CN114456124B - New cilastatin hapten, artificial antigen, antibody and preparation method and application thereof - Google Patents
New cilastatin hapten, artificial antigen, antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of food detection, and particularly provides a novel halimant, an artificial antigen, an antibody, a preparation method and application thereof, wherein the preparation method is used for preparing two types of novel halimant, namely a hapten CET1 and a hapten CET2, the hapten CET1 is used for preparing the artificial antigen CET1, the artificial antigen CET2-OVA is further used for detecting novel halimant, the minimum detection limit of the antibody on the novel halimant is 0.22ng/mL, the half inhibition concentration is 6.57ng/mL, the antibody is used for establishing an immunoassay method of the novel halimant, and the rapid and accurate detection of the novel halimant is realized.
Description
Technical Field
The invention relates to the field of food detection, in particular to a new listata hapten, an artificial antigen, an antibody, a preparation method and application thereof.
Background
Cetillistat, also known as cetiristat, cetilistat, cetiristat Li Sida, is a human lipase inhibitor originally developed by Alizyme, UK, for the treatment of obesity and diabetes caused by obesity, and was licensed to the Wuta pharmaceutical company in Japan by Alizyme, 2003 and marketed in Japan at 9 months 2013. The medicine has the advantages of no action on nervous system, no influence on other enzyme activities of gastrointestinal tract, no absorption, no blood intake, no appetite suppression, and no need of diet restriction. The phenomenon of oil-carrying during defecation (a large amount of oil drops float on the water surface) can occur 24 hours after the medicine is taken, namely, undigested fat is removed. The mechanism of action is to inactivate enzymes by covalent bond formation with active serine sites of gastric and pancreatic lipases in the stomach and small intestinal lumen, the inactivated enzymes being unable to hydrolyze fats, primarily triglycerides, in food 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 taking of the cilistat can cause adverse reactions such as intestinal tract action, fatty diarrhea, increased anus defecation and the like. Other digestive tract adverse reactions also include oily spotting, gastrointestinal exhaustion with stool, greasy (oily) stool, even fecal incontinence, and the like.
At present, the new listat is not approved by domestic market, so that no excessive report and research on the new listat detection method in China exist. Yu Zhou detected neolistata by HPLC-PDA (Yu Z, wan C.A simple and convenient method for simultaneous determination of four major species of illegal additives in slimming health food [ J ]. Journal of Liquid Chromatography & Related Technologies,2010,33 (4): 452-461.); qin Jun, et al, also used high performance liquid chromatography HPLC (Qin Jun, wang Xianqin, fu Shiling, et al HPLC determination method for Cetilistat and related impurities:, 2015.). However, the high performance liquid chromatography has the characteristics of high detection efficiency, high accuracy, strong anti-interference capability and the like; however, the equipment and instruments required for detection are expensive, high in cost, complicated in sample pretreatment and require professional operators, and the requirements of mass sample field detection are not met.
Compared with the existing detection method, the immunodetection method based on antigen-antibody specific molecular recognition has the advantages of being more advantageous in the field detection aspect, has the characteristics of rapidness, sensitivity, simplicity, convenience and the like, is low in cost, and has lower requirements on the skills of operators. However, the key point of the development of the immunoassay method is to design a proper neorisstat hapten and prepare an antibody with high sensitivity and strong specificity, but no related report on neorisstat hapten, artificial antigen and antibody is found in the prior art.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of a detection method of neorisstat in the prior art and provides a neorisstat hapten, an artificial antigen, an antibody, a preparation method and application thereof.
The invention aims to provide a novel cilastatin hapten.
The invention also aims to provide application of the novel lisat hapten in preparation of novel lisat artificial antigen.
The invention also aims to provide a novel artificial antigen.
The invention also aims to provide application of the novel human antigen to preparation of novel human antibodies.
The invention also aims to provide a novel orlistat antibody.
The invention also aims to provide an immunoassay method for detecting the novel lisat.
The invention also aims to provide a kit for detecting the novel lisat.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a neorisstat hapten which is hapten CET1 or hapten CET2, wherein the structural formula of the hapten CET1 is shown as a formula (I),
the hapten CET1 is named by a system naming method as follows: 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),
the hapten CET2 is named by a system naming method as follows: 4- (((hexadecyloxy) carbonyl) amino) benzoic acid.
The preparation method of hapten CET1 comprises the following steps:
s1, mixing a mixed solution of 2-amino-5-methylbenzoic acid and pyridine dichloromethane with 3-chloropropyl chloroformate at the temperature of a reaction system of not more than 20 ℃, fully reacting, removing solvent dichloromethane and unreacted pyridine, dissolving reactants by dichloromethane and water, controlling the temperature to be 4-6 ℃, regulating the pH value to be 1-2, filtering precipitated solids, washing the solids with water, and drying the solids to obtain an intermediate product 1;
s2, controlling the temperature of a reaction system to be not more than 20 ℃, mixing the methylene dichloride solution of the intermediate product 1 with phosphorus oxychloride, and then heating and refluxing to fully react. The temperature of the reaction system is controlled to be not more than 5 ℃, water is added to separate reactants, a dichloromethane layer is purified, filtered, dichloromethane is removed, crystallization and purification are carried out, and an intermediate product 2 is obtained by drying;
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 water phase, concentrating organic phase, separating and purifying to obtain the neorivastat hapten CET1.
Preferably, the molar ratio of the 2-amino-5-methylbenzoic acid, 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 mixed solution of 2-amino-5-methylbenzoic acid and pyridine in dichloromethane is mixed with 3-chloropropyl chloroformate in the step S1, wherein the temperature of the mixed solution of 2-amino-5-methylbenzoic acid and pyridine in dichloromethane is reduced to 15 ℃ and then the mixed solution is mixed with 3-chloropropyl chloroformate; the mixing is performed by dripping 3-chloropropyl chloroformate into the mixed solution.
Preferably, the solvent dichloromethane and unreacted pyridine are removed in step S1 by spin evaporation.
Preferably, the control temperature in step S1 is 5 ℃.
Preferably, the pH adjustment in step S1 is performed with 10% by volume hydrochloric acid.
Preferably, the molar ratio of the intermediate product 1 to phosphorus oxychloride in the step S2 is 1:2-4.
Further preferably, the molar ratio of intermediate 1 to phosphorus oxychloride in step S2 is 1:3.
Preferably, sodium hydride is added to remove water when the methylene dichloride solution of the intermediate product 1 is mixed with phosphorus oxychloride in the step S2, and benzophenone is used as an indicator to indicate the water removal effect.
Preferably, the step S2 of mixing the dichloromethane solution of the intermediate product 1 with phosphorus oxychloride is to control the temperature of the dichloromethane solution of the intermediate product 1 to 15 ℃ and then slowly dropwise add the phosphorus oxychloride.
Preferably, the step S2 of adding water to separate the reactants is adding primary water; the dichloromethane layer is purified by washing the dichloromethane layer with water, then with saturated sodium bicarbonate solution, then with saturated saline solution, and then adding anhydrous sodium sulfate and activated carbon to remove excess moisture and impurities.
Preferably, step S2 removes dichloromethane from the purified dichloromethane layer by distillation under reduced pressure.
Preferably, the crystallization and purification in the step S2 are to dissolve the reactant with methylene dichloride removed in toluene, and then to carry out fractional cooling crystallization, filtration and washing.
Further preferably, the condition of the fractional cooling crystallization is that the temperature is firstly reduced to 14-16 ℃, the stirring crystallization is carried out for 2.5-3 hours, then the temperature is continuously reduced to 0 ℃, and the stirring crystallization is carried out for 2.5-3 hours.
Further preferably, the condition of the fractional cooling crystallization is that the temperature is firstly reduced to 15 ℃, the crystallization is stirred for 3 hours, then the temperature is continuously reduced to 0 ℃, and the crystallization is stirred for 3 hours.
Preferably, the drying in step S2 is drying under reduced pressure at 45 ℃ to constant weight.
Preferably, the molar ratio of the intermediate 2, 11-aminoundecanoic acid, potassium hydroxide and sodium iodide in the step S3 is 1:1-2:1-3:0.1-0.5.
It is further preferred that the molar ratio of intermediate 2, 11-aminoundecanoic acid, potassium hydroxide, and sodium iodide in step S3 is 1:1.2:2:0.2.
Preferably, the condition of the sufficient reaction in the step S3 is that the reaction is carried out for 10 to 12 hours at a temperature of between 90 and 95 ℃.
Further preferably, the condition of the sufficient reaction in step S3 is heating to 90℃for reaction for 12 hours.
Preferably, in the separation and purification step S3, the concentrated organic phase is applied to a silica gel column, and ethyl acetate and petroleum ether are used in a volume ratio of 4-6: 1, eluting 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:
the structural formula of the intermediate product 2 is as follows:
the preparation method of hapten CET2 comprises the following steps:
the temperature of the reaction system is controlled to be less than 20 ℃, the methylene dichloride mixed solution of para aminobenzoic acid and pyridine is mixed with cetyl chloroformate for full reaction, then the solvent methylene dichloride and unreacted pyridine are removed, reactants are dissolved by methylene dichloride and water, the temperature is controlled to be 4-6 ℃, the pH value is regulated to be 1-2, the precipitated solid is filtered, and the solid is washed with water and dried to obtain the neorisstat hapten CET2.
Preferably, the molar ratio of the para-aminobenzoic acid to the pyridine to the cetyl chloroformate is 1:2-4:1-1.5.
Further preferably, the molar ratio of para-aminobenzoic acid, pyridine and cetyl chloroformate is 1:3:1.2.
Preferably, the methylene chloride mixed solution of the para-aminobenzoic acid and the pyridine is cooled to 15 ℃ and then mixed with cetyl chloroformate.
Preferably, the solvent dichloromethane and unreacted pyridine are removed by means of rotary evaporation.
Preferably, the pH adjustment is performed using 10% by volume hydrochloric acid.
The use of the hapten CET1 and/or hapten CET2 for the preparation of novel artificial antigens is also within the scope of the invention.
A novel artificial antigen is obtained by coupling hapten CET1 or hapten CET2 with carrier protein, the structural formula of artificial antigen CET1 obtained by coupling hapten CET1 with carrier protein is shown as formula (III), wherein P is carrier protein,
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 the carrier protein,
preferably, the carrier protein (P) is any one or more of bovine serum albumin (Bovine serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), lactoferrin (LF) or chicken Ovalbumin (OVA).
According to the preparation method of the artificial antigen CET1 or the artificial antigen CET2, hapten CET1 or hapten CET2 is utilized to couple carrier protein through an active ester method.
As a specific embodiment of the above 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 for 2-4 hours in a dark place to obtain hapten CET1 activating solution;
(2) Adding a carrier protein to the PBS buffer;
(3) Slowly and dropwise adding the hapten CET1 activation solution in the step (1) into the carrier protein solution in the step (2), and reacting for 12 hours at 4 ℃;
(4) And (3) dialyzing the reaction solution obtained in the step (3) by using PBS buffer solution to obtain the artificial antigen CET1.
Preferably, the molar ratio of hapten CET1, NHS and EDC in step (1) is 1:1-2:1.5-2.5.
More preferably, the molar ratio of hapten CET1, NHS to EDC in step (1) is 1:1.75:2.25.
Preferably, the mass 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 ratio of the carrier protein to PBS buffer in step (2) is 10 mg/1 mL.
Preferably, the mass ratio of hapten CET1 in step (1) to carrier protein in step (2) is 1-2:1-4.
More preferably, the mass ratio of hapten CET1 in step (1) to carrier protein in step (2) is 1:3.
The preparation method of the artificial antigen CET2 is the same as that of the artificial antigen CET1.
The application of the novel human antigen to preparation of the novel human antigen is also within the protection scope of the invention.
A novel artificial antigen combination comprising an immunogen and a coating antigen, the immunogen being derived from said hapten CET1 conjugated carrier protein, i.e. artificial antigen CET1; the coating antigen is the novel artificial antigen.
Preferably, the coating antigen is derived from the hapten CET2 conjugated carrier protein, i.e.the artificial antigen CET2.
Further preferably, the immunogen is derived from the hapten CET1 conjugated carrier protein Lactoferrin (LF), artificial antigen CET1-LF; the coating antigen is obtained from the CET2 coupling carrier protein chicken Ovalbumin (OVA), namely artificial antigen CET2-OVA.
The use of the artificial antigen combination in the preparation of antibodies to and/or detection of neorisstat is also within the scope of the present invention.
The novel orlistat antibody is prepared by immunizing animals with artificial antigen CET1 obtained by coupling hapten CET1 with carrier protein.
Preferably, the neorisstat antibody is prepared from an artificial antigen CET1-LF immunized animal obtained by coupling the hapten CET1 with the carrier protein Lactoferrin (LF).
Preferably, the neorisetah antibody is a monoclonal antibody or a polyclonal antibody.
A preparation method of a novel orlistat polyclonal antibody utilizes an artificial antigen CET1 obtained by coupling a hapten CET1 with a carrier protein to immunize an experimental animal.
Preferably, the artificial antigen CET1-LF obtained by coupling the hapten CET1 with the carrier protein Lactoferrin (LF) is used for immunizing experimental animals.
As a specific embodiment of the above method, the preparation method of the neorisstat polyclonal antibody comprises the following steps:
(1) Using the artificial antigen CET1 to be matched with an immune adjuvant to immunize an experimental animal;
(2) In the first immunization, the artificial antigen CET1 is emulsified with an equal volume of complete Freund's adjuvant to immunize New Zealand white rabbits;
(3) A second immunization after 4 weeks followed by booster immunization every 3 weeks.
(4) After four times of booster immunization, the heart is taken out blood and separated to obtain serum, namely the polyclonal antibody.
Preferably, the artificial antigen CET1 is an artificial antigen CET1-LF obtained by coupling the hapten CET1 with the carrier protein Lactoferrin (LF).
The application of the novel orlistat antibody in detecting novel orlistat and/or preparing a kit for detecting novel orlistat is also within the protection scope of the invention.
An immunoassay method for detecting neolisat takes the neolisat artificial antigen as an antigen and takes an antibody prepared by an artificial antigen CET1 immune animal obtained by coupling a hapten CET1 with a carrier protein as a detection antibody for detection; the immunoassay method is a method of non-diagnostic therapeutic interest.
Preferably, the immunoassay method uses artificial antigen CET2 obtained by coupling hapten CET2 with carrier protein as antigen.
Further preferably, the immunoassay method uses an artificial antigen CET2-OVA of which carrier protein is chicken Ovalbumin (OVA) as an antigen, and uses an antibody prepared by immunizing an animal with an artificial antigen CET1-LF of which carrier protein is Lactoferrin (LF) as an immunogen as a detection antibody.
Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensor, immune colloidal gold, and the like.
A kit for detecting neorisstat, comprising the neorisstat artificial antigen and the neorisstat 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 of the hapten CET1 coupled with the carrier protein.
Further preferably, the kit comprises an antibody prepared from an artificial antigen CET2-OVA obtained by coupling the hapten CET2 with a carrier protein chicken Ovalbumin (OVA) and an artificial antigen CET1-LF of coupling the hapten CET1 with a carrier protein Lactoferrin (LF).
Preferably, the kit further comprises one or more of an enzyme label plate, a cilostat standard, an enzyme conjugate, a chromogenic solution, a stop solution or a wash solution.
Preferably, the kit comprises the novel artificial antigen coated ELISA plate, a novel standard product of the novel artificial antigen, an enzyme conjugate, a chromogenic solution, a stop 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 neorisstat hapten, hapten CET1 and hapten CET2, wherein artificial antigen CET1-LF prepared by coupling hapten CET1 with Lactoferrin (LF) is used as an immunogen, a neorisstat antibody is further prepared, and artificial antigen CET2-OVA prepared by coupling hapten CET2 with chicken Ovalbumin (OVA) is used as a coating antigen; the hapten CET1 and the to-be-detected object of the invention have high overlapping degree of the skeleton structure of the novel listat, the immunogenicity of the novel listat artificial antigen CET1-LF is effectively improved, the structural difference between the artificial antigens 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 for the neolisat is 0.22ng/mL, and the half inhibition concentration IC 50 The quantitative detection range is 0.78-55.26 ng/mL, the detection sensitivity is high, and the linear range is wide; the cross reaction rate of the orlistat is 100%, and the orlistat is not cross-reacted; the antibody of the invention has the characteristics of simplicity, convenience, rapidness, strong specificity, wide linear range and high sensitivity; the purpose of rapidly and accurately detecting the novel lisat can be realized by utilizing the novel lisat artificial antigen and the novel lisat artificial antibody.
Drawings
FIG. 1 is a synthetic scheme for hapten CET1 of the present invention.
FIG. 2 is a synthetic scheme for hapten CET2 of the present invention.
FIG. 3 is a UV scan of haptens CET1, LF, CET1-LF of the invention.
FIG. 4 is an ultraviolet scan of hapten CET2, OVA, CET2-OVA of the present invention.
FIG. 5 is a standard curve of an indirect competition ELISA using the novel cilastatin polyclonal antibody of the invention.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 Synthesis and identification of New risetah hapten
1. Synthesis and identification of neolistata hapten CET1
1.1 Synthesis of neolistata hapten CET1
2-amino-5-methylbenzoic acid (1 mmoL) and pyridine (3 mmoL) are taken and dissolved in methylene dichloride, stirred until the mixture is clarified, cooled to 15 ℃, and then 3-chloropropyl chloroformate (1.2 mmoL) is dropwise added, the temperature is controlled to be less than 20 ℃, and the reaction is carried out for 2 hours at room temperature after the dropwise addition is finished. After the reaction is finished, removing dichloromethane and unreacted pyridine by rotary evaporation, adding 750g of dichloromethane, 250g of water, cooling by ice water, controlling the temperature to be 5 ℃, dropwise adding hydrochloric acid with the volume fraction of 10%, adjusting the pH to 1-2, precipitating a large amount of solids, filtering, washing the solids by water, and drying to obtain an intermediate product 1.
Intermediate 1 (1 mmoL) is added into a reaction bottle, dichloromethane is dissolved (sodium hydride is used for removing water, benzophenone is used as an indicator for indicating the water removal effect), ice water is cooled, the temperature of a reaction system is controlled to 15 ℃, phosphorus oxychloride (3 mmoL) is slowly added dropwise, the temperature of the system is controlled to be not more than 20 ℃, and after the dropwise addition is finished, the reaction is fully performed by heating and refluxing. After the reaction, cooling with ice water, controlling the system temperature not to exceed 5 ℃, slowly dropwise adding primary water, continuously stirring for half an hour, layering, washing a dichloromethane layer with saturated sodium bicarbonate solution, washing with saturated saline solution, finally washing with anhydrous sodium sulfate 10g, stirring active carbon 5g at room temperature for one hour, filtering, distilling to remove dichloromethane under reduced pressure, 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 (1 mmoL) and 11-aminoundecanoic acid (1.2 mmoL), adding potassium hydroxide (2 mmoL) and sodium iodide (0.2 mmoL) and dissolving with anhydrous N, N-Dimethylformamide (DMF), heating to 90 ℃ for reacting for 12 hours, adding water after the reaction is finished, adding 1 mole/L hydrochloric acid to adjust the pH to be neutral, extracting with ethyl acetate, oscillating, standing, removing water phase, concentrating an organic phase, loading on a silica gel column, and using ethyl acetate and petroleum ether according to the volume ratio of 5: and (1) eluting, separating and purifying by using the mixed solvent to obtain the cilastatin hapten CET1. The synthetic route for hapten CET1 is shown in FIG. 1.
1.2 identification of the neolistata hapten CET1
Nuclear magnetic identification of hapten CET 1: 1 H 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 of hapten CET1 resulted in the following: MS: c (C) 23 H 34 N 2 O 5 ：418.25，ESI-[M-H] - ：417.3。
The structural formula of hapten CET1 is shown as formula (I):
hapten CET1 is named by 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 neolistata hapten CET2
2.1 Synthesis of neolistat hapten CET2
Weighing para aminobenzoic acid (1 mmoL) and pyridine (3 mmoL), dissolving in dichloromethane, stirring to clarify, cooling to 15 ℃, dropwise adding cetyl chloroformate (1.2 mmoL), controlling the temperature to be less than 20 ℃, and reacting for 2 hours at room temperature after the dropwise adding is finished. After the reaction is finished, removing dichloromethane and unreacted pyridine by rotary evaporation, adding dichloromethane, water, cooling with ice water, controlling the temperature to be 5 ℃, dropwise adding hydrochloric acid with volume fraction of 10%, adjusting the pH to 1-2, precipitating a large amount of solid, filtering, washing the solid with water, and drying to obtain the neorivastat hapten CET2. The synthetic route pattern for hapten CET2 is shown in FIG. 2.
2.2 identification of the neolistata hapten CET2
Nuclear magnetic identification of hapten CET 2: 1H NMR (600 MHz, DMSO-d 6) δ9.98 (s, 1H), 8.12-8.07 (m, 2H), 7.86 (d, J=8.6 Hz, 2H), 1.62 (p, J=6.8 Hz, 2H), 1.47-1.08 (m, 29H), 0.85 (t, J=6.9 Hz, 3H).
The mass spectrum of hapten CET2 resulted in: MS: c (C) 24 H 39 NO 4 ：405.29，ESI-[M-H] - ：404.3。
The structural formula of hapten CET2 is shown as a formula (II):
hapten CET2 is named by systematic nomenclature: 4- (((hexadecyloxy) carbonyl) amino) benzoic acid.
EXAMPLE 2 Synthesis and identification of novel human antigens
1. Synthesis of novel lisi artificial antigen
Hapten CET1 or hapten CET2 prepared in example 1 is conjugated to Lactoferrin (LF) or chicken Ovalbumin (OVA) by an active ester method.
3.3mg (0.008 mmol) of hapten CET1 prepared in example 1, 0.014mmol of NHS and 0.018mmol of EDC are weighed and dissolved in 50-200 mu L of DMF, and stirred at room temperature for 2-4 hours in a dark place to obtain hapten activating solution; 10mg of LF was added to 1mL of PBS buffer (0.01 mol/L, pH=7.4); slowly and dropwise adding the hapten activating solution into a PBS buffer solution of LF, and reacting for 12 hours at 4 ℃; then, the artificial antigen CET1 is obtained after dialysis for 3 days and 3 times per day by using PBS buffer solution, and is split into centrifuge tubes for storage at the temperature of minus 20 ℃ for use.
Wherein, the formula of PBS buffer solution: na (Na) 2 HPO 4 ·12H 2 O 2.90g，NaCl 8.50g，KCl 0.20g，KH 2 PO 4 0.20g, distilled water was added to a volume of 1000mL.
The preparation of artificial antigen CET2 is identical to artificial antigen CET1, except that the carrier protein is chicken ovalbumin.
2. Identification of novel human antigens
Ultraviolet scanning is carried out on LF, hapten CET1 and synthetic artificial antigens CET1-LF. The ultraviolet scan results are shown in fig. 3.
The LF, hapten CET1 and CET1-LF are respectively subjected to ultraviolet (200-350 nm) scanning identification, and the absorption curve of the CET1-LF is found to be obviously different from that of carrier protein LF by comparing the highest absorption values of all substances before and after coupling, the hapten CET1 has a characteristic peak at 240nm and 300nm, and after coupling with LF, the absorption peak of the CET1-LF is obviously higher than that of LF at 240nm and 300nm, and the absorption curve of the CET1-LF is obviously shifted relative to that of the hapten CET1. Since the small molecular components such as unreacted medicines and the like are completely dialyzed and removed in the dialysis process of the coupling reaction, the characteristic peak of the medicines, which appears in the coupling product, is contributed by the medicine molecules combined by the proteins, so that the reaction product is a complex of carrier protein LF and hapten CET1, and the successful coupling of CET1-LF is demonstrated.
Ultraviolet scanning was performed on OVA, hapten CET2 and the synthesized CET2-OVA described above. The ultraviolet scan results are shown in fig. 4.
The OVA, hapten CET2 and CET2-OVA are respectively subjected to ultraviolet (200-350 nm) scanning identification, and the absorption curve of the CET2-OVA is found to be obviously different from that of the carrier protein OVA by comparing the highest absorbance values of all substances before and after coupling, the hapten CET2 has a characteristic peak at 240nm and 260nm respectively, and after coupling of the OVA, the absorption peak of the CET2-OVA is obviously higher than that of the OVA at 240nm and 260nm and is obviously displaced relative to the curve of the hapten CET2. Since the small molecule components such as unreacted drugs are completely removed by dialysis in the dialysis process of the coupling reaction, the drug characteristic peak of the coupling product is contributed by the protein-bound drug molecules, so that the reaction product is a complex of carrier protein OVA and hapten CET2, and the successful coupling of CET2-OVA is demonstrated.
Example 3 preparation of antibodies
1. Preparation of polyclonal antibodies
CET1-LF prepared in example 2 was used as immunogen and immunoadjuvant (incomplete Freund's adjuvant for the first immunization and Freund's incomplete adjuvant for the later booster immunization) were emulsified uniformly in a volume ratio of 1:1, and the body weight of the immunized New Zealand white rabbit was 2.5-3 kg. The immunization was performed a second time after 4 weeks, followed by a booster immunization every 3 weeks. The ear margin vein was bled 1 week after the third boost and serum titers were determined using an indirect competition ELISA. When the potency no longer increases, the ear margin vein is used to boost the immunity. After one week heart blood was collected and serum was obtained from the collected blood in the following manner: the mixture is incubated at 37 ℃ for 0.5 to 1 hour, then kept still at 4 ℃ for overnight, the separated serum is sucked by a suction pipe, and then centrifuged at 3000 to 5000rpm for 10 minutes at 4 ℃ to obtain the supernatant. The antiserum is purified to polyclonal antibody by ammonium sulfate precipitation method, and frozen at-20deg.C for use.
Example 4 optimization of the combination of Nelistat immunogen and coating antigen
The invention also prepares artificial antigen CET1-BSA taking Bovine Serum Albumin (BSA) as carrier protein and artificial antigen CET1-OVA taking chicken Ovalbumin (OVA) as carrier protein according to the preparation method of CET1-LF of the embodiment 2, and the coupling is successful.
The prepared CET1-BSA and CET1-LF prepared in example 2 were used as immunogens, and neorisstat polyclonal antibodies prepared by immunizing New Zealand white rabbits according to the method of example 3 were subjected to coating antigen screening to prepare CET1-OVA and CET2-OVA prepared in example 2 as coating antigens, and the titers and inhibition ratios of antisera obtained by immunizing New Zealand white rabbits were detected by ELISA.
The specific operation steps are as follows:
(1) Diluting the coating original CET1-OVA and CET2-OVA with coating solution (0.05M carbonate buffer solution, pH 9.6) to the concentration of 250ng/mL, coating a 96-well ELISA plate, adding 100 mu L of the coating solution into each well, incubating overnight in a constant-temperature water bath at 37 ℃, discarding the coating solution, and washing 2 times with PBST (0.01M PBS,0.06%Tween-20 (v/v));
(2) Adding 120 μl of sealing solution (1 wt% fish gelatin protein) into each hole, sealing at 37deg.C for 3 hr, discarding sealing solution, clapping the plate, and oven drying at 37deg.C;
(3) The prepared neorisstat polyclonal antibody was diluted to 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000 with PBST, while blank wells (replaced with PBST) were set; 1mg/mL of the orlistat drug was diluted 1000-fold to 1 μg/mL with PBST;
the titers are as follows: adding 50 mu L of PBST into each hole, diluting the obtained neorisstat polyclonal antibody by double ratio, sequentially adding 50 mu L of the neorisstat polyclonal antibody into each hole, and replacing the last hole with 50 mu L of PBST without adding the antibody;
inhibition column: adding 50 mu L of neorisstat medicine into each hole, then diluting the double ratio to obtain neorisstat polyclonal antibody, sequentially adding 50 mu L of neorisstat polyclonal antibody into each hole, adding no antibody into the last hole, and replacing the last hole with 50 mu L of PBST; incubating at 37deg.C for 40min, washing for 5 times, and clapping the plate;
(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 a color development solution, and incubating at 37 ℃ for color development for 10min;
(6) 10% H is added 2 SO 4 The reaction was terminated and OD values were read at 450 nm;
the potency is OD 450 The dilution of the antiserum was about 1.0.
Inhibition ratio = (OD value of potency-OD value of inhibition)/OD value of inhibition × 100%
The titers and inhibition rates of the antisera of the 4-group immunogen and coating antigen combinations are shown in table 1.
Table 14 titers and inhibition rates of antisera of group immunogen and coating antigen combinations
As can be seen from Table 1, different novel human antigens have certain titers as antisera generated by immunized New Zealand white rabbits, and the obtained antisera has different degrees of inhibition effects on a target analyte of novel Listat. Wherein, the antiserum potency 1:512000 and inhibition ratio 90.31% shown by the immunogen and coating antigen structure combination of the number 4 are the optimal combination; under the combination, the neorisstat antibody not only can specifically identify a target analyte neorisstat, but also has good antibody sensitivity; the antigen and coating antigen combinations of numbers 4 are optimal because the antiserum titers and inhibition rates are higher than those of the antigen and coating antigen combinations of numbers 1, 2 and 3. CET1-LF is used as immunogen and CET2-OVA is used as coating antigen.
Example 5 establishment of an Indirect competitive ELISA assay for New Listat
1. Experimental method
An indirect competition ELISA method to detect neolisat comprising the steps of:
(1) The artificial antigen CET2-OVA prepared in example 2 was used as a coating source, diluted to 62.5ng/mL with a coating solution, coated with 96-well ELISA plates, added with 100. Mu.L per well, and incubated overnight (12 h) at 37 ℃;
(2) Removing the coating liquid, washing twice, and beating to dryness;
(3) 120 mu L of blocking solution (namely 1wt% of fish skin collagen) is added into each hole, and the mixture is blocked for 3 hours at 37 ℃;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30min, taking out, and bagging for standby by self-sealing;
(5) The PBST of example 4 was used at 1: the preparation of the polyclonal antibody of the new listat prepared in the example 3 is diluted 4000 times, and the new listat drug is diluted 6000ng/mL, 1000ng/mL, 166.67ng/mL, 27.78ng/mL, 4.63ng/mL, 0.77ng/mL, 0.13ng/mL, 0.02ng/mL and 0.004ng/mL;
(6) Adding 50 mu L of a drug diluent of the to-be-detected cilostat (three groups are parallel) into each row, adding 50 mu L of a polyclonal antibody diluent of the cilostat prepared in the example 3 per hole, incubating for 40min at 37 ℃, washing for five times, and beating to dryness;
(7) Adding goat anti-rabbit secondary antibody-HRP (5000-fold dilution) 100 μl/well, incubating at 37deg.C for 30min, washing five times, and drying;
(8) Adding a developing solution, and developing for 10min at 100 mu L per hole;
(9) 50. Mu.L of 10% H was added 2 SO 4 The reaction was stopped and the OD was read at 450 nm.
2. Experimental results
The standard curve of the indirect competition ELISA for detecting the neorisstat drug is shown in FIG. 5, and as can be seen from FIG. 5, the standard curve of the indirect competition ELISA for detecting the neorisstat drug has a typical S-shaped curve, has good detection sensitivity, and is used for detecting the half inhibition concentration (IC 50 ) The quantitative detection range is 0.78-55.26 ng/mL, and the lowest detection Limit (LOD) is 0.22ng/mL; the invention proves that the antibody for detecting the novel lisat prepared by the invention can meet the detection requirement.
Example 6 evaluation of specificity for detection of antibodies to neolisat
1. Experimental method
The specificity of the antibodies for detecting the neolisat is determined by cross-reaction experiments of the neolisat antibodies with neolisat drugs and analogues thereof, the specificity of the antibodies being expressed in terms of cross-reactivity (CR), the smaller the cross-reactivity, the stronger the specificity. The preparation method comprises respectively subjecting orlistat and its analogue orlistat to multiple dilution, and measuring by indirect competition ELISA method, and obtaining analogue IC by the same procedure as in example 5 50 Values, the cross-reactivity (CR) of cilirta was calculated according to the following formula,
CR(％)＝IC 50 (New Listat)/IC 50 (analog) ×100%
2. Experimental results
The results of cross-reactions of the polyclonal antibodies to cilostat prepared in example 3 with cilostat drugs and analogues thereof are shown in table 2,
TABLE 2 Cross-reaction results of New Listat polyclonal antibodies with New Listat and analogues thereof
Note that: NR represents no reaction
As is clear from Table 2, the cross-reactivity of the polyclonal antibody for detecting neolisat to neolisat was 100%, and IC 50 6.57ng/mL, no crossover to the new listata analog orlistat; the method has the advantages that the antibody for detecting the neorisstat has high recognition capability and strong specificity on the neorisstat, can effectively eliminate the interference of the neorisstat analogue orlistat on the detection of the neorisstat, and can be specially used for detecting the neorisstat.
EXAMPLE 7 development of a kit for detecting New lisat
1. Composition of the kit
A kit for detecting neolisat comprising the following parts:
(1) Preparing an ELISA plate coated with a coating source: the novel risc artificial antigen CET2-OVA prepared in example 2 is used as a coating source, the coating source is diluted to 31.25 mug/L by using a coating buffer solution, 100 mug/L is added into each hole, the mixture is incubated overnight at 37 ℃ in a dark place, liquid in the hole is poured out, the mixture is washed for 2 times by using a washing solution for 30 seconds each time, the mixture is dried by beating, 200 mug of sealing solution is then added into each hole, the mixture is incubated for 2 hours at 25 ℃ in a dark place, the liquid in the hole is poured out, and the mixture is vacuum-sealed by using an aluminum film for storage after drying; the coating buffer solution is carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, the sealing solution is phosphate buffer solution with the pH value of 7.1-7.5 and contains casein with the mass ratio of 1-3 wt% and 0.1-0.3 mol/L;
(2) New listata standard solution: 8 concentration gradients of 1000 μg/L,200 μg/L,40 μg/L,8 μg/L,1.6 μg/L,0.32 μg/L,0.064 μg/L,0.0128 μg/L, respectively;
(3) The neorisstat polyclonal antibody prepared in example 3;
(4) Enzyme conjugate: horseradish peroxidase-labeled neorisstat polyclonal antibody prepared in example 3;
(5) Substrate color development liquid: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(6) The termination liquid is 2mol/L H 2 SO 4 ；
(7) The washing liquid is pH 7.4 and contains 0.5-1.0% Tween-20, 0.01-0.03% sodium azide preservative and 0.1-0.3 mol/L phosphate buffer solution, wherein the percentages are weight volume percentages.
2. Sample detection
And numbering corresponding micropores of the sample and the standard substance in sequence, making 2 holes of each sample and each standard substance in parallel, and recording the positions of the standard holes and the sample holes. The enzyme conjugate concentrate is diluted with an enzyme conjugate diluent in a 1:10 volume ratio (i.e., one portion of enzyme conjugate concentrate is added to 10 portions of enzyme conjugate diluent, ready for use) as desired. Adding 50 mu L of standard substance/sample into corresponding microwells, adding 50 mu L of enzyme conjugate working solution, gently shaking and mixing, and placing the mixture in a light-shielding environment at 25 ℃ for reaction for 30min after covering with a cover plate film. Spin-drying the liquid in the holes, and adding 250 mu L/hole of washing working solution; washing for 4-5 times, and at intervals of 10s each time, pouring out the washing liquid in the plate holes, and drying by using absorbent paper (after drying, the unused gun heads can be used for puncturing by the bubbles which are not clear). Adding 50 mu L/hole of substrate color development liquid A, adding 50 mu L/hole of substrate color development liquid B, gently shaking and uniformly mixing, and placing the mixture in a light-shielding environment at 25 ℃ for reaction for 10min after a cover plate film is used; adding 50 mu L of stop solution per hole, gently shaking and mixing, setting an enzyme label instrument and 450nm, and measuring the OD value of each hole.
3. Analysis of detection results
The percent absorbance of the 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 values of the first standard (0 μg/L) and multiplied by 100%. The standard graph is plotted with the percent absorbance of the standard as the ordinate and the logarithm of the concentration of the cilastat standard (μg/L) as the abscissa. Substituting the percentage absorbance of the sample into a standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the dilution factor corresponding to the standard curve to obtain the actual concentration of the new lisat in the sample.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (6)
1. A novel cilastatin hapten is characterized in that the structural formula of the hapten is shown as a formula (I),
2. a novel cilastatin hapten is characterized in that the structural formula of the hapten is shown as a formula (II),
3. the method for preparing the neorisstat hapten according to claim 1, which comprises the following steps:
s1, controlling the temperature of a reaction system to be not more than 20 ℃, mixing a mixed solution of 2-amino-5-methylbenzoic acid and pyridine dichloromethane with 3-chloropropyl chloroformate, fully reacting, removing solvent dichloromethane and unreacted pyridine, dissolving reactants by using dichloromethane and water, controlling the temperature to be 4-6 ℃, adjusting the pH to 1-2, filtering precipitated solids, washing the solids with water, and drying to obtain an intermediate product 1;
s2, controlling the temperature of a reaction system to be not more than 20 ℃, mixing a dichloromethane solution of the intermediate product 1 with phosphorus oxychloride, and then heating and refluxing for full reaction; the temperature of the reaction system is controlled to be not more than 5 ℃, water is added to separate reactants, a dichloromethane layer is purified, filtered, dichloromethane is removed, crystallization and purification are carried out, and an intermediate product 2 is obtained by drying;
s3, dissolving intermediate products 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;
the structural formula of the intermediate product 1 is
/>
The structural formula of the intermediate product 2 is
4. The preparation process of new listat hapten includes the steps of mixing mixed solution of p-aminobenzoic acid and pyridine with cetyl chloroformate, reaction, eliminating solvent dichloromethane and unreacted pyridine, dissolving reactant in dichloromethane and water at 4-6 deg.c, regulating pH to 1-2, filtering to separate out solid, water washing and stoving.
5. Use of a neorisstat hapten according to claim 1 or 2 for the preparation of a neorisstat artificial antigen.
6. A combination of novel human antigens comprising an immunogen and a coating antigen, said immunogen being derived from the novel human hapten conjugated lactoferrin of claim 1; the coating antigen is obtained by coupling neorisestat hapten with chicken egg albumin according to claim 2.
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| US4410634A (en) * | 1979-10-26 | 1983-10-18 | Dynasciences Corporation | Method of passively adsorbing immuno-reactive haptens to solid phases |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US4410634A (en) * | 1979-10-26 | 1983-10-18 | Dynasciences Corporation | Method of passively adsorbing immuno-reactive haptens to solid phases |
| CN112040980A (en) * | 2018-04-06 | 2020-12-04 | 瑞泽恩制药公司 | Leptin receptor agonist antibodies for the treatment of metabolic dysfunction or hypoleptin |
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| UV spectrophotometric method development and validation for determination of cetilistat in API and in pharmaceutical dosage form.《International Journal for Pharmaceutical Research Scholars》.2018,第7卷(第1期),第1-8页. * |
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