CN111943882B - Pythium hapten, artificial antigen and antibody as well as preparation method and application thereof - Google Patents
Pythium hapten, artificial antigen and antibody as well as preparation method and application thereof Download PDFInfo
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- CN111943882B CN111943882B CN202010708519.0A CN202010708519A CN111943882B CN 111943882 B CN111943882 B CN 111943882B CN 202010708519 A CN202010708519 A CN 202010708519A CN 111943882 B CN111943882 B CN 111943882B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/52—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring condensed with a ring other than six-membered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
Abstract
The invention discloses a procymidone hapten, an artificial antigen and an antibody as well as a preparation method and application thereof, wherein the procymidone hapten with two different structures is provided, wherein the procymidone hapten with the structure shown in a formula (I) is used as a procymidone immunization hapten, so that the characteristic structure of procymidone is retained to the maximum extent, the immunogenicity of the procymidone hapten is obviously enhanced, and the procymidone hapten also has carboxyl which can be coupled with carrier protein; the procymidone hapten with the structure shown in the formula (I) is coupled with carrier protein to obtain the procymidone immunizing antigen to immunize animals, so that the procymidone immunizing antigen is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, the sensitivity of the procymidone antibodies can reach 0.025 mu g/L through detection, the cross reaction rate with other organochlorine pesticides is low, and a foundation is provided for the subsequent establishment of various immunoassay methods of the procymidone.
Description
Technical Field
The invention belongs to the field of food safety detection. More specifically, the invention relates to procymidone haptens, artificial antigens and antibodies, and preparation methods and applications thereof.
Background
Procymidone is a broad-spectrum systemic low-toxicity fungicide and is mainly used for preventing and treating gray mold, sclerotinia, brown rot, big spot and scab of plants such as fruits, vegetables and flowers. Pythium is beneficial to strong stimulation, can normally synthesize and metabolize skin and eyes, can interfere or inhibit the endocrine system of substances once entering a human body, and has influence on the health of the human body. The national standard GB 2763 and 2019 maximum limit of pesticide residue in food safety national standard food stipulate that the limit of the residue of procymidone in vegetables and fruits is 0.2-10 mg/kg.
At present, the methods for analyzing and detecting procymidone residues at home and abroad mainly comprise a high performance liquid chromatography, a gas chromatography-mass spectrometry and the like, but all have the defects of complicated sample pretreatment, long detection time, expensive instruments and the like, can not be widely applied in China, and do not meet the requirements of on-site detection on accurate detection and screening of a large number of samples at low cost in a short time. The immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has many advantages compared with detection methods such as instruments and the like. Therefore, the immunoassay provides a new analysis and detection method for the research of the procymidone residues.
When an immunological detection method is established and the detection method is applied to detect the residual quantity of the procymidone, the key technology is that an antibody with strong specificity and high sensitivity can be obtained, and the goal is to realize the purpose on the premise that a proper procymidone hapten is synthesized and prepared.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the hapten which can furthest reserve the characteristic structure of procymidone and has a connecting arm with a certain length and the preparation method of the hapten; the artificial antigen prepared by the hapten and the antibody with high detection sensitivity and strong specificity; and the use of such haptens.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a procymidone hapten which has a structure represented by formula (I) or formula (II):
wherein n is-CH2The number of groups, n is an integer of 0 to 4.
The invention provides two kinds of procymidone haptens with different structures, wherein the procymidone hapten with the structure shown in the formula (I) is used as a procymidone immunizing hapten, the characteristic structure of procymidone is retained to the maximum extent, an antigenic determinant of the hapten can be better exposed, the immunogenicity of the procymidone hapten is obviously enhanced, and the procymidone hapten also has carboxyl which can be coupled with carrier protein; the procymidone hapten with the structure shown in the formula (I) is coupled with the carrier protein to obtain the procymidone immunizing antigen to immunize animals, so that the procymidone immunizing antigen is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, and provides a basis for subsequently establishing various immunoassay methods of procymidone. In the invention, the procymidone hapten with the structure shown in the formula (II) is used as the procymidone coating hapten, which is beneficial to improving the sensitivity of immunoassay.
In a second aspect, the present invention provides a method for preparing the above-mentioned procymidone hapten, comprising the following steps:
1) reacting 3, 5-dichloro-4-fluoronitrobenzene with water under alkaline condition
Nucleophilic substitution reaction is carried out to obtain an intermediate 1, wherein the intermediate 1 has a structural formula
Wherein n is-CH2The number of groups, n is an integer of 0-4;
2) carrying out hydrogenation reduction reaction on the intermediate 1 under the catalytic action of palladium carbon to obtain an intermediate 2, wherein the intermediate 2 has a structural formula
3) And (3) carrying out cyclization reaction on the intermediate 2, 1, 2-dimethylcyclopropane-1, 2 dicarboxylic acid and oxalyl chloride to obtain the procymidone hapten with the structure shown in the formula (I).
Further, the step 1) comprises the following steps: dissolving 3, 5-dichloro-4-fluoronitrobenzene in ethanol, adding NaOH, stirring, and adding
Fully stirring, reacting for 4 hours at 60 ℃, after the reaction is finished, performing rotary evaporation to remove ethanol, adding water, extracting with ethyl acetate, collecting a water phase, adjusting the pH of the water phase to 5 with hydrochloric acid, extracting with ethyl acetate, drying with anhydrous sodium sulfate, and evaporating to dryness to obtain an intermediate 1; wherein, the 3, 5-dichloro-4-fluoronitrobenzene,
And sodium hydroxide in a ratio of 1:1: 2; the above-mentioned
Preferred are glycine, alanine, aminobutyric acid, aminopentanoic acid, aminocaproic acid.
Further, the step 2) comprises the following steps: and (3) adding methanol to dissolve and clarify the intermediate 1, slowly adding palladium carbon, fully and uniformly mixing, extracting air, introducing hydrogen, stirring at room temperature for 6 hours, after the reaction is finished, removing the palladium carbon by suction filtration, and evaporating to dryness by rotary evaporation to obtain an intermediate 2.
Further, the step 3) comprises the following steps: dissolving 1, 2-dimethylcyclopropane-1, 2-dicarboxylic acid in dichloromethane, keeping the temperature constant at 0-5 ℃ for 30min, adding oxalyl chloride, fully stirring, adding N, N-dimethylformamide, and continuously stirring at 0-5 ℃ for 3 h; adding the intermediate 2, adding triethylamine, performing reflux reaction for 8 hours, after the reaction is finished, performing rotary evaporation to remove an organic solvent, adding water, extracting with ethyl acetate, evaporating to dryness, purifying with a silica gel column, and eluting and separating with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the procymidone hapten with the structure shown in formula (I); wherein the mass ratio of the intermediate 2, 1, 2-dimethylcyclopropane-1, 2 dicarboxylic acid and oxalyl chloride is 1:1: 2.
Further, the preparation method of the procymidone hapten comprises the following steps: providing a compound 1, wherein the compound 1 has a structural formula
The compound 1 reacts with succinic anhydride to obtain the procymidone hapten with the structure shown in the formula (II).
Further, the specific operation is as follows: dissolving the compound 1 with pyridine, adding succinic anhydride, heating in an oil bath, reacting at 75 ℃ for 6h, performing rotary evaporation to remove pyridine after the reaction is finished, adding water and hydrochloric acid, extracting with ethyl acetate, collecting an organic phase, adding anhydrous sodium sulfate into the organic phase, drying and evaporating to dryness, purifying with a silica gel column, eluting and separating with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the procymidone hapten with the structure shown in the formula (II); wherein the mass ratio of compound 1 and succinic anhydride is 1: 1.
According to the invention, the preparation method of the procymidone hapten is reasonably designed according to the structural characteristics of procymidone, the used raw materials are easy to obtain, the reaction operation is simpler, the reaction condition is easy to control, and the purity and the yield of the prepared procymidone hapten are higher.
In a third aspect, the invention provides a procymidone artificial antigen, which comprises a procymidone immune antigen and a procymidone envelope antigen; the procymidone immunogen is formed by coupling a procymidone hapten with a structure shown in a formula (I) and a carrier protein, and the procymidone coating antigen is formed by coupling a procymidone hapten with a structure shown in a formula (II) and a carrier protein.
The Pythium immungen antigen is formed by coupling a Pythium hapten with a structure shown in a formula (I) and a carrier protein, the influence of the carrier protein on the electronic arrangement and the space structure of a small molecule is minimized by an introduced connecting arm, an antigenic determinant of the hapten can be better exposed, the immunogenicity of the hapten is improved, and the titer, the specificity and the affinity of an obtained antibody are better when animals are immunized by using the Pythium immungen antigen.
In the invention, the procymidone coated antigen is formed by coupling the procymidone hapten with the structure shown in the formula (II) and carrier protein, and is favorable for improving the detection sensitivity.
In the field, haptens with the same structure are usually coupled with different carrier proteins to prepare an immune antigen and a coating antigen, while the procymidone immune antigen and the procymidone coating antigen are prepared by creatively coupling two procymidone haptens with different structures with the carrier proteins respectively, so that the method is more favorable for establishing an indirect competitive immunoassay method for determining procymidone with high sensitivity.
In a fourth aspect, the invention provides a procymidone antibody, which is obtained by immunizing animals with the procymidone immunizing antigen and can perform specific immunoreaction with procymidone.
Further, the procymidone antibody is a monoclonal antibody or a polyclonal antibody. In addition, the procymidone antibody can be prepared by adopting a conventional method in the field.
In a specific embodiment, the procymidone antibody is a murine monoclonal antibody specific for a procymidone immunizing antigen against a procymidone hapten of the structure represented by formula (i) above.
The procymidone antibody obtained by adopting the procymidone hapten with the structure shown in the formula (I) and the carrier protein coupled to form the procymidone immunizing antigen has better titer, specificity and affinity.
In a fifth aspect, the invention provides an application of the procymidone antibody in detecting procymidone residues.
The invention induces immune animals to generate antibodies through the procymidone immune antigen, thereby being used in procymidone immunoassay analysis.
The procymidone immunoassay comprises but is not limited to a procymidone ELISA kit, a procymidone colloidal gold test strip and a procymidone time-resolved fluorescence test strip.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention provides two kinds of procymidone haptens with different structures, wherein the procymidone hapten with the structure shown in the formula (I) is used as a procymidone immunizing hapten, the characteristic structure of procymidone is retained to the maximum extent, an antigenic determinant of the hapten can be better exposed, the immunogenicity of the procymidone hapten is obviously enhanced, and the procymidone hapten also has carboxyl which can be coupled with carrier protein; the procymidone hapten with the structure shown in the formula (I) is coupled with carrier protein to obtain the procymidone immunizing antigen to immunize animals, so that the procymidone immunizing antigen is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, the sensitivity of the procymidone antibodies can reach 0.025 mu g/L through detection, the cross reaction rate with other organic chlorine pesticides is low, and a foundation is provided for the subsequent establishment of various immunoassay methods of procymidone. In the invention, the procymidone hapten with the structure shown in the formula (II) is used as the procymidone coating hapten, which is favorable for improving the sensitivity of immunoassay.
According to the invention, the preparation method of the procymidone hapten is reasonably designed according to the structural characteristics of procymidone, the used raw materials are easy to obtain, the reaction operation is simpler, the reaction condition is easy to control, and the purity and the yield of the prepared procymidone hapten are higher.
In the invention, the procymidone hapten and the procymidone envelope antigen are creatively prepared by respectively coupling the procymidone hapten with two different structures and the carrier protein, and the method is more favorable for establishing the indirect competitive immunoassay method for determining procymidone with high sensitivity.
Drawings
FIG. 1 is a scheme for synthesizing a procymidone hapten with a structure shown in formula (I) of the invention
FIG. 2 is a scheme for synthesizing the procymidone hapten with the structure shown in formula (II) of the invention
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.
Example 1
A hapten for the immunization of the pythium aphanidermatum comprises the following steps:
1) dissolving 2.08g of 3, 5-dichloro-4-fluoronitrobenzene in 80mL of ethanol, adding 0.8g of NaOH, stirring, adding 1.05g of aminobutyric acid, fully stirring, reacting at 60 ℃ for 4 hours, removing the ethanol by rotary evaporation after the reaction is finished, adding 100mL of water, extracting with 70mL of ethyl acetate, collecting a water phase, adjusting the pH of the water phase to 5 by using 6mol/L hydrochloric acid, extracting with ethyl acetate, drying by using anhydrous sodium sulfate, and evaporating to obtain an intermediate 1;
2) adding 90mL of methanol into the intermediate 1 for dissolving and clarifying, slowly adding 0.5g of palladium carbon, fully and uniformly mixing, extracting air, introducing hydrogen, stirring at room temperature for 6 hours, after the reaction is finished, removing the palladium carbon by suction filtration, and performing rotary evaporation and evaporation to dryness to obtain an intermediate 2;
3) dissolving 1.58g of 1, 2-dimethylcyclopropane-1, 2-dicarboxylic acid in 100mL of dichloromethane, keeping the temperature constant at 0-5 ℃ for 30min, adding 2.48g of oxalyl chloride, fully stirring, adding 0.5mL of N, N-dimethylformamide, and continuing stirring at 0-5 ℃ for 3 h; adding all the intermediate 2, adding 2.8mL of triethylamine, carrying out reflux reaction for 8h, after the reaction is finished, removing the organic solvent by rotary evaporation, adding 120mL of water, extracting by using 100mL of ethyl acetate, evaporating to dryness, purifying by using a silica gel column, and eluting and separating by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the hapten for the pythium utilization immunity.
Example 2
A hapten for the Pythium ultimum immunization is prepared by the following steps:
1) dissolving 2.08g of 3, 5-dichloro-4-fluoronitrobenzene in 80mL of ethanol, adding 0.8g of NaOH, stirring, adding 0.76g of glycine, fully stirring, reacting at 60 ℃ for 4 hours, removing the ethanol by rotary evaporation after the reaction is finished, adding 100mL of water, extracting with 70mL of ethyl acetate, collecting a water phase, adjusting the pH of the water phase to 5 by using 6mol/L hydrochloric acid, extracting with ethyl acetate, drying by using anhydrous sodium sulfate, and evaporating to obtain an intermediate 1;
2) adding 90mL of methanol into the intermediate 1 for dissolving and clarifying, slowly adding 0.5g of palladium carbon, fully and uniformly mixing, extracting air, introducing hydrogen, stirring at room temperature for 6 hours, after the reaction is finished, removing the palladium carbon by suction filtration, and performing rotary evaporation and evaporation to dryness to obtain an intermediate 2;
3) dissolving 1.58g of 1, 2-dimethylcyclopropane-1, 2-dicarboxylic acid in 100mL of dichloromethane, keeping the temperature constant at 0-5 ℃ for 30min, adding 2.48g of oxalyl chloride, fully stirring, adding 0.5mL of N, N-dimethylformamide, and continuing stirring at 0-5 ℃ for 3 h; and adding all the intermediate 2, adding 2.8mL of triethylamine, carrying out reflux reaction for 8h, after the reaction is finished, carrying out rotary evaporation to remove the organic solvent, adding 120mL of water, extracting with 100mL of ethyl acetate, evaporating to dryness, purifying by using a silica gel column, and eluting and separating by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the hapten for the Pythium ultimum.
Example 3
A hapten for the Pythium ultimum immunization is prepared by the following steps:
1) dissolving 2.08g of 3, 5-dichloro-4-fluoronitrobenzene in 80mL of ethanol, adding 0.8g of NaOH, stirring, adding 0.91g of aminopropionic acid, fully stirring, reacting at 60 ℃ for 4 hours, after the reaction is finished, removing the ethanol by rotary evaporation, adding 100mL of water, extracting with 70mL of ethyl acetate, collecting a water phase, adjusting the pH of the water phase to 5 by using 6mol/L hydrochloric acid, extracting with ethyl acetate, drying by using anhydrous sodium sulfate, and evaporating to dryness to obtain an intermediate 1;
2) adding 90mL of methanol into the intermediate 1 for dissolving and clarifying, slowly adding 0.5g of palladium carbon, fully and uniformly mixing, extracting air, introducing hydrogen, stirring at room temperature for 6 hours, after the reaction is finished, removing the palladium carbon by suction filtration, and carrying out rotary evaporation and evaporation to dryness to obtain an intermediate 2;
3) dissolving 1.58g of 1, 2-dimethylcyclopropane-1, 2-dicarboxylic acid in 100mL of dichloromethane, keeping the temperature constant at 0-5 ℃ for 30min, adding 2.48g of oxalyl chloride, fully stirring, adding 0.5mL of N, N-dimethylformamide, and continuing stirring at 0-5 ℃ for 3 h; and adding all the intermediate 2, adding 2.8mL of triethylamine, carrying out reflux reaction for 8h, after the reaction is finished, carrying out rotary evaporation to remove the organic solvent, adding 120mL of water, extracting with 100mL of ethyl acetate, evaporating to dryness, purifying by using a silica gel column, and eluting and separating by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the hapten for the Pythium ultimum.
Example 4
A hapten for the Pythium ultimum immunization is prepared by the following steps:
1) dissolving 2.08g of 3, 5-dichloro-4-fluoronitrobenzene in 80mL of ethanol, adding 0.8g of NaOH, stirring, adding 1.19g of aminopentanoic acid, fully stirring, reacting at 60 ℃ for 4 hours, after the reaction is finished, removing the ethanol by rotary evaporation, adding 100mL of water, extracting with 70mL of ethyl acetate, collecting a water phase, adjusting the pH of the water phase to 5 by using 6mol/L hydrochloric acid, extracting with ethyl acetate, drying by using anhydrous sodium sulfate, and evaporating to dryness to obtain an intermediate 1;
2) adding 90mL of methanol into the intermediate 1 for dissolving and clarifying, slowly adding 0.5g of palladium carbon, fully and uniformly mixing, extracting air, introducing hydrogen, stirring at room temperature for 6 hours, after the reaction is finished, removing the palladium carbon by suction filtration, and performing rotary evaporation and evaporation to dryness to obtain an intermediate 2;
3) dissolving 1.58g of 1, 2-dimethylcyclopropane-1, 2-dicarboxylic acid in 100mL of dichloromethane, keeping the temperature constant at 0-5 ℃ for 30min, adding 2.48g of oxalyl chloride, fully stirring, adding 0.5mL of N, N-dimethylformamide, and continuing stirring at 0-5 ℃ for 3 h; and adding all the intermediate 2, adding 2.8mL of triethylamine, carrying out reflux reaction for 8h, after the reaction is finished, carrying out rotary evaporation to remove the organic solvent, adding 120mL of water, extracting with 100mL of ethyl acetate, evaporating to dryness, purifying by using a silica gel column, and eluting and separating by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the hapten for the Pythium ultimum.
Example 5
A hapten for the immunization of the pythium aphanidermatum comprises the following steps:
1) dissolving 2.08g of 3, 5-dichloro-4-fluoronitrobenzene in 80mL of ethanol, adding 0.8g of NaOH, stirring, adding 1.34g of aminocaproic acid, fully stirring, reacting at 60 ℃ for 4 hours, removing the ethanol by rotary evaporation after the reaction is finished, adding 100mL of water, extracting with 70mL of ethyl acetate, collecting a water phase, adjusting the pH of the water phase to 5 by using 6mol/L hydrochloric acid, extracting with ethyl acetate, drying by using anhydrous sodium sulfate, and evaporating to dryness to obtain an intermediate 1;
2) adding 90mL of methanol into the intermediate 1 for dissolving and clarifying, slowly adding 0.5g of palladium carbon, fully and uniformly mixing, extracting air, introducing hydrogen, stirring at room temperature for 6 hours, after the reaction is finished, removing the palladium carbon by suction filtration, and performing rotary evaporation and evaporation to dryness to obtain an intermediate 2;
3) dissolving 1.58g of 1, 2-dimethylcyclopropane-1, 2-dicarboxylic acid in 100mL of dichloromethane, keeping the temperature constant at 0-5 ℃ for 30min, adding 2.48g of oxalyl chloride, fully stirring, adding 0.5mL of N, N-dimethylformamide, and continuing stirring at 0-5 ℃ for 3 h; and adding all the intermediate 2, adding 2.8mL of triethylamine, carrying out reflux reaction for 8h, after the reaction is finished, carrying out rotary evaporation to remove the organic solvent, adding 120mL of water, extracting with 100mL of ethyl acetate, evaporating to dryness, purifying by using a silica gel column, and eluting and separating by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the hapten for the Pythium ultimum.
Example 6
A hapten for procymidone coating is prepared by the following steps:
providing a compound 1, wherein the compound 1 has a structural formula
Dissolving 2.99g of compound 1 in 80mL of pyridine, adding 1.1g of succinic anhydride, heating in an oil bath, reacting for 6h at 75 ℃, removing pyridine by rotary evaporation after the reaction is finished, adding 100mL of water and 10mL of 1mol/L hydrochloric acid, extracting with 80mL of ethyl acetate, collecting an organic phase, adding anhydrous sodium sulfate to the organic phase, drying and evaporating to dryness, purifying by using a silica gel column, eluting and separating by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the semi-antigen for the pythium aphanidermatum coating.
Example 7
The preparation method of the procymidone immunogen comprises the following steps:
taking 28mg of the pythium immunogen in example 1, adding 1mL of N, N-dimethylformamide to dissolve, clarifying, adding 19mg of N-hydroxysuccinimide (NHS) and 24.5mg of carbodiimide (EDC), and fully reacting for 1h to obtain a hapten solution A; dissolving 100mg of Human Serum Albumin (HSA) in PB buffer solution to obtain solution B; slowly dripping the solution A into the solution B, stirring at 4 deg.C for 12h, dialyzing and purifying with 0.02mol/L PBS for 3 days, changing the solution 3 times per day to obtain Pythium immunogen, packaging, and storing at-20 deg.C.
Example 8
The preparation method of the procymidone envelope antigen comprises the following steps:
taking 18mg of the procymidone envelope antigen in the embodiment 6, adding 1mL of N, N-dimethylformamide for dissolving, adding 0.1mL of triethylamine and 0.22mL of isobutyl chloroformate, and reacting at 0-5 ℃ for 4h to obtain a hapten solution A; taking 100mg of Ovalbumin (OVA), and adding 0.1mol/L PB buffer solution for dissolving to obtain B solution; slowly dripping the solution A into the solution B, reacting at 4 deg.C for 12h, dialyzing and purifying with 0.02mol/LPBS for 3 days, changing the solution 3 times per day to obtain Pythium ultimum coated antigen, packaging, and storing at-20 deg.C.
Example 9
A procymidone antibody is prepared by the following steps:
1. animal immunization
10 healthy female Balb/c mice (divided into two groups A and B, 5 mice in each group) with 6-8 weeks are taken, emulsified by Freund's complete adjuvant for primary immunization, and injected subcutaneously at the back of the neck and the back at multiple points, wherein the immunization dose of each mouse is 200 mu g of the procymidone immunization antigen in the embodiment 7; then boosting immunity and injecting subcutaneous multi-point on the back of the neck once every two weeks, and emulsifying by Freund incomplete adjuvant; the last immunization uses normal saline to replace Freund's incomplete adjuvant, and is injected in the abdominal cavity, and the injection dosage is the same as the previous times. The specific immunization procedure is shown in table 1.
Table 1 mouse immunization procedure
And (3) for the third time, the fourth time and 7d after the boosting immunization, the tail of the mouse is cut off, blood is taken, and the serum titer of the mouse is measured by an ELISA method, wherein the specific steps are as follows:
(1) diluting the procymidone coating antigen in the embodiment 8 by 1:1000 with 0.05mol/L of carbonate buffer solution with pH9.6, coating an enzyme label plate by 100 mu L of each hole, incubating for 2h at 37 ℃, throwing off the coating solution, washing for 1 time by PBST, and patting dry;
(2) adding 150 mu L of sealing liquid into each hole, reacting at 37 ℃ for 2h, then pouring out the sealing liquid, and patting to dry;
(3) adding 50 mu L of antiserum diluted by PBS in each hole, reacting at 25 ℃ for 30min, then removing the reaction liquid, washing 3-5 times by PBST, and patting dry at intervals of 30s each time;
(4) adding 100 mu L/hole of horseradish peroxidase-labeled goat anti-mouse anti-antibody (1:1000) diluted by PBS, reacting for 30min at 25 ℃, washing for 3-5 times by PBST (PBST), and patting dry at intervals of 30s each time;
(5) adding 50 μ L of substrate developing solution A and B into each well, reacting at 25 deg.C in dark for 15min, adding 50 μ L of 2mol/L H into each well2SO4Terminating the reaction by the solution;
(6) measuring OD value with wavelength of 450nm by enzyme labeling instrument to obtain sample well OD450The titer of positive sera was determined as a dilution factor close to 1.
2. Cell fusion
(1) Preparing feeder cells: the Balb/c mice aged 8-10 weeks are killed by cervical dislocation, soaked in 75% alcohol for 5min, immediately placed in an ultra-clean bench, placed in a flat dish with the abdomen facing upwards or fixed on an anatomical plate. The skin of the abdomen of the mouse is clamped by an ophthalmic forceps, a small opening is cut by scissors, and the peritoneum is not cut to avoid the outflow and pollution of the abdominal cavity fluid. Then blunt dissection was performed up and down with scissors to fully expose the peritoneum. Wiping peritoneum with alcohol cotton ball for sterilization. 5mL of RPMI-1640 basic culture solution was aspirated by a syringe, injected into the abdominal cavity of the mouse, the syringe was gently withdrawn, and the leg and tail of the mouse were shaken several times. The liquid in the abdominal cavity is pumped back by the original syringe and is injected into the centrifuge tube. The operation is repeated for 3-4 times. Centrifuging at 1000r/min for 10min, and discarding the supernatant. Resuspending the cells with 20-50 mL of complete culture medium, adding 100 μ L/well dropwise to the culture plate, and placing in an incubator for later use.
(2) Preparation of splenocytes: and 3d after the boosting immunization, taking an immunized Balb/c mouse, taking the orbit, performing blood sampling, then dislocating and killing, disinfecting in 75% alcohol, taking the spleen, removing connective tissues, preparing a spleen cell suspension, transferring into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging at 1500-2000 r/min for 5min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.
(3) Myeloma cell preparation: taking 3 bottles of myeloma cells with good growth state (the number of living cells is more than 95 percent), completely blowing down the myeloma cells, transferring the myeloma cells into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging at 1500-2000 r/min for 5min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.
(4) Cell mixing: spleen cells and myeloma cells are mixed and centrifuged at 1500-2000 r/min for 5min, wherein the ratio of the spleen cells to the myeloma cells is 8: 1.
(5) Cell fusion: centrifuging the mixed cells, pouring out the supernatant, making the precipitated cell mass into paste, placing the paste in a water bath at 37 ℃, adding 1mL of fusion agent which is polyethylene glycol (PEG)4000 within 1min, acting for 2min, slightly stirring the cells, adding 20mL of serum-free PEG nutrient solution within 4min, centrifuging at 1000r/min for 10min, and discarding the supernatant. The cells were resuspended in 20-50 mL of complete medium, plated on 96-well feeder cells-containing cell culture plates at 100. mu.L per well, and placed in an incubator.
3. Cell line screening
And (3) when the cells grow to 1/3-1/2 of the bottom of the hole, carrying out antibody detection. Screening culture wells with hybridoma cell growth by adopting an ELISA method, wherein the screening comprises two steps: in the first step, positive cell holes are screened by indirect ELISA, in the second step, procymidone is selected as a standard substance, and the inhibition effect of the positive cells is measured by indirect competitive ELISA. And selecting a well with better inhibition on the procymidone standard substance, carrying out subcloning by adopting a limiting dilution method, and carrying out detection by using the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the procymidone monoclonal antibody.
4. Preparation of ascites
Liquid paraffin was injected into Balb/c mice for 6-8 weeks at 500. mu.L/mouse. 10 days later, hybridoma cells in logarithmic growth phase were collected in RPMI-1640 basic medium, counted in a hemocytometer at a cell concentration of 1.0X 10 and a microscope6～1.5×106In the size per mL range. Each mouse was injected with 0.5mL hybridoma cells into the abdominal cavity. Note that after one week the abdomen of the mouse was enlarged, ascites was collected in the abdomen of the mouse with a sterile syringe once every one to two days, and this was repeated until the mouse died naturally. Centrifuging at 4 deg.C for 5min at 5000r/min, collecting supernatant, and removing fat and protein membrane floating on the upper layer of abdominal water.
5. Antibody purification
The monoclonal antibody is purified by an octanoic acid-ammonium sulfate method.
6. Antibody titer determination
And (3) measuring the antibody titer by adopting an indirect ELISA method, and referring to the step 1, measuring the serum titer of animal immunity. The result shows that the titer of the procymidone monoclonal antibody is more than or equal to 100000.
7. Antibody cross-reactivity assay
The indirect competitive ELISA method is adopted for determination, and the result shows that the cross reaction rate of the procymidone monoclonal antibody to procymidone and other organic chlorine pesticides is as follows: the procymidone is 100 percent, and the hexachloro cyclohexane is less than 1 percent of the dichlorprop, the dichlorodiphenyl trichloroethane, the iprodione, the chlorothalonil and the ethofulelene. Therefore, the prepared antibody has better specificity.
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
Claims (10)
1. A procymidone hapten which has a structure shown in a formula (I) or a formula (II):
wherein n is-CH2The number of groups, n is an integer of 0 to 4.
2. The method of preparing a procymidone hapten according to claim 1, comprising the steps of:
1) reacting 3, 5-dichloro-4-fluoronitrobenzene with water under alkaline conditions
Nucleophilic substitution reaction is carried out to obtain an intermediate 1, wherein the intermediate 1 has a structural formula
Wherein n is-CH2The number of groups, n is an integer of 0-4;
2) carrying out hydrogenation reduction reaction on the intermediate 1 under the catalytic action of palladium carbon to obtain an intermediate 2, wherein the intermediate 2 has a structural formula
3) And (3) carrying out cyclization reaction on the intermediate 2, 1, 2-dimethylcyclopropane-1, 2 dicarboxylic acid and oxalyl chloride to obtain the procymidone hapten with the structure shown in the formula (I).
3. The method for preparing a procymidone hapten according to claim 2, wherein the step 1) comprises the following steps: dissolving 3, 5-dichloro-4-fluoronitrobenzene in ethanol, adding NaOH, stirring, and adding
Fully stirring, reacting for 4 hours at 60 ℃, after the reaction is finished, performing rotary evaporation to remove ethanol, adding water, extracting with ethyl acetate, collecting a water phase, adjusting the pH of the water phase to 5 with hydrochloric acid, extracting with ethyl acetate, drying with anhydrous sodium sulfate, and evaporating to dryness to obtain an intermediate 1; wherein, the 3, 5-dichloro-4-fluoronitrobenzene,
And sodium hydroxide in a ratio of 1:1: 2.
4. The method for preparing a procymidone hapten according to claim 2, wherein the step 2) comprises the following steps: and (3) adding methanol to dissolve and clarify the intermediate 1, slowly adding palladium carbon, fully and uniformly mixing, extracting air, introducing hydrogen, stirring at room temperature for 6 hours, after the reaction is finished, removing the palladium carbon by suction filtration, and evaporating to dryness by rotary evaporation to obtain an intermediate 2.
5. The method for preparing a procymidone hapten according to claim 2, wherein the step 3) comprises the following steps: dissolving 1, 2-dimethylcyclopropane-1, 2-dicarboxylic acid in dichloromethane, keeping the temperature constant at 0-5 ℃ for 30min, adding oxalyl chloride, fully stirring, adding N, N-dimethylformamide, and continuously stirring at 0-5 ℃ for 3 h; adding the intermediate 2, adding triethylamine, performing reflux reaction for 8 hours, after the reaction is finished, performing rotary evaporation to remove an organic solvent, adding water, extracting with ethyl acetate, evaporating to dryness, purifying with a silica gel column, and eluting and separating with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the procymidone hapten with the structure shown in formula (I); wherein the mass ratio of the intermediate 2, 1, 2-dimethylcyclopropane-1, 2 dicarboxylic acid and oxalyl chloride is 1:1: 2.
6. The method of preparing a procymidone hapten according to claim 1, comprising the steps of: providing a compound 1, said compound 1 having the formula
The compound 1 reacts with succinic anhydride to obtain the procymidone hapten with the structure shown in the formula (II).
7. The method of claim 6, comprising the steps of: dissolving the compound 1 with pyridine, adding succinic anhydride, heating in an oil bath, reacting at 75 ℃ for 6h, performing rotary evaporation to remove pyridine after the reaction is finished, adding water and hydrochloric acid, extracting with ethyl acetate, collecting an organic phase, adding anhydrous sodium sulfate into the organic phase, drying and evaporating to dryness, purifying with a silica gel column, eluting and separating with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 to obtain the procymidone hapten with the structure shown in the formula (II); wherein the mass ratio of compound 1 to succinic anhydride is 1: 1.
8. A procymidone artificial antigen, wherein the procymidone artificial antigen comprises a procymidone immunity antigen and a procymidone envelope antigen; the procymidone immunogen is formed by coupling a procymidone hapten with a structure shown in a formula (I) and a carrier protein, and the procymidone coating antigen is formed by coupling a procymidone hapten with a structure shown in a formula (II) and a carrier protein, wherein the structure is shown in the formula (I) and the carrier protein are shown in claim 1.
9. A procymidone antibody which is obtained by immunizing an animal with the procymidone immunization antigen of claim 8 and which specifically immunoreacts with procymidone.
10. Use of the procymidone antibody of claim 9 in detecting procymidone residues.
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| CN109956895A (en) * | 2019-03-14 | 2019-07-02 | 深圳市易瑞生物技术股份有限公司 | A kind of procymidone haptens and its synthetic method and application |
| CN110819597A (en) * | 2019-10-11 | 2020-02-21 | 江苏权正检验检测有限公司 | Hybridoma cell strain secreting procymidone monoclonal antibody and application thereof |
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| JP2001281250A (en) * | 2000-03-30 | 2001-10-10 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Method for detecting low-molecule compound using antigen/antibody reaction |
| CN109956895A (en) * | 2019-03-14 | 2019-07-02 | 深圳市易瑞生物技术股份有限公司 | A kind of procymidone haptens and its synthetic method and application |
| CN110819597A (en) * | 2019-10-11 | 2020-02-21 | 江苏权正检验检测有限公司 | Hybridoma cell strain secreting procymidone monoclonal antibody and application thereof |
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