CN114456124A - Cetilistat hapten, artificial antigen and antibody as well as preparation methods and applications thereof - Google Patents

Cetilistat hapten, artificial antigen and antibody as well as preparation methods and applications thereof Download PDF

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CN114456124A
CN114456124A CN202111668048.6A CN202111668048A CN114456124A CN 114456124 A CN114456124 A CN 114456124A CN 202111668048 A CN202111668048 A CN 202111668048A CN 114456124 A CN114456124 A CN 114456124A
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cetilistat
hapten
antibody
artificial antigen
cet1
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CN114456124B (en
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雷红涛
潘康亮
王子安
全琦琪
李向梅
沈兴
王锦
关甜
徐振林
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South China Agricultural University
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/04—1,3-Oxazines; Hydrogenated 1,3-oxazines
    • C07D265/12—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems
    • C07D265/14—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D265/24—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems condensed with one six-membered ring with hetero atoms directly attached in positions 2 and 4
    • C07D265/26—Two oxygen atoms, e.g. isatoic anhydride
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    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/26—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring
    • C07C271/28—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring to a carbon atom of a non-condensed six-membered aromatic ring
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    • 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
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    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity

Abstract

The invention belongs to the technical field of food detection, and particularly provides a CeL-II hapten, an artificial antigen, an antibody and preparation methods and applications thereof, two CeL-II hapten, CET1 and CET2 are prepared, CET1 is used to obtain CET1, a specific antibody for detecting CeL-II is further prepared, CET2-OVA is used as an envelope antigen, the minimum detection limit of the antibody on CeL-II is 0.22ng/mL, the half-inhibition concentration of the antibody is 6.57ng/mL, the antibody has good sensitivity and specificity, an immunoassay method of CeL-II is established by using the antibody, and the rapid and accurate detection of CeL-II is realized.

Description

Cetilistat hapten, artificial antigen and antibody as well as preparation methods and applications thereof
Technical Field
The invention relates to the field of food detection, in particular to a Cetilistat hapten, an artificial antigen and an antibody as well as a preparation method and application thereof.
Background
Cetilistat (Cetilistat), also known as Cetilistat, is a human lipase inhibitor, originally developed by Alizyme, uk, for the treatment of obesity and diabetes caused by obesity, and Cetilistat was granted to wutian pharmaceutical company, japan in 2003 and was marketed in japan in 9 months in 2013. The medicine has the advantages of no action on nervous system, no influence on other enzyme activity of gastrointestinal tract, no absorption, no appetite suppression, and no restriction on diet. After the medicine is applied, the phenomenon of defecation with oil (a large amount of oil drops float on the water surface) can occur 24 hours, namely undigested fat is removed. The mechanism of action is to inactivate enzymes by forming covalent bonds with the active serine sites of gastric and pancreatic lipases in the gastric and small intestinal lumens, which are unable to hydrolyze fats in food, primarily triglycerides, to absorbable free fatty acids and monoacylglycerols. Undigested triglycerides are not absorbed by the body, thereby reducing caloric intake and controlling body weight. The long-term administration of the cetilistat can cause adverse reactions such as intestinal function, fat diarrhea, anal defecation increase and the like. Other adverse reactions in the digestive tract include oily spots, gastrointestinal gas with stool, fatty (oily) stool, even fecal incontinence, etc.
At present, the new libastat is not approved by domestic marketing, so that domestic methods for detecting the new libastat are not reported and researched too much. Yu Zhou detects Cetilistat by HPLC-PDA method (Yu Z, Wan C.A. simple and continuous method for simultaneous analysis of four major sites of antibiotic additives in grouting fourth. J. Journal of Liquid Chromatography & Related Technologies,2010,33(4): 452-); the Qin Jun et al also used HPLC (Qin Jun, Wang Xian, Fu Shi Ling, et al. HPLC determination method for Cetilistat and related experiments: 2015.) for the determination of Cetilistat. However, the high performance liquid chromatography has the characteristics of high detection efficiency, high accuracy, strong anti-interference capability and the like; however, instruments and equipment required for detection are expensive, high in cost, complex in sample pretreatment, and required to be operated by professionals, and the requirements of field detection of large-batch samples are not met.
Compared with the existing detection method, the immunodetection method based on antigen-antibody specific molecule recognition has more advantages in the field detection aspect, has the characteristics of rapidness, sensitivity, simplicity and convenience and the like, and has low cost and lower requirement on the skill of operators. However, the key to the development of the immunoassay method is to design a suitable cetilistat hapten and prepare an antibody with high sensitivity and strong specificity, but related reports on the cetilistat hapten, artificial antigen and antibody are not found in the prior art.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of a method for detecting the cetilistat in the prior art, and provides a cetilistat hapten, an artificial antigen, an antibody, a preparation method and application thereof.
The invention aims to provide a cetilistat hapten.
The invention also aims to provide the application of the cetilistat hapten in preparing the cetilistat artificial antigen.
The invention also aims to provide the artificial antigen of the new libestat.
The invention also aims to provide application of the artificial antigen of the new libestat in preparing the artificial antibody of the new libestat.
The invention also aims to provide a novel antibody of the Cetilistat.
The invention also aims to provide an immunoassay method for detecting the cetilistat.
The invention also aims to provide a kit for detecting the cetilistat.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a CeT1 hapten or CET2 hapten of CeT, wherein the structural formula of the CET1 is shown in a formula (I),
Figure BDA0003448831230000021
the hapten CET1 is named by adopting a systematic nomenclature: 11- ((3- ((6-methyl-4-oxo-4H-benzo [ d ] [1,3] oxazin-2-yl) oxy) propyl) amino) undecanoic acid;
the structural formula of the hapten CET2 is shown as a formula (II),
Figure BDA0003448831230000031
the hapten CET2 is named by adopting a systematic nomenclature: 4- (((hexadecyloxy) carbonyl) amino) benzoic acid.
The preparation method of the hapten CET1 comprises the following steps:
s1, controlling the temperature of a reaction system to be not more than 20 ℃, mixing a dichloromethane mixed solution of 2-amino-5-methylbenzoic acid and pyridine with chloroformic acid-3-chloropropyl ester, fully reacting, removing a solvent dichloromethane and unreacted pyridine, dissolving reactants with dichloromethane and water, controlling the temperature to be 4-6 ℃, adjusting the pH to 1-2, filtering precipitated solid, washing the solid with water, and drying to obtain an intermediate product 1;
s2, controlling the temperature of the reaction system to be not more than 20 ℃, mixing the dichloromethane solution of the intermediate product 1 with phosphorus oxychloride, and then heating, refluxing and fully reacting. Controlling the temperature of a reaction system to be not more than 5 ℃, adding water to separate the reactants, purifying a dichloromethane layer, filtering, removing dichloromethane, crystallizing, purifying and drying to obtain an intermediate product 2;
s3, dissolving the intermediate product 2, 11-aminoundecanoic acid, potassium hydroxide and sodium iodide in anhydrous N, N-Dimethylformamide (DMF), fully reacting, adding water, adjusting pH to be neutral, extracting with ethyl acetate, removing a water phase, concentrating an organic phase, separating and purifying to obtain the CeT 1.
Preferably, the molar ratio of the 2-amino-5-methylbenzoic acid, the pyridine and the 3-chloropropyl chloroformate in the step S1 is 1: 2-3: 1-2.
Further preferably, the molar ratio of 2-amino-5-methylbenzoic acid, pyridine and 3-chloropropyl chloroformate in step S1 is 1:3: 1.2.
Preferably, the step S1 of mixing the dichloromethane mixed solution of 2-amino-5-methylbenzoic acid and pyridine with 3-chloropropyl chloroformate is to reduce the temperature of the dichloromethane mixed solution of 2-amino-5-methylbenzoic acid and pyridine to 15 ℃ and then mix with 3-chloropropyl chloroformate; the mixing mode is that chloroformic acid-3-chloropropyl ester is dripped into the mixed solution.
Preferably, the solvent dichloromethane and unreacted pyridine are removed in step S1 by rotary evaporation.
Preferably, the control temperature of step S1 is 5 ℃.
Preferably, the pH adjustment in step S1 is performed by using 10% volume fraction hydrochloric acid.
Preferably, the molar ratio of the intermediate product 1 to the phosphorus oxychloride in the step S2 is 1: 2-4.
Further preferably, the molar ratio of the intermediate product 1 to the phosphorus oxychloride in the step S2 is 1: 3.
Preferably, sodium hydride is added to remove water when the dichloromethane solution of the intermediate product 1 is mixed with phosphorus oxychloride in step S2, and benzophenone is used as an indicator to indicate the effect of water removal.
Preferably, the dichloromethane solution of the intermediate product 1 is mixed with the phosphorus oxychloride in the step S2 by controlling the temperature of the dichloromethane solution of the intermediate product 1 to be 15 ℃ and then slowly adding the phosphorus oxychloride dropwise.
Preferably, the step of adding water to stratify the reactants in step S2 is adding primary water; the dichloromethane layer was purified by washing the dichloromethane layer with water, then with a saturated sodium bicarbonate solution, then with a saturated brine, and then adding anhydrous sodium sulfate and activated carbon to remove excess water and impurities.
Preferably, step S2 adopts reduced pressure distillation to remove dichloromethane in the purified dichloromethane layer.
Preferably, the crystallization purification in step S2 is to dissolve the reactant with dichloromethane removed in toluene, fractionally cool and crystallize, filter and wash.
Further preferably, the condition of fractional cooling crystallization is that the temperature is firstly reduced to 14-16 ℃, stirred and crystallized for 2.5-3 h, then continuously reduced to 0 ℃, and stirred and crystallized for 2.5-3 h.
Further preferably, the condition of fractional cooling crystallization is that the temperature is firstly reduced to 15 ℃, stirred and crystallized for 3 hours, then the temperature is continuously reduced to 0 ℃, and stirred and crystallized for 3 hours.
Preferably, the drying in step S2 is drying at 45 ℃ under reduced pressure to constant weight.
Preferably, the molar ratio of the intermediate product 2, the 11-aminoundecanoic acid, the potassium hydroxide and the sodium iodide in the step S3 is 1: 1-2: 1-3: 0.1-0.5.
Further preferably, the molar ratio of the intermediate product 2, 11-aminoundecanoic acid, potassium hydroxide, and sodium iodide in step S3 is 1:1.2:2: 0.2.
Preferably, the sufficient reaction condition in the step S3 is heating to 90-95 ℃ for reaction for 10-12 h.
Further preferably, the condition for sufficient reaction in step S3 is heating to 90 ℃ for 12 h.
Preferably, in the step S3, the separation and purification are performed by passing the concentrated organic phase through a silica gel column, and mixing the concentrated organic phase with ethyl acetate and petroleum ether at a volume ratio of 4-6: and (1) eluting by using the mixed solvent, thereby separating and purifying.
Further preferably, the volume ratio of ethyl acetate to petroleum ether is 5: 1.
the structural formula of the intermediate product 1 is as follows:
Figure BDA0003448831230000051
the structural formula of the intermediate product 2 is as follows:
Figure BDA0003448831230000052
the preparation method of the hapten CET2 comprises the following steps:
controlling the temperature of a reaction system to be less than 20 ℃, mixing a dichloromethane mixed solution of p-aminobenzoic acid and pyridine with cetyl chloroformate, fully reacting, then removing a solvent dichloromethane and unreacted pyridine, dissolving reactants with dichloromethane and water, controlling the temperature to be 4-6 ℃, adjusting the pH to 1-2, filtering precipitated solid, washing the solid with water, and drying to obtain the CeT2 of the tilistat hapten.
Preferably, the molar ratio of the p-aminobenzoic acid to the pyridine to the cetyl chloroformate is 1:2 to 4:1 to 1.5.
Further preferably, the molar ratio of p-aminobenzoic acid, pyridine and hexadecyl chloroformate is 1:3: 1.2.
Preferably, the dichloromethane mixed solution of the para aminobenzoic acid and the pyridine is cooled to 15 ℃, and then mixed with the cetyl chloroformate.
Preferably, the solvent dichloromethane and unreacted pyridine are removed by rotary evaporation.
Preferably, the pH adjustment is performed using 10% by volume hydrochloric acid.
The application of the hapten CET1 and/or the hapten CET2 in preparing the artificial antigen of the neolistat is also in the protection scope of the invention.
The artificial antigen of the Cet1 is obtained by coupling the hapten CET1 or the hapten CET2 with carrier protein, the structural formula of the artificial antigen CET1 obtained by coupling the hapten CET1 with the carrier protein is shown in a formula (III), wherein P is the carrier protein,
Figure BDA0003448831230000061
the structural formula of the artificial antigen CET2 obtained by coupling the hapten CET2 with carrier protein is shown as a formula (IV), wherein P is carrier protein,
Figure BDA0003448831230000062
preferably, the carrier protein (P) is any one or more of Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Lactoferrin (Lactoferrin, LF), or chicken ovalbumin (ovabunin, OVA).
The preparation method of the artificial antigen CET1 or the artificial antigen CET2 utilizes hapten CET1 or hapten CET2 to couple carrier protein by an active ester method.
As a specific embodiment of the method, the preparation method of the artificial antigen CET1 comprises the following steps:
(1) dissolving hapten CET1 and N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in N, N-Dimethylformamide (DMF), and stirring at room temperature in a dark place for 2-4 h to obtain hapten CET1 activation solution;
(2) adding carrier protein into PBS buffer solution;
(3) slowly and dropwise adding the hapten CET1 activating solution in the step (1) into the carrier protein solution in the step (2), and reacting for 12 hours at 4 ℃;
(4) and (4) dialyzing the reaction solution obtained in the step (3) by using a PBS buffer solution to obtain the artificial antigen CET 1.
Preferably, the molar ratio of the hapten CET1, NHS and EDC in the step (1) is 1: 1-2: 1.5-2.5.
More preferably, the molar ratio of the haptens CET1, NHS to EDC in step (1) is 1:1.75: 2.25.
Preferably, the mass-to-volume ratio of the carrier protein to the PBS buffer in the step (2) is 8-11 mg: 0.5-1.5 mL.
More preferably, the mass-to-volume ratio of the carrier protein to the PBS buffer in step (2) is 10mg:1 mL.
Preferably, the mass ratio of the hapten CET1 in the step (1) to the carrier protein in the step (2) is 1-2: 1-4.
More preferably, the mass ratio of hapten CET1 in step (1) to the carrier protein in step (2) is 1: 3.
The preparation method of the artificial antigen CET2 is the same as the artificial antigen CET 1.
The application of the artificial antigen of the neolistat in preparing the neolistat antibody is also within the protection scope of the invention.
A new linaglistat artificial antigen combination comprises an immunogen and a coating antigen, wherein the immunogen is obtained by coupling hapten CET1 with a carrier protein, namely artificial antigen CET 1; the coating antigen is the artificial antigen of the Cetilistat.
Preferably, the coating antigen is obtained by coupling the hapten CET2 with a carrier protein, namely artificial antigen CET 2.
Further preferably, the immunogen is obtained by coupling the hapten CET1 with a carrier protein Lactoferrin (LF), namely artificial antigen CET 1-LF; the coating antigen is obtained by coupling the CET2 with carrier protein chicken Ovalbumin (OVA), namely artificial antigen CET 2-OVA.
The application of the artificial antigen combination in preparing the Cetilistat antibody and/or detecting Cetilistat is also within the protection scope of the invention.
The CeT1 artificial antigen is obtained by coupling the hapten CET1 with carrier protein and is used for immunizing animals to obtain the CeT1 antibody.
Preferably, the Cetiramistat antibody is prepared by immunizing an animal with an artificial antigen CET1-LF obtained by coupling the hapten CET1 with a carrier protein Lactoferrin (LF).
Preferably, the cetilistat antibody is a monoclonal antibody or a polyclonal antibody.
A preparation method of a CeT1 artificial antigen polyclonal antibody is obtained by coupling the hapten CET1 with carrier protein.
Preferably, experimental animals are immunized by using an artificial antigen CET1-LF obtained by coupling the hapten CET1 with a carrier protein Lactoferritin (LF).
As a specific embodiment of the above method, the preparation method of the cetilistat polyclonal antibody comprises the following steps:
(1) immunizing experimental animals by using the artificial antigen CET1 matched with an immune adjuvant;
(2) in the first immunization, the artificial antigen CET1 is emulsified with an equal volume of complete Freund adjuvant to immunize a New Zealand white rabbit;
(3) the second immunization was performed after 4 weeks, followed by booster immunizations every 3 weeks.
(4) After four times of booster immunization, the heart was bled and separated to obtain serum, i.e., polyclonal antibody.
Preferably, the artificial antigen CET1 is the artificial antigen CET1-LF obtained by coupling the hapten CET1 with a carrier protein Lactoferrin (LF).
The application of the Cetilistat antibody in the detection of Cetilistat and/or the preparation of a Cetilistat detection kit is also within the protection scope of the invention.
An immunoassay method for detecting Cetilistat comprises the steps of taking an artificial antigen of CeT1 coupled with a carrier protein as an antigen, and taking an antibody prepared by immunizing an animal with the artificial antigen CET1 obtained by coupling the hapten CET1 with the carrier protein as a detection antibody for detection; the immunoassay method is a non-diagnostic therapeutic objective method.
Preferably, the immunoassay method takes an artificial antigen CET2 obtained by coupling hapten CET2 with a carrier protein as an antigen.
Further preferably, the immunoassay method takes an artificial antigen CET2-OVA with a carrier protein of chicken Ovalbumin (OVA) as an antigen and takes an artificial antigen CET1-LF with a carrier protein of Lactoferrin (LF) as an immunogen to immunize an animal to obtain an antibody which is used as a detection antibody for detection.
Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensing, immunocolloidal gold, and the like.
A kit for detecting Cetilistat comprises the Cetilistat artificial antigen and the Cetilistat antibody.
Preferably, the kit comprises an artificial antigen CET2 obtained by coupling the hapten CET2 with a carrier protein and an antibody prepared by immunizing an animal with the artificial antigen CET1 obtained by coupling the hapten CET1 with the carrier protein.
Further preferably, the kit comprises an artificial antigen CET2-OVA obtained by coupling the hapten CET2 with a carrier protein, namely chicken Ovalbumin (OVA), and an antibody prepared by immunizing an animal with the artificial antigen CET1-LF obtained by coupling the hapten CET1 with a carrier protein, namely Lactoferrin (LF).
Preferably, the kit further comprises one or more of an enzyme label plate, a cetilistat standard substance, an enzyme conjugate, a developing solution, a stop solution or a washing solution.
Preferably, the kit comprises the ELISA plate coated by the artificial antigen of the Cetilistat, a Cetilistat standard substance, an enzyme conjugate, a developing solution, a stopping solution and a concentrated washing solution.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides two kinds of CeT1 and CET2 as hapten, CET1 is used for coupling artificial antigen CET1-LF prepared from Lactoferrin (LF) as immunogen, and then a CeT2 is used for coupling artificial antigen CET2-OVA prepared from Ovalbumin (OVA) as coating antigen; the hapten CET1 and the new libstat to be detected have high overlapping degree of the skeleton structure, so that the immunogenicity of the new libstat artificial antigen CET1-LF is effectively improved, the structural difference between the artificial antigen CET1-LF and the hapten CET1 is larger, larger steric hindrance is formed, and the antibody affinity is further improved;
(2) meanwhile, the antibody obtained by the invention has high titer, strong specificity and high affinity, the lowest detection limit LOD of the neolistat is 0.22ng/mL, and the half-inhibition concentration IC50The detection sensitivity is high, and the linear range is wide; the cross reaction rate to the cetilistat is 100 percent, and the cetilistat analogue orlistat has no cross reaction; the antibody of the invention has the characteristics of simplicity, rapidness, strong specificity, wide linear range and high sensitivity; the aim of quickly and accurately detecting the cetilistat can be achieved by using the artificial antigen and the antibody of the cetilistat.
Drawings
FIG. 1 is a synthetic scheme of the hapten CET1 of the invention.
FIG. 2 is a synthetic scheme of the hapten CET2 of the invention.
FIG. 3 is a UV scan of the haptens CET1, LF, CET1-LF of the invention.
FIG. 4 is a UV scan of the haptens CET2, OVA, CET2-OVA of the invention.
FIG. 5 is a standard curve of indirect competitive ELISA with the polyclonal antibody of Cetilistat of the present invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Synthesis and characterization of Cetilistat hapten
1. Synthesis and identification of CeT1 hapten of Cetiramistat
1.1 Synthesis of CeT1 hapten of Cetilistat
Dissolving 2-amino-5-methylbenzoic acid (1mmoL) and pyridine (3mmoL) in dichloromethane, stirring until the mixture is clear, cooling to 15 ℃, dropwise adding chloroformic acid-3-chloropropyl ester (1.2mmoL), controlling the temperature to be less than 20 ℃, and reacting at room temperature for 2 hours after the dropwise addition. And after the reaction is finished, removing dichloromethane and unreacted pyridine by rotary evaporation, adding 750g of dichloromethane, 250g of water, cooling with ice water, controlling the temperature to be 5 ℃, dropwise adding hydrochloric acid with the volume fraction of 10%, adjusting the pH value to 1-2, separating out a large amount of solid, filtering, washing the solid with water, and drying to obtain an intermediate product 1.
Adding the intermediate product 1(1mmoL) into a reaction bottle, dissolving dichloromethane (sodium hydride is used for removing water, benzophenone is used as an indicator to indicate the effect of water removal), cooling by ice water, controlling the temperature of a reaction system to be 15 ℃, slowly dropwise adding phosphorus oxychloride (3mmoL), controlling the temperature of the system to be not more than 20 ℃, heating and refluxing after dropwise adding are finished, and fully reacting. After the reaction is finished, cooling with ice water, controlling the temperature of the system to be not more than 5 ℃, slowly dropwise adding primary water, continuously stirring for half an hour, layering, washing a dichloromethane layer with water, washing with saturated sodium bicarbonate solution, finally washing with saturated salt solution, adding 10g of anhydrous sodium sulfate and 5g of activated carbon, stirring for one hour at room temperature, filtering, distilling under reduced pressure to remove dichloromethane, adding 200mL of toluene, stirring, cooling to 15 ℃, stirring for crystallization for 3 hours, continuously cooling to 0 ℃, stirring for crystallization for 3 hours, filtering, washing a solid with a proper amount of toluene, and drying under reduced pressure (45 ℃) to constant weight to obtain an intermediate product 2.
Taking intermediate 2(1mmoL) and 11-aminoundecanoic acid (1.2mmoL), adding potassium hydroxide (2mmoL) and sodium iodide (0.2mmoL), dissolving with anhydrous N, N-Dimethylformamide (DMF), heating to 90 deg.C for reaction for 12h, adding water after the reaction is finished, adding 1moL/L hydrochloric acid to adjust pH to neutrality, extracting with ethyl acetate, shaking, standing, removing water phase, concentrating organic phase, loading on silica gel column, and mixing with ethyl acetate and petroleum ether at volume ratio of 5: 1, eluting, separating and purifying the mixed solvent to obtain the cetilistat hapten CET 1. The synthetic scheme for hapten CET1 is shown in FIG. 1.
1.2 identification of CeT1 hapten for Cetilistat
Nuclear magnetic identification of hapten CET 1:1H NMR(600MHz,Chloroform-d)δ7.91(d,J＝2.0Hz,1H),7.53(dd,J＝8.4,2.0Hz,1H),7.31(d,J＝8.3Hz,1H),4.42(t,J＝6.7Hz,2H),2.42(s,3H),1.80(h,J＝8.0,7.4Hz,2H),1.49–1.21(m,21H),0.88(t,J＝7.0Hz,3H).
the mass spectrum result of hapten CET1 is: MS: c23H34N2O5：418.25，ESI-[M-H]-：417.3。
Hapten CET1 has the structural formula shown in formula (I):
Figure BDA0003448831230000111
hapten CET1 was named using the systematic nomenclature: 11- ((3- ((6-methyl-4-oxo-4H-benzo [ d ] [1,3] oxazin-2-yl) oxy) propyl) amino) undecanoic acid.
2. Synthesis and identification of CeT2 hapten of Cet
2.1 Synthesis of CeT2 hapten of Cetilistat
Weighing p-aminobenzoic acid (1mmoL) and pyridine (3mmoL), dissolving in dichloromethane, stirring to clarify, cooling to 15 deg.C, adding dropwise cetyl chloroformate (1.2mmoL), controlling temperature to be less than 20 deg.C, and reacting at room temperature for 2 hr. And after the reaction is finished, removing dichloromethane and unreacted pyridine by rotary evaporation, adding dichloromethane, water and ice water for cooling, controlling the temperature to be 5 ℃, dropwise adding hydrochloric acid with the volume fraction of 10%, adjusting the pH to 1-2, separating out a large amount of solid, filtering, washing the solid with water, and drying to obtain the CeT2 hapten of the neolinastat. The synthetic scheme for hapten CET2 is shown in FIG. 2.
2.2 identification of CeT2 hapten for Cetilistat
Nuclear magnetic identification of hapten CET 2: 1H NMR (600MHz, DMSO-d6) δ 9.98(s,1H), 8.12-8.07 (m,2H),7.86(d, J ═ 8.6Hz,2H),1.62(p, J ═ 6.8Hz,2H), 1.47-1.08 (m,29H),0.85(t, J ═ 6.9Hz,3H).
The mass spectrum result of hapten CET2 is: MS:C24H39NO4：405.29，ESI-[M-H]-：404.3。
hapten CET2 has the structural formula shown in formula (II):
Figure BDA0003448831230000112
hapten CET2 was named using the systematic nomenclature: 4- (((hexadecyloxy) carbonyl) amino) benzoic acid.
Example 2 Synthesis and characterization of artificial antigen of Cetilistat
1. Synthesis of artificial antigen of Cetilistat
The hapten CET1 or the hapten CET2 prepared in example 1 was coupled with Lactoferrin (LF) or chicken Ovalbumin (OVA) by an active ester method.
Weighing 3.3mg (0.008mmol) of hapten CET1 prepared in example 1, dissolving 0.014mmol NHS and 0.018mmol EDC in 50-200 muL DMF, and stirring at room temperature in the dark for 2-4 h to obtain a hapten activation solution; 10mg of LF was added to 1mL of PBS buffer (0.01mol/L, pH 7.4); slowly and dropwise adding the hapten activating solution into a PBS (phosphate buffer solution) of LF (low frequency) to react for 12 hours at 4 ℃; and dialyzing with PBS buffer solution for 3 days, 3 times per day, and after dialysis, obtaining artificial antigen CET1, subpackaging in centrifuge tubes, and storing at-20 deg.C for use.
The formula of the PBS buffer solution is as follows: na (Na)2HPO4·12H2O 2.90g，NaCl 8.50g，KCl 0.20g，KH2PO40.20g, adding distilled water to a constant volume of 1000 mL.
The artificial antigen CET2 is prepared by the same method as the artificial antigen CET1, and only the difference is that the carrier protein is chicken ovalbumin.
2. Identification of artificial antigen of Cetilistat
UV scanning was performed on LF, hapten CET1 and the synthetic artificial antigen CET1-LF described above. The results of the uv scan are shown in figure 3.
The method comprises the steps of respectively carrying out ultraviolet (200-350 nm) scanning identification on LF, hapten CET1 and CET1-LF, and comparing the highest absorbance values of the substances before and after coupling to find that the absorption curve of CET1-LF is obviously different from that of carrier protein LF, the hapten CET1 has a characteristic peak at 240nm and 300nm respectively, and after coupling LF, the absorption peak of CET1-LF is obviously higher than that of LF at 240nm and 300nm and has obvious displacement relative to the curve of hapten CET 1. Since the unreacted small molecular components such as the drug and the like are completely dialyzed and removed in the dialysis process of the coupling reaction, the characteristic peak of the drug appearing in the coupling product is contributed by the protein-bound drug molecule, so that the reaction product is a compound of the carrier protein LF and the hapten CET1, and the success of CET1-LF coupling is proved.
UV scanning was performed on OVA, hapten CET2 and the synthetic CET2-OVA described above. The results of the uv scan are shown in figure 4.
Ultraviolet (200-350 nm) scanning identification is carried out on OVA, hapten CET2 and CET2-OVA respectively, and the highest absorbance values of the substances before and after coupling are compared, so that the absorption curve of CET2-OVA is obviously different from that of carrier protein OVA, hapten CET2 has a characteristic peak at 240nm and 260nm respectively, after OVA coupling, the absorption peak of CET2-OVA is obviously higher than that of OVA at 240nm and 260nm, and the curve of the CET2 is obviously shifted relative to that of hapten CET. Since the unreacted small molecular components such as the drug and the like are completely dialyzed and removed in the dialysis process of the coupling reaction, the characteristic peaks of the drug presented by the coupling product are contributed by the drug molecules bound by the protein, so that the reaction product is a compound of the carrier protein OVA and the hapten CET2, and the success of CET2-OVA coupling is proved.
EXAMPLE 3 preparation of antibodies
1. Preparation of polyclonal antibodies
CET1-LF prepared in example 2 is used as an immunogen and is uniformly emulsified with an immune adjuvant (incomplete Freund's adjuvant is used for the first immunization and incomplete Freund's adjuvant is used for the subsequent booster immunization) according to the volume ratio of 1:1, and the weight of the immunized rabbit is 2.5-3 kg. Multiple subcutaneous injections were administered to the neck and back, 4 weeks later for a second immunization, followed by boosts every 3 weeks apart. Blood was taken from the ear peripheral vein 1 week after the third booster immunization and serum titers were determined using indirect competition ELISA. When the titer no longer increased, the marginal ear vein was used for boosting. Blood was collected from the heart one week later, and the manner in which the collected blood was used to obtain serum was: carrying out warm bath at 37 ℃ for 0.5-1 h, standing overnight at 4 ℃, sucking the precipitated serum by using a suction tube, centrifuging at 3000-5000 rpm at 4 ℃ for 10min, and taking the supernatant. The antiserum is purified to obtain polyclonal antibody by ammonium sulfate precipitation method, and is frozen at-20 deg.C for use.
Example 4 combinatorial optimization of the immunogen and the coatingen of Cetilistat
The invention also prepares artificial antigen CET1-BSA with Bovine Serum Albumin (BSA) as carrier protein and artificial antigen CET1-OVA with chicken Ovalbumin (OVA) as carrier protein according to the preparation method of CET1-LF of example 2, and the coupling is successful.
The prepared CET1-BSA and the CET1-LF prepared in example 2 were used as immunogens, and the CeT1-OVA and the CeT2-OVA prepared in example 2 were prepared as coating antigens by screening the coating antigens against the CeT1-LF prepared in example 3 by immunizing white New Zealand rabbits, and the titers and inhibition ratios of the antiserum obtained by immunizing white New Zealand rabbits were examined by ELISA.
The specific operation steps are as follows:
(1) diluting the original CET1-OVA and CET2-OVA respectively with coating solution (0.05M carbonate buffer solution, pH 9.6) to a concentration of 250ng/mL, coating 96-well enzyme-labeled plates, adding 100 μ L of each well, incubating overnight in a constant temperature water bath at 37 ℃, discarding the coating solution, and washing with PBST (0.01M PBS, 0.06% Tween-20(v/v)) for 2 times;
(2) adding 120 μ L of sealing solution (1 wt% of fish glue protein) into each well, sealing at 37 deg.C for 3 hr, discarding sealing solution, clapping, and oven drying at 37 deg.C in drying oven for use;
(3) the prepared Cetilistat polyclonal antibody is diluted by PBST to be 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000 and 1:512000, and blank control holes (replaced by PBST) are arranged at the same time; 1mg/mL of the Cetilistat drug is diluted 1000 times by PBST to be 1 mug/mL;
the potency is listed as: firstly adding 50 mu L of PBST into each hole, then diluting the PBST by a multiple ratio to obtain a Cetilistat polyclonal antibody, and sequentially adding the Cetilistat polyclonal antibody into the holes according to 50 mu L of each hole, wherein the antibody is not added into the last hole and is replaced by 50 mu L of PBST;
inhibition column: adding 50 mu L of the Cetilistat drug into each hole, diluting the mixture by a multiple ratio to obtain Cetilistat polyclonal antibody, and sequentially adding the Cetilistat polyclonal antibody into the holes according to 50 mu L of each hole, wherein the antibody is not added into the last hole and is replaced by 50 mu L of PBST; incubating at 37 deg.C for 40min, washing for 5 times, and clapping;
(4) adding goat anti-rabbit secondary antibody Ig-HRP (5000-fold dilution), incubating for 30min at 37 ℃, washing for 5 times, and clapping;
(5) adding color developing solution, and incubating and developing at 37 deg.C for 10 min;
(6) adding 10% of H2SO4The reaction was stopped and the OD read at 450 nm;
the potency is OD450The dilution ratio of the antiserum is about 1.0.
Inhibition rate (titer OD value-inhibited OD value)/inhibited OD value 100%
The titers and inhibition rates of the antisera of the 4 groups of immunogens and coatinggen combinations are shown in Table 1.
TABLE 14 titers and inhibition rates of antisera with combinations of immunogens and coatinggens
Figure BDA0003448831230000141
As can be seen from Table 1, different artificial antigens of Cetilistat have certain titer as antiserum produced by immunized New Zealand white rabbits, and the obtained antiserum has different degrees of inhibitory effect on target analyte Cetilistat. Wherein the antiserum titer shown by the combination of the immunogen and the coating antigen structure in the number 4 is 1:512000 and the inhibition rate is 90.31 percent, and the combination is the optimal combination; under the combination, the Cetilistat antibody can specifically recognize target analyte Cetilistat, and the sensitivity of the Cetilistat antibody is good; the antiserum titer and the inhibition rate are higher than those of the immunogen and coatingen combinations of the numbers 1, 2 and 3, so the immunogen and coatingen structural combination of the number 4 is the optimal combination. Namely CET1-LF as immunogen and CET2-OVA as coating antigen.
Example 5 establishment of Indirect competitive ELISA detection method for Cetilistat
1. Experimental methods
An indirect competitive ELISA method for detecting cetilistat, which comprises the following steps:
(1) using the artificial antigen CET2-OVA prepared in example 2 as a coating antigen, diluting the antigen to 62.5ng/mL by using a coating solution, coating a 96-well enzyme label plate, adding 100 mu L of the antigen to each well, and incubating the antigen overnight at 37 ℃ (12 h);
(2) discarding the coating solution, washing twice, and patting to dry;
(3) adding 120 μ L of blocking solution (1 wt% fish skin collagen) into each well, and blocking at 37 deg.C for 3 hr;
(4) removing the sealing liquid, clapping, drying at 37 ℃ for 30min, taking out, and packaging with a self-sealing bag for later use;
(5) using the PBST of example 4 at 1: the Cetilistat polyclonal antibody prepared in example 3 is diluted 4000 times, and the Cetilistat drug is diluted to 6000ng/mL, 1000ng/mL, 166.67ng/mL, 27.78ng/mL, 4.63ng/mL, 0.77ng/mL, 0.13ng/mL, 0.02ng/mL and 0.004 ng/mL;
(6) adding 50 μ L of the drug diluent of the new libestat to be detected (three groups are parallel) into each row, adding 50 μ L/hole of the polyclonal antibody diluent of the new libestat prepared in the embodiment 3, incubating for 40min at 37 ℃, washing for five times, and patting dry;
(7) adding 100 μ L/well of goat anti-rabbit secondary antibody-HRP (5000-fold dilution), incubating at 37 deg.C for 30min, washing for five times, and patting dry;
(8) adding color development solution, wherein each well is 100 μ L, and developing for 10 min;
(9) 50 μ L of 10% H was added2SO4The reaction was stopped with solution and the OD read at 450 nm.
2. Results of the experiment
The standard curve of indirect competitive ELISA for detecting the drug of Cetilistat is shown in FIG. 5. As can be seen from FIG. 5, the standard curve of indirect competitive ELISA for detecting the drug of Cetilistat has a typical sigmoidal curve, has good detection sensitivity, and is used for detecting the half Inhibitory Concentration (IC) of the antibody of Cetilistat50) 6.57ng/mL, the quantitative detection range of 0.78-55.26 ng/mL, and the lowest limit of detection (LOD) of 0.22 ng/mL; the antibody for detecting the Cetilistat, which is prepared by the invention, can meet the detection requirement.
Example 6 evaluation of specificity of antibody for detecting Cetilistat
1. Experimental methods
The specificity of the antibody for detecting the cetilistat is determined by carrying out a cross reaction experiment on the cetilistat antibody and the cetilistat medicine and the analogue thereof, wherein the specificity of the antibody is expressed by cross reaction rate (CR), and the smaller the cross reaction rate is, the stronger the specificity is. The tilistat and its analog orlistat were diluted in multiples and measured by indirect competitive ELISA, as in example 5, to obtain IC of the analog50The cross-reactivity ratio (CR) of Cetilistat was calculated according to the following formula,
CR(％)＝IC50(Cetilistat)/IC50(analogue). times.100%
2. Results of the experiment
The cross-reaction results of the Cetilistat polyclonal antibody prepared in example 3 and Cetilistat drugs and their analogues are shown in Table 2,
TABLE 2 results of cross-reaction of the Cetilistat polyclonal antibody with Cetilistat and its analogs
Figure BDA0003448831230000161
Note: NR represents no reaction
As can be seen from Table 2, the cross-reactivity of the polyclonal antibody for detecting Cetilistat with Cetilistat was 100%, IC506.57ng/mL, no crossover to the New Linlistat analog orlistat; the antibody for detecting the cetilistat has high recognition capability and strong specificity on the cetilistat, can effectively eliminate the interference of the cetilistat serving as a cetilistat analogue on the detection of the cetilistat, and can be specially used for detecting the cetilistat.
Example 7 development of a kit for detecting Cetilistat
1. Composition of the kit
The kit for detecting the cetilistat comprises the following parts:
(1) preparing an enzyme label plate coated with a coating antigen: taking the artificial antigen CET2-OVA of the Cetiramistat prepared in the example 2 as a coating antigen, diluting the coating antigen to 31.25 mu g/L by using a coating buffer solution, adding 100 mu L of the coating antigen into each hole, incubating overnight in a dark place at 37 ℃, pouring out liquid in the holes, washing for 2 times by using a washing solution, drying for 30s each time, then adding 200 mu L of a sealing solution into each hole, incubating for 2h in a dark place at 25 ℃, pouring out liquid in the holes, drying, and storing in a vacuum sealing way by using an aluminum film; the coating buffer solution is a carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, and the confining solution is a phosphate buffer solution with the pH value of 7.1-7.5 and containing casein and 0.1-0.3 mol/L in a mass ratio of 1-3 wt%;
(2) cetilistat standard solution: 8 concentration gradients of 1000. mu.g/L, 200. mu.g/L, 40. mu.g/L, 8. mu.g/L, 1.6. mu.g/L, 0.32. mu.g/L, 0.064. mu.g/L, 0.0128. mu.g/L respectively;
(3) the cetilistat polyclonal antibody prepared in example 3;
(4) enzyme conjugate: horseradish peroxidase-labeled cetilistat polyclonal antibody prepared in example 3;
(5) substrate color developing solution: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(6) the stop solution is 2mol/L H2SO4；
(7) The washing liquid has a pH value of 7.4, and contains 0.5-1.0% of tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages.
2. Sample detection
And numbering the corresponding micropores of the samples and the standard products in sequence, making 2 holes in parallel for each sample and standard product, and recording the positions of the standard holes and the sample holes. The enzyme conjugate concentrate was diluted with the enzyme conjugate diluent at a 1:10 volume ratio as needed (i.e., one portion of the enzyme conjugate concentrate was added to 10 portions of the enzyme conjugate diluent and was ready for formulation). Adding 50 mu L of standard substance/sample into corresponding micropores, adding 50 mu L of working solution of the enzyme conjugate, gently shaking and mixing uniformly, covering a cover plate with a cover plate, and reacting for 30min in a dark environment at 25 ℃. Spin-drying the liquid in the holes, and adding 250 mu L/hole of washing working solution; and (4) fully washing for 4-5 times, splashing the washing liquid in the plate hole at intervals of 10s every time, and patting the washing liquid by using absorbent paper (the washing liquid is not broken by clear bubbles and can be eaten and is not punctured by a used gun head). Adding 50 mu L/hole of substrate color development liquid A, adding 50 mu L/hole of substrate color development liquid B, lightly oscillating, mixing, covering with cover plate, and reacting at 25 deg.C in dark environment for 10 min; adding 50 mu L of stop solution into each hole, slightly oscillating and uniformly mixing, setting an enzyme-labeling instrument and a position of 450nm, and measuring the OD value of each hole.
3. Analysis of detection results
The percent absorbance of a standard or sample is equal to the average of the absorbance values of the standard or sample (double well) divided by the average of the absorbance values of the first standard (0 μ g/L) and multiplied by 100%. And drawing a standard curve graph by taking the percent absorbance of the standard substance as a vertical coordinate and taking the logarithm of the concentration (mu g/L) of the Cetilistat standard substance as a horizontal coordinate. And substituting the percent absorbance of the sample into the standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the new lysstat in the sample.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. The Cetilistat hapten is characterized in that the structural formula of the hapten is shown as a formula (I),
Figure FDA0003448831220000011
2. the Cetilistat hapten is characterized in that the structural formula of the hapten is shown as a formula (II),
Figure FDA0003448831220000012
3. the process for preparing the cetilistat hapten of claim 1, which comprises the following steps:
s1, controlling the temperature of a reaction system to be not more than 20 ℃, mixing a dichloromethane mixed solution of 2-amino-5-methylbenzoic acid and pyridine with chloroformic acid-3-chloropropyl ester, fully reacting, removing a solvent dichloromethane and unreacted pyridine, dissolving reactants with dichloromethane and water, controlling the temperature to be 4-6 ℃, adjusting the pH to 1-2, filtering precipitated solid, washing the solid with water, and drying to obtain an intermediate product 1;
s2, controlling the temperature of the reaction system to be not more than 20 ℃, mixing the dichloromethane solution of the intermediate product 1 with phosphorus oxychloride, and then heating, refluxing and fully reacting; controlling the temperature of a reaction system to be not more than 5 ℃, adding water to separate the reactants, purifying a dichloromethane layer, filtering, removing dichloromethane, crystallizing, purifying and drying to obtain an intermediate product 2;
s3, dissolving the intermediate product 2, 11-aminoundecanoic acid, potassium hydroxide and sodium iodide in anhydrous N, N-dimethylformamide, fully reacting, adding water, adjusting pH to be neutral, extracting with ethyl acetate, removing a water phase, concentrating an organic phase, separating and purifying to obtain the compound.
4. The method for preparing the cetilistat hapten of claim 2, wherein the temperature of a reaction system is controlled to be less than 20 ℃, a dichloromethane mixed solution of p-aminobenzoic acid and pyridine is mixed with cetyl chloroformate to be fully reacted, then a solvent dichloromethane and unreacted pyridine are removed, dichloromethane and water are used for dissolving reactants, the temperature is controlled to be 4-6 ℃, the pH value is adjusted to 1-2, the precipitated solid is filtered, and the solid is washed with water and dried to obtain the cetilistat hapten.
5. Use of the cetilistat hapten of claim 1 or 2 in the preparation of an artificial antigen of cetilistat.
6. An artificial antigen of neolistat, which is obtained by coupling the hapten of neolistat according to claim 1 or 2 to a carrier protein.
7. A artificial antigen combination of cetilistat, comprising an immunogen and a coating antigen, wherein the immunogen is obtained by coupling the hapten and a carrier protein according to claim 1; the coatingen is the artificial antigen of cetilistat of claim 6.
8. A Cetilistat antibody prepared by immunizing an animal with the artificial antigen obtained by coupling the Cetilistat hapten with a carrier protein according to claim 1.
9. An immunoassay method for detecting cetilistat, characterized in that the cetilistat artificial antigen of claim 6 is used as an antigen, and the cetilistat antibody of claim 8 is used as a detection antibody.
10. A kit for detecting cetilistat, comprising the cetilistat artificial antigen of claim 6 and the cetilistat antibody of claim 8.
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