WO2023102750A1 - Haptène de fenfluramine, son procédé de préparation et son utilisation, antigène artificiel d'haptène de fenfluramine et anticorps d'haptène de fenfluramine - Google Patents

Haptène de fenfluramine, son procédé de préparation et son utilisation, antigène artificiel d'haptène de fenfluramine et anticorps d'haptène de fenfluramine 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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English (en)
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

La présente invention concerne un haptène de fenfluramine, son procédé de préparation et son utilisation, un antigène artificiel de fenfluramine et un anticorps de fenfluramine. Selon la présente invention, deux haptènes, à savoir l'haptène FB et l'haptène FN, sont préparés, l'antigène artificiel FB-LF est obtenu au moyen d'un couplage de l'haptène FB à la protéine porteuse LF, un anticorps spécifique pour la détection de fenfluramine est en outre préparé, et l'antigène artificiel FN-OVA est utilisé en tant que revêtement. L'anticorps a une bonne sensibilité et une bonne spécificité vis-à-vis de la fenfluramine, avec une concentration inhibitrice semi-maximale de 10,22 ng/mL, une limite de détection la plus faible de 0,63 ng/mL et une plage de détection quantitative de 1,76-59,13 ng/mL, n'a pas de réaction croisée avec les analogues de fenfluramine, et peut détecter de manière spécifique la fenfluramine. La présente invention concerne un procédé de dosage immunologique pour la fenfluramine, qui permet de détecter rapidement et avec précision la fenfluramine.
PCT/CN2021/136186 2021-12-07 2021-12-07 Haptène de fenfluramine, son procédé de préparation et son utilisation, antigène artificiel d'haptène de fenfluramine et anticorps d'haptène de fenfluramine WO2023102750A1 (fr)

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Citations (1)

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CN112321443A (zh) * 2020-10-22 2021-02-05 广州万孚生物技术股份有限公司 一种右丙氧芬人工半抗原及其制备方法和应用

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CN112321443A (zh) * 2020-10-22 2021-02-05 广州万孚生物技术股份有限公司 一种右丙氧芬人工半抗原及其制备方法和应用

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