CN112250641A - Hydrochlorothiazide hapten, artificial antigen, antibody and preparation method and application thereof - Google Patents
Hydrochlorothiazide hapten, artificial antigen, antibody and preparation method and application thereof Download PDFInfo
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- CN112250641A CN112250641A CN202011037271.6A CN202011037271A CN112250641A CN 112250641 A CN112250641 A CN 112250641A CN 202011037271 A CN202011037271 A CN 202011037271A CN 112250641 A CN112250641 A CN 112250641A
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- hydrochlorothiazide
- hapten
- antibody
- formula
- artificial antigen
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Abstract
The invention discloses a hydrochlorothiazide hapten, an artificial antigen, an antibody, a preparation method and application thereof. The invention prepares various hydrochlorothiazide haptens, couples carrier protein to further obtain artificial antigen, separates and purifies to obtain hydrochlorothiazide antibody, and finally obtains the best combination of the antibody and the coated artificial antigen by screening immune artificial antigen and the coated artificial antigen with various structures. The antibody has high sensitivity and high specificity recognition capability on the dihydrochlorothiazide, the half inhibition concentration is 12.27ng/mL, and a core raw material is provided for establishing an immunodetection method of the specific dihydrochlorothiazide. On the basis, the antibody is utilized to establish an enzyme linked immunosorbent assay kit and a colloidal gold test strip for detecting the illegally added dihydrocathelicidin in the health-care food, has the characteristics of short detection time, simple operation, high sensitivity, high accuracy and the like, and realizes the specific detection of the illegally added dihydrocathelicidin in the health-care food.
Description
Technical Field
The invention relates to the technical field of antigen-antibody detection, in particular to a hydrochlorothiazide hapten, an artificial antigen, an antibody and a preparation method and application thereof.
Background
Hydrochlorothiazide (HCT), also known as Hydrochlorothiazide, has moderate diuretic and antihypertensive effects, acts on edematous diseases, can excrete excessive sodium and water in the body, reduces the extracellular volume, has the effect of eliminating edema, and can be used for treating hypertension, central and renal diabetes insipidus and renal calculus.
Statistically, the number of adult hypertensive patients worldwide has increased from 5 hundred million 9400 in 1975 to 11 hundred million 3000 million in 2015. From 1979 to 2016, the number of obese adult women increased from 6 to 3 billion 9 million and obese men increased from 3 to 1 million to 2 to 8 million and 1 million worldwide. Under the large background, the demand of society for health-care food for assisting weight loss and lowering blood pressure is greatly increased. In order to gain violence, some illegal molecules illegally add HCT into the antihypertensive health-care food or the weight-losing health-care food so as to achieve the effect of quickly lowering blood pressure or the effect of reducing weight in a short time. The health food with the illegally added HCT is unknowingly taken, which may cause hyperuricemia and cause adverse reactions of a cardiovascular system and an endocrine system; the old patients are more likely to suffer from hypotension, electrolyte disorder and renal function damage when taking the medicine. Therefore, it is necessary to monitor and detect HCT in health food.
At present, the detection methods of HCT content in health food in China mainly comprise liquid chromatography-tandem mass spectrometry, high performance liquid chromatography, electrochemical sensors, capillary electrophoresis, infrared spectroscopy and the like. The detection methods have expensive instruments, poor portability and complex sample pretreatment, and need professional technical personnel to operate, so that the requirements of modern detection on rapidness and accuracy cannot be met. The immunoassay detection method can detect the target object according to color development by the principle of specific combination of antigen and antibody, is rapid, sensitive and accurate, has low cost and simple and convenient operation, makes up the defects of an instrumental analysis method, and has detection sensitivity meeting the requirements.
The key to establishing immunoassay methods is the ability to produce antibodies with high affinity and high specificity for small molecule compounds. Because the small molecular compound is hapten and has no immunogenicity, the small molecular compound can not directly induce an organism to generate a specific antibody, and only through proper chemical modification, a connecting arm with an active group at the upper end part is arranged at a certain position of a hapten molecular structure, and then the connecting arm is coupled with a macromolecular carrier to form an artificial antigen, the organism can be induced to generate immune response to generate the specific antibody. Therefore, the design of immune hapten molecules and the development of a simple and quick method for detecting HCT are very important.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a hydrochlorothiazide hapten.
The second purpose of the invention is to provide the dihydroculotine artificial antigen.
It is a third object of the invention to provide hydrochlorothiazide antibodies.
The fourth purpose of the invention is to provide a preparation method of hydrochlorothiazide hapten.
The fifth purpose of the invention is to provide the application of hydrochlorothiazide hapten.
The sixth purpose of the invention is to provide ELISA and colloidal gold immunochromatography kit for detecting hydrochlorothiazide.
The purpose of the invention is realized by the following technical scheme:
the structural formula of the hydrochlorothiazide hapten is shown as a formula (I)
Shown, or as formula (II)
Shown, or as formula (III)
Shown, or as formula (IV)
As shown.
The dihydroculothia hapten of formula (I) is designated by the systematic nomenclature: 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid (i.e.: 4- ((6-chloro-1, 1-dioxido-7-subst aAmoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid).
The dihydrocuvatipine hapten of the formula (II) is named by a systematic nomenclature: 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid (i.e., 2- (6-chloro-1, 1-dioxido-7-sulfo-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid).
The dihydroculothia hapten of formula (III) is designated by the systematic nomenclature: 6, 6 '- (6-chloro-1, 1-dioxine-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazine-2, 4(3H) -diyl) dihexanoic acid (i.e., 6' - (6-chloro-1, 1-dioxido-7-sulfomethyl-2H-benzol [ e ] [1, 2, 4] thiadiazide-2, 4(3H) -diyl) dihexanoic acid.
The dihydroculothia hapten of formula (IV) is designated by the systematic nomenclature: 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propanoic acid (i.e., 2- (6-chloro-1, 1-dioxido-7-sulfomethyl-2, 3-dihydo-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propanoic acid).
The hydrochlorothiazide hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid and the hydrochlorothiazide molecular skeleton structure have high overlapping degree, so that immune induction is facilitated, and the immunogenicity of the hydrochlorothiazide antigen 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-carrier protein conjugate can be effectively improved.
The present invention also provides the dihydrocuckutidine hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid, 6' - (6-chloro-1, 1-dioxin-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazin-2, 4(3H) -diyl) dihexanoic acid, The preparation method of 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propionic acid is characterized in that an imino functional group on a hexahydro-keurithia six-membered heterocyclic ring is used as an arm extension site, and four dihydro-keurithia haptens are obtained by respectively reacting with 4- (2-bromoethyl) benzoic acid, bromoacetic acid, 6-bromohexanoic acid and 2-bromopropionic acid.
Specifically, when preparing the semi-antigen of the formula (I), the method comprises the following steps:
hydrochlorothiazide is dissolved in 20mL of anhydrous acetonitrile in K2CO3Adding 4- (2-bromoethyl) benzoic acid as a catalyst, carrying out reflux reaction at 40-80 ℃ for 5 hours, removing the solvent, extracting the reaction solution, collecting an organic phase, extracting the water phase with ethyl acetate, collecting the organic phase, and adding anhydrous Na2SO4Drying, filtering, distilling the filtrate under reduced pressure to remove solvent, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (1: 1, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain yellow oily substance to obtain hapten of formula (I).
When preparing the hapten of formula (II), the method comprises the following steps:
hydrochlorothiazide is dissolved in 20mL of anhydrous acetonitrile in K2CO3Adding bromoacetic acid as a catalyst, performing reflux reaction at 40-80 ℃ for 5 hours, removing the solvent, extracting the reaction solution, collecting an organic phase, extracting a water phase with ethyl acetate, collecting the organic phase, and adding anhydrous Na2SO4Drying, filtering, distilling the filtrate under reduced pressure to remove solvent, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (5: 4, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain white powder to obtain hapten of formula (II).
When preparing the hapten of formula (III), the method comprises the following steps:
hydrochlorothiazide is dissolved in 20mL of anhydrous acetonitrile in K2CO3Adding 6-bromohexanoic acid as a catalyst, performing reflux reaction at 60-80 ℃ for 5 hours, removing the solvent, extracting the reaction solution, collecting an organic phase, extracting the water phase with ethyl acetate, collecting the organic phase, and adding anhydrous Na2SO4Drying, filtering, distilling the filtrate under reduced pressure to remove solvent, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (5: 6, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain white solid powder to obtain hapten of formula (III).
When preparing the hapten of formula (IV), the method comprises the following steps:
hydrochlorothiazide is dissolved in 20mL anhydrous acetonitrileIn K2CO3Adding 2-bromopropionic acid as a catalyst, performing reflux reaction at 40-80 ℃ for 5 hours, removing the solvent, extracting the reaction solution, collecting an organic phase, extracting the water phase with ethyl acetate, collecting the organic phase, and adding anhydrous Na2SO4Drying, filtering, distilling the filtrate under reduced pressure to remove solvent, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (7: 8, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain yellow powder to obtain hapten of formula (IV).
When the semi-antigen is prepared, the acetonitrile is used as a solvent, the acetonitrile is easy to remove, the simplicity of treatment reaction is improved, the bromoacid is used for directly carrying out one-step reaction, the time cost is greatly reduced, the temperature is moderate, the generation of byproducts is avoided, and the yield is greatly improved.
Specifically, the preparation method of the hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid comprises the following steps:
dissolving 1mol of hydrochlorothiazide in 20mL of anhydrous acetonitrile, and adding 2mol of K2CO3Adding 1.2mol of 4- (2-bromoethyl) benzoic acid as a catalyst, carrying out reflux reaction at 40-80 ℃ under magnetic stirring, monitoring the reaction by TLC (thin layer chromatography), using petroleum ether-ethyl acetate (1: 1, v/v) as a developing agent, quenching the reaction by using ice water after 5h of reaction, and removing the solvent by reduced pressure distillation. Extracting the reaction liquid with ethyl acetate and water, and collecting an organic phase; extracting the water phase with ethyl acetate, collecting organic phase, mixing organic phases, and adding anhydrous Na2SO4Drying and filtering, removing solvent from the filtrate by distillation under reduced pressure, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (1: 1, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain yellow oily substance, wherein the oily substance is dihydrocurtiothiazole hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ]][1，2，4]Thiadiazin-4-yl) methyl) benzoic acid.
Specifically, the preparation method of the hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazine-4-yl) acetic acid comprises the following steps:
dissolving 1mol of hydrochlorothiazide in 20mL of anhydrous acetonitrile, and adding 2mol of K2CO3Adding 1.2mol of bromoacetic acid as a catalyst, carrying out reflux reaction at 40-80 ℃ under magnetic stirring, monitoring the reaction by TLC, using petroleum ether-ethyl acetate (2: 3, v/v) as a developing agent, quenching the reaction by using ice water after 5h of reaction, and removing the solvent by reduced pressure distillation. Extracting the reaction solution with ethyl acetate and water, collecting organic phase, extracting water phase with ethyl acetate, collecting organic phase, mixing organic phases, and extracting with anhydrous Na2SO4Drying and filtering, removing solvent from the filtrate by distillation under reduced pressure, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (2: 3, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain white powder, wherein the white powder is dihydrocoxsackiethia hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ]][1，2，4]Thiadiazin-4-yl) acetic acid.
Specifically, the preparation method of the hapten 6, 6' - (6-chloro-1, 1-dioxin-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazine-2, 4(3H) -diyl) dihexanoic acid comprises the following steps:
dissolving 1mol of hydrochlorothiazide in 20mL of anhydrous acetonitrile, and adding 2mol of K2CO3Adding 1.2mol of 6-bromohexanoic acid as a catalyst, carrying out reflux reaction at 60-80 ℃ under magnetic stirring, monitoring the reaction by TLC, using petroleum ether-ethyl acetate (5: 6, v/v) as a developing agent, quenching the reaction by using ice water after 5h of reaction, and removing the solvent by reduced pressure distillation. Extracting the reaction liquid with ethyl acetate and water, and collecting an organic phase; extracting the water phase with ethyl acetate, collecting organic phase, mixing organic phases, and adding anhydrous Na2SO4Drying and filtering, removing solvent from the filtrate by reduced pressure distillation, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (5: 6, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain white solid powder, wherein the white solid powder is dihydrocudrartiothiazole 6, 6' - (6-chloro-1, 1-dioxin-7-sulfamoyl-2H-benzo [ e ]][1，2，4]Thiadiazine-2, 4(3H) -diyl) dihexanoic acid.
Specifically, the preparation method of the hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazine-4-yl) propionic acid comprises the following steps:
dissolving 1mol of hydrochlorothiazide in 20mL of anhydrous acetonitrile, and adding 2mol of K2CO3Adding 1.2mol of 2-bromopropionic acid as a catalyst, carrying out reflux reaction at 40-80 ℃ under magnetic stirring, monitoring the reaction by TLC, using petroleum ether-ethyl acetate (7: 8, v/v) as a developing agent, quenching the reaction by using ice water after 5h of reaction, and removing the solvent by reduced pressure distillation. Extracting the reaction liquid with ethyl acetate and water, and collecting an organic phase; extracting the water phase with ethyl acetate, collecting organic phase, mixing organic phases, and adding anhydrous Na2SO4Drying and filtering, removing solvent from the filtrate by distillation under reduced pressure, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (7: 8, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain white solid powder, wherein the white solid powder is dihydrocudrartiothiazole-2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ]][1，2，4]Thiadiazin-4-yl) propionic acid.
The invention also provides a hydrochlorothiazide artificial antigen which is obtained by coupling the hapten and carrier protein, wherein the carrier protein is Bovine Serum Albumin (BSA) or chicken Ovalbumin (OVA).
When the hydrochlorothiazide 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid is coupled with the carrier protein, the specific structure of the hydrochlorothiazide 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid protrudes out of the surface of the carrier protein, and an epitope serving as a carrier is exposed to an animal immune system, so that a foundation is laid for obtaining an antibody with high specificity and high quality.
The invention also provides a preparation method of the artificial antigen, which is prepared by adopting an active ester method and specifically comprises the following steps:
s1, dissolving the hapten shown in the formula (I), the formula (II), the formula (III) or the formula (IV) in anhydrous DMF respectively, adding NHS and EDC, stirring for 4-6 h at 4 ℃, and marking as solution A;
s2, dissolving carrier protein in carbonate buffer solution, marking as solution B, dropwise adding the solution A into the solution B, and reacting for 6-10 h at 4 ℃;
and S3, dialyzing with phosphate buffer solution at 4 ℃ for 3 days, changing the dialyzate for 3 times every day, and obtaining the hydrochlorothiazide artificial antigen after dialysis.
Preparation of the carbonate buffer (0.1M, pH 9.6): weigh 1.59g NaCO3And 2.93g NaHCO3And using deionized water to fix the volume to 1000 mL.
Preparation of the phosphate buffer solution (0.01M, pH 7.4): na (Na)2HPO4·12H2O 2.90g，NaCl 8.50g， KCl 0.20g，KH2PO40.20g, and adding deionized water to make the volume reach 1000 mL.
Preferably, the feeding ratio of the dihydroculothia artificial antigen, the NHS and the EDC of S1 is 1: 1-2; the molar ratio of the carrier protein to the hapten is 1: 60-80.
More preferably, the feeding ratio of the dihydrobenzothiadiazole artificial antigen, NHS and EDC of S1 is 1: 1.5.
The invention also provides the application of the hydrochlorothiazide artificial antigen, which is to prepare a hydrochlorothiazide antibody by taking the hydrochlorothiazide artificial antigen as an immunogen or take the hydrochlorothiazide artificial antigen as an envelope antigen; the antibody includes a monoclonal antibody, a polyclonal antibody, or a genetically engineered antibody.
The invention also provides a hydrochlorothiazide antibody which is prepared from the hydrochlorothiazide artificial antigen.
Specifically, when the antibody is prepared, the method for preparing the dihydroculotidine antibody comprises the following steps:
immunizing New Zealand white rabbits with the artificial antigen of dihydrocuckoo hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid coupled BSA, mixing and emulsifying 1mg/mL artificial antigen solution with an equal amount of adjuvant, mixing and emulsifying the initial immunization with Freund's complete adjuvant, immunizing, emulsifying the subsequent booster immunization with immunogen and Freund's incomplete adjuvant, and immunizing five times at intervals of 3 weeks, wherein the immunization dose is 0.5 mL/rabbit; blood was collected on the tenth day after the fifth immunization, and the antibody was purified by the octanoic acid-ammonium sulfate method.
The invention also provides application of the hydrochlorothiazide antibody in detecting hydrochlorothiazide.
The invention also provides a kit for detecting hydrochlorothiazide, which comprises the hydrochlorothiazide hapten, the hydrochlorothiazide artificial antigen or the hydrochlorothiazide antibody.
The invention also provides a method for detecting hydrochlorothiazide, which takes a conjugate of hydrochlorothiazide hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazine-4-yl) acetic acid and chicken ovalbumin as an envelope antigen, takes the hydrochlorothiazide antibody as a detection antibody, and adopts an indirect competitive ELISA method for detection.
Compared with the prior art, the invention has the following beneficial effects:
the invention firstly provides a plurality of hydrochlorothiazide haptens, namely 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e)][1，2，4]The thiadiazine-4-yl) methyl) benzoic acid has high overlapping degree with the skeleton structure of the hydrochlorothiazide to be detected, effectively improves the immunogenicity of the hydrochlorothiazide hapten-carrier protein conjugate, has larger structural difference with the hapten, forms larger steric hindrance, further improves the affinity of the antibody, uses the artificial antigen obtained by the hydrochlorothiazide hapten as immunogen to immunize a New Zealand white rabbit, and adopts an octanoic acid-ammonium sulfate method to purify the antibody to obtain the antibody. The obtained antibody has high titer, strong specificity and high affinity, and the LOD of the antibody to hydrochlorothiazide established by the antibody is 0.26ng/mL and IC50The detection sensitivity is high, and the linear range is wide, wherein the detection sensitivity is 12.27ng/mL, the quantitative detection range is 1.07-140.39 ng/mL; the antibody of the invention has the characteristics of simplicity, convenience, rapidness, strong specificity, wide linear range and high sensitivity, and has good application prospect and wide development space in the rapid and effective detection of hydrochlorothiazide.
Drawings
FIG. 1 is a UV scan of the artificial antigen 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-BSA;
FIG. 2 is a UV scan of the artificial antigen 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-OVA;
FIG. 3 is a scheme of hapten synthesis;
FIG. 4 is a graph of a standard DHK ELISA;
FIG. 5 is a schematic view of a dihydroculotione colloidal gold immunochromatographic test strip for visual judgment;
FIG. 6 is a schematic side view of a dihydroculotidine immunochromatographic strip; wherein, 1-PVC soleplate; 2-sample pad; 3-a conjugate pad; 4-NC film; 5-detection line (T-line); 6-Mass control line (line C); 7-absorbent pad.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 preparation of hydrochlorothiazide hapten
Synthesis of monohydroxathiin hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid
The resulting path is shown in fig. 3.
The synthesis steps comprise:
dissolving 1mol of hydrochlorothiazide in 20mL of anhydrous acetonitrile, and adding 2mol of K2CO3As a catalyst, 1.2mol of 4- (2-bromo) was addedEthyl) benzoic acid, performing reflux reaction at 40-80 ℃ under magnetic stirring, monitoring the reaction by TLC, using petroleum ether-ethyl acetate (1: 1, v/v) as a developing agent, quenching the reaction by using ice water after 5h of reaction, and removing the solvent by reduced pressure distillation. Extracting the reaction liquid with ethyl acetate and water, and collecting an organic phase; extracting the water phase with ethyl acetate, collecting organic phase, mixing organic phases, and adding anhydrous Na2SO4Drying and filtering, removing solvent from the filtrate by distillation under reduced pressure, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (1: 1, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain yellow oily substance, wherein the oily substance is dihydrocurtiothiazole hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ]][1，2，4]Thiadiazin-4-yl) methyl) benzoic acid. The structural formula is as follows:
synthesis of di, dihydrocuckoo hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid
The synthesis steps comprise:
dissolving 1mol of hydrochlorothiazide in 20mL of anhydrous acetonitrile, and adding 2mol of K2CO3Adding 1.2mol of bromoacetic acid as a catalyst, carrying out reflux reaction at 40-80 ℃ under magnetic stirring, monitoring the reaction by TLC, using petroleum ether-ethyl acetate (2: 3, v/v) as a developing agent, quenching the reaction by using ice water after 5h of reaction, and removing the solvent by reduced pressure distillation. Extracting the reaction solution with ethyl acetate and water, collecting organic phase, extracting water phase with ethyl acetate, collecting organic phase, mixing organic phases, and extracting with anhydrous Na2SO4Drying and filtering, removing solvent from the filtrate by distillation under reduced pressure, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (2: 3, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain white powder, wherein the white powder is dihydrocoxsackiethia hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ]][1，2，4]Thiadiazin-4-yl) acetic acid. The structural formula is as follows:
synthesis of trihydrockothiazolidine hapten 6, 6' - (6-chloro-1, 1-dioxine-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazine-2, 4(3H) -diyl) dihexanoic acid
The synthesis steps comprise:
dissolving 1mol of hydrochlorothiazide in 20mL of anhydrous acetonitrile, and adding 2mol of K2CO3Adding 1.2mol of 6-bromohexanoic acid as a catalyst, carrying out reflux reaction at 60-80 ℃ under magnetic stirring, monitoring the reaction by TLC, using petroleum ether-ethyl acetate (5: 6, v/v) as a developing agent, quenching the reaction by using ice water after 5h of reaction, and removing the solvent by reduced pressure distillation. Extracting the reaction liquid with ethyl acetate and water, and collecting an organic phase; extracting the water phase with ethyl acetate, collecting organic phase, mixing organic phases, and adding anhydrous Na2SO4Drying and filtering, removing solvent from the filtrate by reduced pressure distillation, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (5: 6, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain white solid powder, wherein the white solid powder is dihydrocudrartiothiazole 6, 6' - (6-chloro-1, 1-dioxin-7-sulfamoyl-2H-benzo [ e ]][1，2，4]Thiadiazine-2, 4(3H) -diyl) dihexanoic acid. The structural formula is as follows:
synthesis of tetrahydrocuckoo hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propionic acid
The synthesis steps comprise:
dissolving 1mol of hydrochlorothiazide in 20mL of anhydrous acetonitrile, and adding 2mol of K2CO3Adding 1.2mol of 2-bromopropionic acid as a catalyst, carrying out reflux reaction at 40-80 ℃ under magnetic stirring, monitoring the reaction by TLC, using petroleum ether-ethyl acetate (7: 8, v/v) as a developing agent, quenching the reaction by using ice water after 5h of reaction, and removing the solvent by reduced pressure distillation. Extracting the reaction liquid with ethyl acetate and water, and collecting an organic phase; the water phase is extracted by ethyl acetate again,collecting organic phase, combining organic phase, and adding anhydrous Na2SO4Drying and filtering, removing solvent from the filtrate by distillation under reduced pressure, purifying the residue by silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (7: 8, v/v) solution as mobile phase, and concentrating the filtrate under reduced pressure to obtain white solid powder, wherein the white solid powder is dihydrocudrartiothiazole-2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ]][1，2，4]Thiadiazin-4-yl) propionic acid. The structural formula is as follows:
example 2 identification of hydrochlorothiazide haptens
Identification of monohydroxathiin hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid
The hydrogen spectrum nuclear magnetism results are as follows: 1H NMR (600MHz, MeOD) (600M1H), 8.03(d, J ═ 8.2Hz, 2H), 7.50 (d, J ═ 8.2Hz, 2H), 6.99(s, 1H), 4.77(s, 2H), 4.22(s, 2H).
The mass spectrometry results were as follows: ESI-MS: m/z [ M-H]-429.9(C15H14ClN3O6S2)。
As can be seen from the mass spectrum results, 429.9 is the anion molecular peak of hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid, the calculated relative molecular mass is 431.0, and the molecular mass is consistent with the actual relative molecular mass, which indicates that the dihydrochlorothiazide hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid is successfully prepared.
Identification of Dihydroxathiin hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid
The hydrogen spectrum nuclear magnetism results are as follows:1H NMR(600MHz，MeOD)δ8.18(s，1H)，6.97(s，1H)，5.34(t，J＝4.7 Hz，1H)，5.05(s，2H)，3.55(s，2H)。
the mass spectrometry results were as follows: ESI-MS: m/z [ M-H]-353.8(C9H10ClN3O6S2)。
As can be seen from the mass spectrum results, 353.80 is the anion molecular peak of hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid, the calculated relative molecular mass is 354.97, which is consistent with the actual relative molecular mass, and the result shows that the dihydrocyclothia hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid is successfully prepared.
Identification of trihydroclothiazepine hapten 6, 6' - (6-chloro-1, 1-dioxine-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazine-2, 4(3H) -diyl) dihexanoic acid
The hydrogen spectrum nuclear magnetism results are as follows: 1H NMR (600MHz, MeOD) δ 8.23(s, 1H), 7.74-7.70(m, 1H), 7.64-7.59 (m, 1H), 6.99(s, 1H), 4.29(t, J ═ 6.6Hz, 2H), 2.88(t, J ═ 6.9, 3.5Hz, 4H), 2.25(t, J ═ 7.4, 6.4 Hz, 4H), 1.48-1.44(m, 6H), 1.33-1.29(m, 6H). carbon spectrum nuclear magnetic results are as follows: 13C NMR (151MHz, MeOD). delta.176.06(s), 149.82(s), 136.17(s), 133.28(s), 130.95(s), 128.47(s), 118.14(s), 65.26(s), 42.32(s), 42.13(s), 33.34(s), 30.32(s), 28.91(s), 28.80(s), 25.80(s), 24.16(s), 18.86(s), 12.65(s).
The mass spectrometry results were as follows: ESI-MS: m/z [ M-H]-524.1(C19H28ClN3O8S2)。
As can be seen from the mass spectrum result, 523.9 is the anion molecular peak of hapten BP, the calculated relative molecular mass is 525.10, which is consistent with the actual relative molecular mass, and the result shows that the dihydrocurtiothioantigen 6, 6' - (6-chloro-1, 1-dioxin-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazine-2, 4(3H) -diyl) dihexanoic acid is successfully prepared.
Identification of tetrahydrocudracet hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propionic acid
The hydrogen spectrum nuclear magnetism results are as follows: 1H NMR (600MHz, MeOD) δ 8.18(s, 1H), 6.92(s, 1H), 5.12(q, J ═ 11.3Hz, 2H), 4.39(q, J ═ 7.2Hz, 1H), 4.29(t, J ═ 6.6Hz, 1H), 3.35(s, 1H), 1.41(d, J ═ 7.6Hz, 3H).
The mass spectrometry results were as follows: ESI-MS: m/z [ M-H]-367.60(C10H12ClN3O6S2)。
As can be seen from the mass spectrum result, 367.60 is the anion molecular peak of the hapten BP, the calculated relative molecular mass is 368.99, and the relative molecular mass is consistent with the actual relative molecular mass, which indicates that the dihydrochlorothiazide hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazine-4-yl) propionic acid is successfully prepared.
EXAMPLE 3 preparation of Artificial antigen for hydrochlorothiazide
Synthesis of artificial antigen
A method for preparing hydrochlorothiazide artificial antigen comprises the following steps:
the bishydrokethiuraemic hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid, 6, 6' - (6-chloro-1, 1-dioxin-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazin-2, 4(3H) -diyl) dihexanoic acid prepared in example 1, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propionic acid is respectively coupled with bovine serum albumin BSA (bovine albumin) and egg white albumin OVA (ovalbumin) by an active ester method, the haptens are respectively dissolved in 200 mu L of anhydrous DMF, 1.5eq NHS and 1.5eq EDC are added into the solution to react for 4 hours at 4 ℃, and the solution is marked as solution A; dropwise adding the solution A into a PBS buffer solution of carrier protein, and continuously reacting for 8 hours at 4 ℃; dialyzing the reaction solution at 4 deg.C for 3 days, changing the dialyzate 3 times per day, dialyzing for 9 times, and collecting the solution in the dialysis bag to obtain artificial antigen of hydrochlorothiazide 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-BSA, 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-OVA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-BSA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-OVA, 6, 6 '- (6-chloro-1, 1-dioxine-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazin-2, 4(3H) -diyl) dihexanoic acid-BSA, 6, 6' - (6-chloro-1, 1-dioxine-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazine-2, 4(3H) -two base) two caproic acid-OVA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazine-4-base) propionic acid-BSA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazine-4-base) propionic acid-OVA and other 8 kinds of artificial antigen.
The two hydrochlorothiazide artificial antigens (see example 5 in detail) which are optimally combined are characterized in that the structural formulas are respectively shown as a formula (VI) and a formula (VII):
structurally, the dihydrocuurine hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid has a benzene ring structure, the arm is separated from the reaction site by a methylene, the hapten 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid is a straight-chain arm, the benzene ring is also separated from the reaction site by a methylene, the benzene ring forms steric hindrance and greatly stimulates the immune activity, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, the 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid has a simple structure, and is matched with hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid to form a high-affinity and strong-specificity antibody as a coating structure.
Identification of artificial antigens
The artificial antigen 4- ((6-chloro-1, l-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-BSA was subjected to UV scanning, and the results are shown in FIG. 1.
Respectively carrying out ultraviolet (150-500 nm) scanning identification on 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid, BSA and an artificial antigen 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-BSA, wherein the concentrations of the three are 1mg/mL, and by comparing the highest absorbance values of the substances before and after coupling, the absorption curve of the artificial antigen is obviously different from that of a carrier protein, and the BSA only has a characteristic peak at 280nm, the 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid has a characteristic peak at 290nm, after the coupling reaction, the 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-BSA has a characteristic peak at 270nm, and a significant shift can be seen in comparison of the curves of the three, so that the reaction products are carrier protein BSA and 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid, and the coupling is successful.
The artificial antigen 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-OVA was subjected to UV scanning, and the results are shown in FIG. 2.
2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid, OVA and artificial antigen 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-OVA are respectively subjected to ultraviolet (150-500 nm) scanning identification, wherein the concentrations of the three are all 1mg/mL, and the absorption curve of the artificial antigen is obviously different from that of a carrier protein by comparing the highest absorbance values of the substances before and after coupling, the OVA has a characteristic peak only at 280nm, and 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid has a characteristic peak at 300nm, after the coupling reaction, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-OVA has a characteristic peak at 270nm, and a remarkable shift can be seen by comparing the three curves, so that the reaction products are carrier protein OVA and 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid, and the coupling is successful.
EXAMPLE 4 preparation of hydrochlorothiazide antibody
First, animal immunization
Taking a healthy New Zealand white rabbit with the weight of 1.5-2 kg as an experimental animal, taking artificial antigen of dihydrocretinib, namely 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-BSA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-BSA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propionic acid-BSA is taken as immunogen, subcutaneous injection is carried out on the back of the neck of a big white rabbit, 1mg/mL of artificial antigen solution is respectively mixed and emulsified with an equal amount of adjuvant, the primary immunization is mixed and emulsified with Freund's complete adjuvant and then immunized, the subsequent boosting immunization is carried out by emulsifying the immunogen and the Freund's incomplete adjuvant and five times of immunization, each time is separated by 3 weeks, and the immunization dose is 0.5 mL/rabbit; during the period, a small amount of blood is taken from the ear marginal vein for antibody quality identification, blood is taken from the tenth day after the fifth immunization, and the antibody is purified by adopting an octanoic acid-ammonium sulfate method.
Purification of di, dihydrocuckoo hapten 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid antibody (caprylic acid-ammonium sulfate method):
(1) centrifuging rabbit serum at 12000rpm for 15min at 4 deg.C, and removing precipitate;
(2) mixing 1 volume of rabbit serum with 2 volumes of acetate buffer (pH 4.8), stirring at room temperature, and dropwise adding n-octanoic acid in an amount of 75 μ L n-octanoic acid/mL rabbit serum;
(3) stirring and mixing for 30min at room temperature, standing for 2h at 4 ℃ to fully precipitate;
(4) centrifuging at 12000r/min for 15min at 4 deg.C, and removing precipitate;
(5) after the supernatant was filtered through a sand core funnel or a 125 μ M nylon net, 1/10 volumes of PBS buffer (0.1M, pH 7.4) were added, the pH was adjusted to 7.4 with 2M sodium hydroxide, and the total solution volume was calculated;
(6) adding 0.277g/mL ammonium sulfate in ice bath within 30min to obtain 45% saturated solution;
(7) standing at 4 deg.C for more than 1 hr, centrifuging at 12000rpm at 4 deg.C for 15min, and removing supernatant;
(8) the pellet was dissolved in PBS buffer (0.1M, pH 7.4) and dialyzed for three days.
Example 5 hydrochlorothiazide immunogen and coating antigen combination optimization
The dihydrocuvatoxin artificial antigen 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-BSA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-BSA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propionic acid-BSA were separately immunized against New Zealand rabbit, the prepared antibody is subjected to coating antigen screening of all structures, and the titer and the inhibition rate of the prepared antibody are detected by ELISA and are shown in table 1.
The specific operation steps are as follows:
(1) dihydrokeritin antibody was diluted with PBST at 1: 1000, 1: 2000, 1: 4000, 1: 8000, 1: 16000, 1: 32000, 1: 64000, 1: 128000, 1: 256000, with a blank control well (replaced with PBST);
(2) the dihydrocuckuti artificial antigen 4- ((6-chloro-1, 1-dioxy-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) methyl) benzoic acid-OVA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-OVA, 6, 6' - (6-chloro-1, 1-dioxin-7-sulfamoyl-2H-benzo [ e ] [1, 2, 4] thiadiazin-2, 4(3H) -diyl) dihexanoic acid-OVA, 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) propionic acid-OVA was diluted with coating solution (0.05M carbonate buffer, pH 9.6) to a concentration of 125ng/mL, coated with 96-well microplate, 100. mu.L per well, incubated overnight in a 37 ℃ constant temperature water bath, the coating solution was discarded, and washed 2 times with PBST (0.01M PBS, 0.06% Tween-20 (v/v));
(3) adding 120 μ L of sealing solution (1% 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;
(4) diluting 1mg/mL dihydroculothia 1000-fold with PBST to 1000 ng/mL;
(5) adding 50 μ L of dihydrocudracet dilution (three groups in parallel) to each row, adding 50 μ L of PBST dilution/well, incubating at 37 deg.C for 40min, and washing 5 times;
(6) adding goat anti-rabbit secondary antibody IgG-HRP (5000-fold dilution), incubating for 30min at 37 ℃, washing for 5 times, and clapping;
(7) adding color development liquid for developing for 10 min;
(8) add 50. mu.L of 10% H2SO4The reaction was stopped and the OD read at 450 nm;
the experimental results are as follows: the results of the detection of the titer inhibition ratios of antiserum obtained by immunizing the New Zealand white rabbits are shown in Table 1, and it can be seen that the immunized rabbits all generate rabbit polyclonal antibodies, the obtained antiserum has different degrees of inhibition effects on the target analyte dihydrochellothia, and the antibody inhibition ratio of the No. 1 New Zealand white rabbits is the highest and 88.2%, which indicates that the specificity recognition of the antibody dihydrochellothia is the strongest, and further indicates that the immune artificial antigen and the coated artificial antigen of the No. 1 are the best combination.
Inhibition rate (OD value of titer-OD value of inhibition)/OD value of inhibition 100%;
TABLE 1 potency and inhibition data for the optimal combination of three immunogens for hydrochlorothiazide and coatinggen
EXAMPLE 6 establishment of Standard Curve for hydrochlorothiazide ELISA
First, ELISA detection
(1) Dihydrokeurothiazole antibody was diluted 1: 64000 with PBST while blank control wells (substituted with PBST);
(2) diluting the hydrochlorothiazide 2- (6-chloro-1, 1-dioxide-7-sulfamoyl-2, 3-dihydro-4H-benzo [ e ] [1, 2, 4] thiadiazin-4-yl) acetic acid-OVA artificial antigen with a coating solution to a concentration of 125ng/mL, coating a 96-well enzyme label plate, adding 100 mu L of the enzyme label plate to each well, incubating in a constant-temperature water bath tank at 37 ℃ overnight, discarding the coating solution, and washing with PBST (0.01M PBS, 0.06% Tween-20(v/v)) for 2 times;
(3) adding 120 μ L of sealing solution (1% 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;
(4) diluting 1ug/mL dihydroculothia by 5 times and then 3 times with PBST, and diluting the dihydroculothia to 1000.00, 200.00, 66.67, 22.22, 7.41, 2.47 and 0.82 ng/mL;
(5) adding 50 μ L of dihydrocudracet dilution (three groups in parallel) to each row, adding 50 μ L of PBST dilution/well, incubating at 37 deg.C for 40min, and washing 5 times;
(6) adding goat anti-rabbit secondary antibody IgG-HRP (5000-fold dilution), incubating for 30min at 37 ℃, washing for 5 times, and clapping;
(7) adding color development liquid for developing for 10 min;
(8) add 50. mu.L of 10% H2SO4The reaction was stopped and the OD read at 450 nm;
second, result in
Concentration of hydrochlorothiazide standard substance to abscissa, B/B0(OD of well to which hydrochlorothiazide was added450OD of wells without hydrochlorothiazide450) For the ordinate, an indirect competition standard curve is established. The ELISA standard curve chart of hydrochlorothiazide is shown in FIG. 4, and it can be seen that the standard curve is S-shaped, the linear correlation is good, the lowest detection limit of hydrochlorothiazide is 0.26ng/mL, IC50The detection sensitivity is high, and the linear range is wide, wherein the detection sensitivity is 12.27ng/mL, and the linear range is 1.07-140.39 ng/mL.
Example 7 Dihydrokeurothia colloidal gold Rapid test method
First, preparation of gold-labeled antibody and gold-labeled conjugate pad
Colloidal gold suspension with average diameter of 40nm was prepared by reducing chloroauric acid with trisodium citrate. Under reflux, 100mL of 0.01% chloroauric acid solution was heated to boiling, and 1.1mL of 1% trisodium citrate was added rapidly with constant stirring. Heating and stirring were continued for 5min when the reaction solution became reddish-red in color. After cooling to room temperature, 0.05% sodium azide was added and stored at 4 ℃.
The colloidal gold is labeled with 0.2mol of K before being labeled with the antibody2CO3The solution was adjusted to pH 8.2 and 30. mu.g of antibody-labeled 1mL of colloidal gold solution was determined by classical NaCl titration. Then, labeling was carried out in an optimum amount, and after 1 hour of labeling, 10% BSA was added with stirring (to make the final BSA concentration 1%), and after 1 hour of incubation, centrifugation was carried out at 10000rpm at 4 ℃ for 25min, and the supernatant was removed. Adding 5% BSA solution with the same volume of colloidal gold solution for resuspension, centrifuging at 4 deg.C and 10000rpm for 25min, and repeating twice. Finally, it was resuspended in 1/5 volumes of colloidal gold solution in TB solution (containing 3% BSA, 3% sucrose, 0.01mol/L sodium borate and 0.05% sodium azide) and stored at 4 ℃. Spraying 4% BSA solution at 8 μ L/cm onto glass wool with XYZ-3000 three-dimensional film spraying instrument, drying at 42 deg.C for 50min in a drying oven, spraying gold-labeled antibody at 6 μ L/cm onto glass wool, drying at 42 deg.C for 50min in the drying oven, and vacuum drying for storage.
II, coupling antigen goat anti-rabbit coated cellulose membrane
An XYZ-3000 three-dimensional film spraying instrument is used for spraying the coating antigen with the concentration of 0.15mg/mL on the lower side of the cellulose film in an amount of 0.8 mu L/cm to serve as a detection line. Goat anti-rabbit IgG at a concentration of 0.24mg/L was sprayed onto the upper side of the cellulose membrane in an amount of 0.8. mu.L/cm using an XYZ-3060 three-dimensional film spraying apparatus as a control line with a two-line interval of 7 mm.
Three, the equipment of quick test paper strip
As shown in FIG. 6, the cellulose film 4 was stuck to the middle portion of the backing sheet 1, and the absorbent pad 7 was stuck to the upper side of the cellulose film 4 so as to overlap the cellulose film 4 by 1 mm. The gold-labeled conjugate pad 3 was stuck under the cellulose film 4 with an overlap of 1 mm. The sample pad 2 is stuck under the gold-labeled conjugate pad 3 with an overlap of 2 mm. The assembled test paper board was cut into test paper strips 3.05mm wide with a cutter.
Fourthly, preparation of detection sample liquid
Adding 100 μ L of liquid health product such as oral liquid into 900 μ L of PBST to obtain sample solution.
And (3) tablet preparation: removing outer sugar coating, grinding into powder, dissolving the powder with 5 times volume of treatment solution (carbonate buffer solution containing 50% methanol, pH 9.6), vortex for 3min, centrifuging at 3000r/min for 6min, collecting supernatant 100 μ L, and adding to PBST 900 μ L to obtain sample solution.
And (3) capsule preparation: removing capsule coat, dissolving the powder with 5 times volume of treatment solution (carbonate buffer solution containing 50% methanol, pH 9.6), vortexing for 3min, centrifuging at 3000r/min for 6min, collecting supernatant 100 μ L, and adding to PBST 900 μ L to obtain sample solution.
1. Rapid test strip detection and judgment
When the sample solution to be tested is added into the test end of the test strip or the test paper card, the solution to be tested drives the object to be tested and the gold-labeled antibody in the gold-labeled conjugate pad 3 to diffuse together to the cellulose membrane 4 through the siphon action, and finally permeates into the end 7 of the water absorption pad. In the diffusion process, if the sample contains the substance to be detected, the substance to be detected is combined with the gold-labeled antibody, so that the antigen binding site on the gold-labeled antibody is occupied, the combination of the gold-labeled antibody and the invisible detection line 5 (the combination of the hapten and the carrier protein) on the cellulose membrane 4 is prevented, and the invisible detection line 5 is not colored or is weakly colored, namely, the detection sample is positive or weakly positive; if the sample to be detected does not exist in the sample, a clear red line is displayed when the gold-labeled antibody meets the invisible detection line 5 in the upward moving process, and the detection sample is negative. Similarly, the gold-labeled antibody also binds to the invisible control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, so that the invisible control line 6 is red. The presence or absence of the color of the invisible control line 6 indicates the validity or invalidity of the test strip, respectively, and the determination result is shown in fig. 5.
2. Determination of detection Limit
A series of standard medicines with concentration are added into blank oral liquid, hard capsules, soft capsules and tablet samples, the samples are pre-treated, the samples are detected by the colloidal gold test strip, and visual detection limit is determined by naked eye qualitative judgment. Specific results are shown in the following table.
Claims (9)
1. The hydrochlorothiazide hapten is characterized in that the structural formula of the hydrochlorothiazide hapten is shown as a formula (I)
Shown, or as formula (II)
Shown, or as formula (III)
Shown, or as formula (IV)
As shown.
2. The method of producing hydrochlorothiazide hapten according to claim 1, wherein the hapten of formula (I) is produced by the steps of:
dissolving dihydrothifluzamide in anhydrous acetonitrile with K2CO3Adding 4- (2-bromoethyl) benzoic acid as a catalyst, performing reflux reaction at 40-80 ℃, removing a solvent, extracting a reaction solution, collecting an organic phase, extracting a water phase with ethyl acetate, collecting the organic phase, combining the reaction solution and the organic phase obtained by extraction in the water phase, drying, filtering and purifying to obtain the hapten of the formula (I);
when preparing the hapten of formula (II), the method comprises the following steps:
dissolving dihydrothifluzamide in anhydrous acetonitrile with K2CO3Adding bromoacetic acid as a catalyst, performing reflux reaction at 40-80 ℃, removing the solvent, extracting the reaction solution, collecting an organic phase, extracting a water phase with ethyl acetate, collecting the organic phase, combining the reaction solution and the organic phase obtained by extraction of the water phase, drying, filtering and purifying to obtain the hapten of the formula (I);
when preparing the hapten of formula (III), the method comprises the following steps:
dissolving dihydrothifluzamide in anhydrous acetonitrile with K2CO3Adding 6-bromohexanoic acid as a catalyst, performing reflux reaction at 60-80 ℃, removing the solvent, extracting the reaction solution, collecting an organic phase, extracting a water phase with ethyl acetate, collecting the organic phase, combining the reaction solution and the organic phase obtained by extraction in the water phase, drying, filtering and purifying to obtain the hapten of the formula (III);
when preparing the hapten of formula (IV), the method comprises the following steps:
dissolving dihydrothifluzamide in anhydrous acetonitrile with K2CO3And (3) adding 2-bromopropionic acid as a catalyst, performing reflux reaction at 40-80 ℃, removing the solvent, extracting the reaction solution, collecting an organic phase, extracting a water phase with ethyl acetate, collecting the organic phase, combining the reaction solution and the organic phase obtained by extraction in the water phase, drying, filtering and purifying to obtain the hapten of the formula (IV).
3. An artificial antigen of hydrochlorothiazide, which is obtained by coupling the hapten of claim 1 with a carrier protein, wherein the carrier protein is bovine serum albumin or chicken egg albumin.
4. The method for producing the artificial antigen according to claim 3, comprising the steps of:
s1, dissolving the hapten shown in the formula (I), the formula (II), the formula (III) or the formula (IV) in anhydrous DMF respectively, adding NHS and EDC, stirring for 4-6 h at 4 ℃, and marking as solution A;
s2, dissolving carrier protein in carbonate buffer solution, marking as solution B, dropwise adding the solution A into the solution B, and reacting for 6-12 h at 4 ℃;
s3, dialyzing at 4 ℃ to obtain the dihydrocathartic artificial antigen.
5. The method for preparing the artificial antigen according to claim 4, wherein the feeding ratio of the dihydrocuckoo artificial antigen, the NHS and the EDC of S1 is 1: 1-2; the molar ratio of the carrier protein to the hapten is 1: 60-80.
6. The use of the hydrochlorothiazide artificial antigen of claim 5, wherein hydrochlorothiazide antibody is prepared using hydrochlorothiazide artificial antigen as an immunogen or hydrochlorothiazide artificial antigen as a coating antigen; the antibody includes a monoclonal antibody, a polyclonal antibody, or a genetically engineered antibody.
7. A hydrochlorothiazide antibody produced from the hydrochlorothiazide artificial antigen of claim 3.
8. Use of the hydrochlorothiazide antibody of claim 7 for detecting hydrochlorothiazide.
9. An ELISA and colloidal gold immunochromatographic kit for detecting hydrochlorothiazide, comprising hydrochlorothiazide hapten according to claim 1, hydrochlorothiazide antigen according to claim 3 or hydrochlorothiazide antibody according to claim 7.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110240576A (en) * | 2019-06-27 | 2019-09-17 | 广东达元绿洲食品安全科技股份有限公司 | Hydrochioro haptens and artificial antigen and its preparation method and application |
| CN112409348A (en) * | 2020-11-16 | 2021-02-26 | 广西大学 | Preparation method and application of hapten and complete antigen for thiamethoxam |
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| CN110240576A (en) * | 2019-06-27 | 2019-09-17 | 广东达元绿洲食品安全科技股份有限公司 | Hydrochioro haptens and artificial antigen and its preparation method and application |
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| CN110240576B (en) * | 2019-06-27 | 2023-04-21 | 广东达元绿洲食品安全科技股份有限公司 | Hydrochlorothiazide hapten and artificial antigen as well as preparation methods and applications thereof |
| CN112409348A (en) * | 2020-11-16 | 2021-02-26 | 广西大学 | Preparation method and application of hapten and complete antigen for thiamethoxam |
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