉明抗体，其特征在于，利用权利要求1所述芬氟拉明半抗原偶联载体蛋白得到的人工抗原免疫动物制备得到。A fenfluramine antibody, characterized in that it is prepared by immunizing animals with an artificial antigen obtained by coupling a fenfluramine hapten to a carrier protein according to claim 1.
  9. 一种检测芬氟拉明的试剂盒，其特征在于，包含权利要求6所述芬氟拉 明人工抗原和权利要求8所述芬氟拉明抗体。A test kit for detecting fenfluramine, characterized in that, comprising the fenfluramine artificial antigen described in claim 6 and the fenfluramine antibody described in claim 8.
  10. 一种检测芬氟拉明的免疫分析方法，其特征在于，以权利要求6所述芬氟拉明人工抗原为抗原，以权利要求8所述芬氟拉明抗体为检测抗体进行检测。An immunoassay method for detecting fenfluramine, characterized in that the fenfluramine artificial antigen described in claim 6 is used as an antigen, and the fenfluramine antibody described in claim 8 is used as a detection antibody for detection.
PCT/CN2021/136186 2021-12-07 2021-12-07 Fenfluramine hapten, and preparation method therefor and use thereof, fenfluramine hapten artificial antigen, and fenfluramine hapten antibody WO2023102750A1 (en)

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CN112321443A (en) * 2020-10-22 2021-02-05 广州万孚生物技术股份有限公司 Dextropropoxyphene artificial hapten as well as preparation method and application thereof

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