CN117263827A - Propamocarb hapten, artificial antigen and application thereof - Google Patents

Propamocarb hapten, artificial antigen and application thereof Download PDF

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Publication number
CN117263827A
CN117263827A CN202311071967.4A CN202311071967A CN117263827A CN 117263827 A CN117263827 A CN 117263827A CN 202311071967 A CN202311071967 A CN 202311071967A CN 117263827 A CN117263827 A CN 117263827A
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propamocarb
hapten
artificial antigen
pad
carrier protein
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韦秀胆
李斌
李韵婷
孙宝林
林晓慧
江林峰
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Licheng Testing And Certification Group Co ltd
GUANGZHOU WANLIAN BIOTECHNOLOGY CO Ltd
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Licheng Testing And Certification Group Co ltd
GUANGZHOU WANLIAN BIOTECHNOLOGY CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C269/06—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups by reactions not involving the formation of carbamate groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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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    • 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
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles

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Abstract

The invention discloses a downy mildewWilliams, artificial antigens and uses thereof. The propamocarb artificial antigen and monoclonal antibody prepared from the propamocarb hapten have strong specificity and IC (integrated circuit) for ELISA (enzyme-linked immunosorbent assay) detection 50 The value was 21.29. Mu.g/L. The invention also provides an immunochromatography test strip for detecting the propamocarb, which is coated with the propamocarb artificial antigen coating antigen and the monoclonal antibody prepared by the invention, the detection lower limit of the propamocarb standard solution is 25 mug/kg, the detection sensitivity in fruit and vegetable samples can reach 100 mug/kg, the rapid, accurate and qualitative detection of the propamocarb is realized, and the method has wide popularization and application values.

Description

Propamocarb hapten, artificial antigen and application thereof
Technical Field
The invention relates to the field of biochemical engineering, in particular to a propamocarb hapten, an artificial antigen and application thereof.
Background
The propamocarb is an aliphatic bactericide with a local systemic effect, has special effects on oomycete fungi, can inhibit the formation of germ cell membranes, and reduces the formation of sporangia and the number of zoospores, thereby achieving the purpose of preventing and treating diseases. The downy mildew, epidemic disease and damping-off of fruit trees have excellent effects, are suitable for foliar spraying and soil treatment, and the downy mildew also has the effect of promoting crop growth. The residual limit of the propamocarb in China is definitely regulated on different samples, the minimum amount of the residual limit is 0.2mg/kg of a broccoli sample, and the residual amount of the residual limit on fruits and vegetables in the United states is 2mg/kg. The research on the rapid detection method of the propamocarb drug has very important significance.
The detection method of the propamocarb mainly comprises instrument detection, and few simple methods suitable for on-site detection are provided, so that great inconvenience is brought to daily detection work. The immunoassay detection technology is a common detection method, is simple to operate and has less time consumption, but the detection effect is determined by the performances of the antigen and the antibody, and the key of the antigen and the antibody is hapten. Therefore, the structural design of hapten is important to obtain antigen and antibody with excellent performance. The propamocarb hapten disclosed in the prior art CN113759120A is introduced with a characteristic structure with an amide bond and a six-membered ring, so that the electron cloud density on the whole molecular structure of the propamocarb is greatly changed, and the preparation of antigen antibodies with high sensitivity and high specificity is not facilitated. The lack of a propamocarb immunoassay detection technology with high sensitivity cannot meet the actual use requirements of the existing market.
Disclosure of Invention
In order to solve the problem that a rapid, sensitive and simple immunodetection method is lacking in the prior art, the invention provides a propamocarb hapten, an artificial antigen and application thereof.
The first object of the present invention is to provide a propamocarb hapten.
The second object of the present invention is to provide a process for preparing a compound having the structural formula (I),
the third object of the invention is to provide the application of the compound with the structural formula shown in the formula (I) in preparing the propamocarb artificial antigen,
a fourth object of the present invention is to provide a propamocarb artificial antigen.
The fifth object of the present invention is to provide a method for preparing the above-mentioned propamocarb artificial antigen.
The sixth object of the invention is to provide the application of the compound with the structural formula shown as the formula (II) in the preparation of the propamocarb antibody,
a seventh object of the present invention is to provide a combination of propamocarb artificial antigens.
The eighth object of the invention is to provide the application of the propamocarb artificial antigen combination in preparation of a kit for detecting propamocarb.
The ninth object of the invention is to provide a kit for detecting propamocarb.
In order to achieve the above object, the present invention is realized by the following means:
the hapten prepared by the invention completely reserves amide ester structural characteristic groups of propamocarb, and the designed coupling arm is a straight chain with active groups, so that the small molecular structure of the prepared artificial antigen completely reserves the original structural characteristics, and the immunogenicity of the antigen is improved.
A propamocarb hapten is characterized in that the structural formula is shown as a formula (I),
the preparation method of the compound with the structural formula shown in the formula (I) comprises the steps of fully reacting propamocarb with bromoacetic acid, and purifying the obtained reactant to obtain the compound;
the structural formula of the propamocarb is as follows:
the bromoacetic acid has the structural formula:
preferably, the molar ratio of propamocarb to bromoacetic acid is 1: (1-3).
More preferably, the molar ratio of propamocarb to bromoacetic acid is 1:2.
preferably, the solvent of the reaction system of propamocarb and bromoacetic acid comprises water.
Preferably, the purification method comprises: drying and washing.
More preferably, the method of drying comprises reduced pressure evaporation to dryness.
More preferably, the method of washing comprises washing with ethyl acetate.
Further preferably, the method of washing comprises washing with ethyl acetate.
Specifically, the preparation method comprises the following steps:
1.0g of propamocarb (CAS: 24579-73-5,5.31 mmol) is taken and put into a 50mL round bottom flask, 10mL of purified water and 1.48g of bromoacetic acid (CAS: 79-08-3, 10.62 mmol) are added in sequence, and the materials are fully and uniformly mixed and reacted for 6-8 hours at 60 ℃; after the reaction, evaporating to dryness under reduced pressure, pulping and washing the precipitate with 50mL ethyl acetate for 3 times to obtain 0.94g of propamocarb hapten.
A propamocarb artificial antigen is obtained by coupling the propamocarb hapten with carrier protein, the structural formula is shown as a formula (II),
wherein the Protein is carrier Protein, and the carrier Protein is bovine serum albumin or hemocyanin.
The preparation method of the propamocarb artificial antigen is characterized in that the propamocarb hapten is coupled with carrier protein by an active ester method.
Preferably, the active ester process comprises the steps of:
fully reacting the propamocarb hapten, dimethylformamide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain hapten activated ester; dissolving carrier protein in phosphate buffer solution to obtain carrier protein solution; and (3) fully reacting the hapten activated ester with the carrier protein solution, and dialyzing to obtain the hapten activated ester.
Specifically, the active ester method comprises the following steps:
dissolving 10mg of propamocarb hapten in 0.2mL of dimethylformamide, stirring fully, adding 5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 5mg of N-hydroxysuccinimide, and stirring at room temperature for 4 hours to obtain hapten activated ester; taking 40mg of carrier protein and fully dissolving in 4mL of PBS solution with the concentration of 0.01mol/L to form a carrier protein solution; slowly dripping hapten activated ester into a carrier protein solution dropwise, stirring while adding, and stirring at room temperature for 16-24 h; and dialyzing the obtained reaction solution with 0.01mol/L PBS solution at room temperature for 3 days, and changing the dialyzate for 3 times per day to remove unreacted micromolecular substances, thereby obtaining the propamocarb artificial antigen.
The application of the compound with the structural formula shown as the formula (II) in the preparation of the propamocarb antibody is also in the protection scope of the invention,
wherein the Protein is carrier Protein, and the carrier Protein is bovine serum albumin or hemocyanin.
A propamocarb antibody is prepared by immunizing animals with the above propamocarb artificial antigen with carrier protein as hemocyanin as immunogen.
Preferably, the propamocarb antibody is a monoclonal antibody, the propamocarb artificial antigen with carrier protein being hemocyanin is used as immunogen to immunize animals to obtain hybridoma cells, the obtained hybridoma cells are cultured, the cells are collected for animal immunization to obtain ascites, and the animal immunization is carried out to obtain the propamocarb antibody.
The propamocarb artificial antigen combination comprises an immunogen and a coating antigen, wherein the immunogen is obtained by coupling the propamocarb hapten with hemocyanin, namely the propamocarb artificial antigen with carrier protein being hemocyanin; the coating antigen is obtained by coupling the propamocarb hapten with bovine serum albumin, namely the carrier protein is the propamocarb artificial antigen of the bovine serum albumin.
The application of the propamocarb artificial antigen combination in preparing a kit for detecting the propamocarb is also in the protection scope of the invention.
An immunochromatography test strip for detecting propamocarb comprises a bottom plate, wherein a gold pad, a sample pad, a coating reaction film and a water absorption pad which are sequentially overlapped are arranged on the bottom plate; the gold sub-pad is a glass fiber pad containing an antibody for detecting propamocarb, the antibody for detecting the propamocarb is prepared from an immunogen immune animal in the propamocarb artificial antigen combination, and colloidal gold is marked; the reaction membrane is a nitrocellulose membrane provided with a detection area and a quality control area, wherein the detection area is coated with the coating antigen in the propamocarb artificial antigen combination, and the quality control area is coated with IgG.
Preferably, the sample pad is soaked with a sample treatment solution of 0.1M PB buffer containing 0.3% wt Tween 20, 1% wt sucrose, 0.5% wt BSA and 0.05% wt sodium azide.
Preferably, the IgG is murine IgG.
Preferably, the antibody for detecting the propamocarb is a monoclonal antibody, the immunogen in the propamocarb artificial antigen combination is used for immunizing animals to obtain hybridoma cells, the obtained hybridoma cells are cultured, the cells are collected for animal immunization to obtain ascites, and the animal immunization is identified and purified to obtain the propamocarb.
More preferably, the antibody for detecting propamocarb is prepared from the monoclonal antibody and colloidal gold according to the mass-volume ratio (5-10 mug): 1mL of the reaction mixture was reacted sufficiently to obtain the product.
Further preferably, the antibody for detecting propamocarb is prepared from the monoclonal antibody and colloidal gold according to a mass-volume ratio of 8 mug: 1mL of the reaction mixture was reacted sufficiently to obtain the product.
Specifically, the preparation method of the immunochromatographic test strip comprises the following steps:
the propamocarb artificial antigen taking carrier protein as bovine serum albumin is taken as a coating source, and a coating buffer solution (0.01M PBS buffer solution containing 1 wt percent of sucrose and 0.05 wt percent of sodium azide, pH=7.6) is used for diluting the concentration to 0.1 mg/mL-0.5 mg/mL; adjusting the concentration of IgG to 0.1 mg/mL-0.5 mg/mL by using a coating buffer solution;
taking a nitrocellulose membrane as a reaction membrane, spraying diluted coating raw material to a detection area on the reaction membrane according to the membrane liquid amount of 0.8 mu L/cm-1.2 mu L/cm, spraying diluted IgG to a control area on the reaction membrane, wherein the interval between the detection area and the control area is 2.5mm, and placing the reaction membrane in a 45 ℃ oven for 12-16 hours to obtain the coating reaction membrane;
soaking a cut blank sample pad with the size of 30cm multiplied by 30cm in a sample treatment solution (0.01M PBT buffer solution containing 0.5%wt polyethylene glycol (PEG), 0.1%wt BSA, 0.1%wt Triton X-100, 1%wt sucrose and 0.05%wt sodium azide) for 5min, taking out, and drying at 37 ℃ for 16h to obtain the sample pad;
dissolving 1g of chloroauric acid with pure water and ultrasound, and then fixing the volume to 100mL to obtain chloroauric acid solution, and keeping the solution at 4 ℃ in a dark place for later use; heating 1m chloroauric acid solution to 100mL of pure water, boiling, adding 0.5mL of 0.06%wt sodium citrate solution, continuously heating for 10min, cooling to room temperature, and adding pure water to complement to 100mL of volume to obtain colloidal gold solution; taking 1mL of colloidal gold solution, adding a proper amount of 0.1mol/L K 2 CO 3 Adding 8 mug of the monoclonal antibody into a colloidal gold solution, reacting for 5 minutes at room temperature, adding 10 mug of 10%wt bovine serum albumin for sealing, fully and uniformly mixing, centrifuging at 12,000rpm for 10 minutes, and discarding all clear liquid to obtain the antibody for detecting propamocarb; re-dissolving 1mL of gold seed diluent (aqueous solution containing 2% wtTris, 5% wt bovine serum albumin, 0.05% wt merthiolate and 5% wt sucrose) to obtain an antibody for detecting propamocarb, uniformly coating the antibody on a glass fiber pad with the area of 5cm multiplied by 5cm, and drying at 37 ℃ for 16 hours to obtain the gold seed pad;
the gold sub-pad, the sample pad, the coating reaction film and the water absorption pad are stuck to the middle part of the PVC board, and the gold sub-pad, the sample pad, the coating reaction film and the water absorption pad are sequentially overlapped, wherein the water absorption pad is adjacent to a control area of the coating reaction film, and the sample pad is adjacent to a detection area of the coating reaction film, so that the test board is obtained; cutting the test paper plate into test strips with the width of 3mm, loading the test strips into the test paper card, and obtaining the sample pad adjacent to the sample adding hole.
The application of any one of the immunochromatographic test strip in preparing a kit for detecting propamocarb is also within the protection scope of the invention.
A kit for detecting propamocarb comprising the above-mentioned propamocarb artificial antigen combination.
Preferably, the kit comprises an immunochromatographic test strip, wherein the immunochromatographic test strip comprises a bottom plate, and a gold pad, a sample pad, a coating reaction membrane and a water absorption pad which are sequentially overlapped are arranged on the bottom plate; the gold sub-pad is a glass fiber pad containing an antibody for detecting propamocarb, the antibody for detecting the propamocarb is prepared from an immunogen immune animal in the propamocarb artificial antigen combination, and colloidal gold is marked; the reaction membrane is a nitrocellulose membrane provided with a detection area and a quality control area, wherein the detection area is coated with the coating antigen in the propamocarb artificial antigen combination, and the quality control area is coated with IgG.
Preferably, the sample pad is soaked with a sample treatment solution of 0.1M PB buffer containing 0.3% wt Tween 20, 1% wt sucrose, 0.5% wt BSA and 0.05% wt sodium azide.
Preferably, the IgG is murine IgG.
Preferably, the antibody for detecting the propamocarb is a monoclonal antibody, the immunogen in the propamocarb artificial antigen combination is used for immunizing animals to obtain hybridoma cells, the obtained hybridoma cells are cultured, the cells are collected for animal immunization to obtain ascites, and the animal immunization is identified and purified to obtain the propamocarb.
More preferably, the antibody for detecting propamocarb is prepared from the monoclonal antibody and colloidal gold according to the mass-volume ratio (5-10 mug): 1mL of the reaction mixture was reacted sufficiently to obtain the product.
Further preferably, the antibody for detecting propamocarb is prepared from the monoclonal antibody and colloidal gold according to a mass-volume ratio of 8 mug: 1mL of the reaction mixture was reacted sufficiently to obtain the product.
Specifically, the preparation method of the immunochromatographic test strip comprises the following steps:
the propamocarb artificial antigen taking carrier protein as bovine serum albumin is taken as a coating source, and a coating buffer solution (0.01M PBS buffer solution containing 1 wt percent of sucrose and 0.05 wt percent of sodium azide, pH=7.6) is used for diluting the concentration to 0.1 mg/mL-0.5 mg/mL; adjusting the concentration of IgG to 0.1 mg/mL-0.5 mg/mL by using a coating buffer solution;
taking a nitrocellulose membrane as a reaction membrane, spraying diluted coating raw material to a detection area on the reaction membrane according to the membrane liquid amount of 0.8 mu L/cm-1.2 mu L/cm, spraying diluted IgG to a control area on the reaction membrane, wherein the interval between the detection area and the control area is 2.5mm, and placing the reaction membrane in a 45 ℃ oven for 12-16 hours to obtain the coating reaction membrane;
soaking a cut blank sample pad with the size of 30cm multiplied by 30cm in a sample treatment solution (0.01M PBT buffer solution containing 0.5%wt polyethylene glycol (PEG), 0.1%wt BSA, 0.1%wt Triton X-100, 1%wt sucrose and 0.05%wt sodium azide) for 5min, taking out, and drying at 37 ℃ for 16h to obtain the sample pad;
dissolving 1g of chloroauric acid with pure water and ultrasound, and then fixing the volume to 100mL to obtain chloroauric acid solution, and keeping the solution at 4 ℃ in a dark place for later use; heating 1m chloroauric acid solution to 100mL of pure water, boiling, adding 0.5mL of 0.06%wt sodium citrate solution, continuously heating for 10min, cooling to room temperature, and adding pure water to complement to 100mL of volume to obtain colloidal gold solution; taking 1mL of colloidal gold solution, adding a proper amount of 0.1mol/L K 2 CO 3 Adding 8 mug of the monoclonal antibody into a colloidal gold solution, reacting for 5 minutes at room temperature, adding 10 mug of 10%wt bovine serum albumin for sealing, fully and uniformly mixing, centrifuging at 12,000rpm for 10 minutes, and discarding all clear liquid to obtain the antibody for detecting propamocarb; re-dissolving 1mL of gold seed diluent (aqueous solution containing 2% wtTris, 5% wt bovine serum albumin, 0.05% wt merthiolate and 5% wt sucrose) to obtain an antibody for detecting propamocarb, uniformly coating the antibody on a glass fiber pad with the area of 5cm multiplied by 5cm, and drying at 37 ℃ for 16 hours to obtain the gold seed pad;
the gold sub-pad, the sample pad, the coating reaction film and the water absorption pad are stuck to the middle part of the PVC board, and the gold sub-pad, the sample pad, the coating reaction film and the water absorption pad are sequentially overlapped, wherein the water absorption pad is adjacent to a control area of the coating reaction film, and the sample pad is adjacent to a detection area of the coating reaction film, so that the test board is obtained; cutting the test paper plate into test strips with the width of 3mm, loading the test strips into the test paper card, and obtaining the sample pad adjacent to the sample adding hole.
Preferably, the sample to be measured is a fruit and vegetable sample. Fruit and vegetable samples that can be detected by the present invention include, but are not limited to, leaf vegetables, rhizome vegetables, and fruits.
Preferably, the crushed sample to be tested is fully mixed with the buffer solution, solid-liquid separation is carried out, and the liquid is collected to obtain the liquid to be tested.
More preferably, the buffer is a phosphate buffer.
More preferably, the solid-liquid separation method includes standing.
Further preferably, the method of solid-liquid separation further comprises centrifugation.
Compared with the prior art, the invention has the following beneficial effects:
the propamocarb artificial antigen and monoclonal antibody prepared from the propamocarb hapten have strong specificity and IC (integrated circuit) for ELISA (enzyme-linked immunosorbent assay) detection 50 The value was 21.29. Mu.g/L. The invention also provides an immunochromatography test strip for detecting the propamocarb, which is coated with the propamocarb artificial antigen coating antigen and the monoclonal antibody prepared by the invention, the detection lower limit of the propamocarb standard solution is 25 mug/kg, the detection sensitivity in fruit and vegetable samples can reach 100 mug/kg, the rapid, accurate and qualitative detection of the propamocarb is realized, and the method has wide popularization and application values.
Drawings
FIG. 1 shows the synthetic route for propamocarb hapten.
FIG. 2 is a mass spectrum of the propamocarb hapten.
FIG. 3 shows the synthetic route for propamocarb artificial antigen.
FIG. 4 is a standard curve of a propamocarb standard solution.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 Synthesis and identification of propamocarb hapten
1. Synthesis of propamocarb hapten
The synthesis route of the propamocarb hapten is shown in figure 1, and the specific steps are as follows:
1.0g of compound a (i.e., propamocarb, CAS:24579-73-5,5.31 mmol) was taken in a 50mL round bottom flask, 10mL of purified water and 1.48g of compound b (i.e., bromoacetic acid, CAS:79-08-3, 10.62 mmol) were added sequentially, thoroughly and uniformly mixed, and reacted at 60℃for 6-8 hours. After the reaction, evaporating to dryness under reduced pressure, pulping and washing the precipitate with 50mL ethyl acetate for 3 times to obtain 0.94g of propamocarb hapten.
2. Identification of propamocarb hapten
The structural formula of the propamocarb hapten is shown as a formula (I):
the mass spectrum results of the propamocarb hapten are shown in fig. 2, and specifically are as follows: ESI-MS:246[ M-1].
EXAMPLE 2 Synthesis of propamocarb-artificial antigen
The structural formula of the propamocarb artificial antigen provided by the invention is shown as a formula (II),
wherein the Protein is a carrier Protein, and the carrier Protein is Bovine Serum Albumin (BSA) or hemocyanin (KLH).
The synthetic route of the propamocarb artificial antigen is shown in fig. 3, taking the propamocarb artificial antigen with carrier protein as BSA as an example, and the specific preparation steps are as follows:
10mg of propamocarb hapten prepared in example 1 is taken and dissolved in 0.2mL of Dimethylformamide (DMF), after stirring fully, 5mg of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 5mg of N-hydroxysuccinimide (NHS) are added, and stirring is carried out for 4 hours at room temperature, thus obtaining hapten activated ester; 40mg BSA is weighed and fully dissolved in 4mL PBS solution with the concentration of 0.01mol/L to form a carrier protein solution; slowly dripping hapten activated ester into a carrier protein solution dropwise, stirring while adding, and stirring at room temperature for 16-24 h; dialyzing the obtained reaction solution with 0.01mol/L PBS solution at room temperature for 3 days, changing the dialyzate for 3 times per day to remove unreacted micromolecular substances, thus obtaining the propamocarb artificial antigen with the carrier protein of BSA, subpackaging, and preserving at 4 ℃ for standby.
According to the above steps, only difference is that KLH is used to replace BSA, thus obtaining the propamocarb artificial antigen with the carrier protein KLH.
EXAMPLE 3 preparation of propamocarb monoclonal antibodies
1. Immunization of animals
The propamocarb artificial antigen prepared in example 2, in which the carrier protein is hemocyanin, was used as an immunogen, and after emulsification with an equal volume of Freund's adjuvant, BALB/C mice were immunized with 50. Mu.g to 100. Mu.g of each mouse. The immunization is carried out at intervals of 2 weeks, 3 times of immunization, the tail venous blood of the mice is adopted to detect the serum titer, and the immunization is enhanced according to the detection result until the antibody titer is no longer increased, and 100 mug of whole antigen is used for subcutaneous enhancement.
2. Hybridoma cell preparation
After 5 days of the last booster immunization, taking the spleen cells of the mice and SP20 cells of human osteosarcoma cells for fusion to obtain fusion cells, screening by using HAT medium, and continuously culturing the fusion cells by using a complete medium instead of the HAT medium after 5 days of screening. Cell supernatants were assayed by ELISA, and cells in wells with strong positives were subjected to limiting dilution cloning. After 3 times of clone culture detection, the cells in the holes which are positive are hybridoma cells secreting monoclonal antibodies.
3. Monoclonal antibody preparation
The hybridoma cells are cultured in an amplifying way and inoculated to the abdominal cavity of a mouse, so that ascites containing the antibody is produced. Purifying the ascites by a caprylic acid-ammonium sulfate precipitation method to obtain the high-purity and high-specificity propamocarb monoclonal antibody.
EXAMPLE 4ELISA Performance evaluation
1. Experimental method
The artificial antigen and monoclonal antibody prepared by the invention are subjected to performance evaluation by adopting an ELISA method, and the method comprises the following steps:
(1) Antigen coating
The propamocarb artificial antigen with the carrier protein of example 2 as bovine serum albumin is used as a coating raw material, the coating raw material is diluted by a coating diluent (carbonate buffer solution, pH 9.6) to obtain a coating raw material diluent with the concentration of 0.3 mug/mL, the coating raw material diluent is added into a polystyrene micro-pore plate according to 100 mug/hole, the coating is carried out at the temperature of 4 ℃ for overnight, the drying is carried out, and PBST is used for washing three times, thus obtaining the coating plate.
(2) Closure
Adding into blocking solution (phosphate buffer solution containing 1% wt BSA) at 280 μl/well, blocking at 37deg.C for 1 hr, spin-drying, washing with PBST three times, drying, and vacuum packaging for storage.
(3) Dilution resistance
The propamocarb monoclonal antibody prepared in example 3 was used as primary antibody, and the primary antibody was diluted with an antibody diluent (phosphate buffer containing 0.05% wt sodium azide, pH 7.4) to give a primary antibody diluent having a concentration of 0.1. Mu.g/mL, which was stored at 4℃for use.
(4) Standard solution preparation
The propamocarb standard was dissolved in 0.01M PBS to give propamocarb standard solutions with concentrations of 0. Mu.g/L, 5. Mu.g/L, 15. Mu.g/L, 45. Mu.g/L, 135. Mu.g/L and 405. Mu.g/L, respectively.
(5) Sample addition and primary antibody incubation
A propamocarb standard solution is added into the coating plate according to 100 mu L/hole, then a primary anti-dilution solution with the concentration of 0.1 mu g/mL is added according to 20 mu L/hole, and the reaction is carried out for 0.5h at 37 ℃ and the drying is carried out.
(6) Wash primary antibody
PBST was added at 280. Mu.L/well, washed 3 times and patted dry.
(7) Second antibody incubation
HRP enzyme-labeled goat anti-mouse IgG enzyme-labeled secondary antibody was added at 100. Mu.L/well, and reacted at 37℃for 0.5h.
(8) Wash secondary antibody
PBST was added at 280. Mu.L/well, washed 3 times and patted dry.
(9) Color development
Adding TMB color development liquid into the mixture according to 100 mu L/hole, and reacting for 15min at 37 ℃; the color development was terminated by adding 1M sulfuric acid at 50. Mu.L/well.
(10) Absorbance measurement
The OD value of each well was measured at a wavelength of 450nm in an microplate reader.
2. Experimental results
TABLE 1ELISA testing of OD values of different concentrations of propamocarb standard solutions
The OD values of the propamocarb standard solutions at different concentrations are shown in table 1. Four-parameter Logistic curve fitting is performed on the data shown in table 1 by ELISA Calc software, a standard curve is drawn and is shown in fig. 4, and a linear equation is as follows:
y＝(A-D)/[1+(x/C)^B]+D，r 2 = 0.99941; where a=2.40612, b= 0.88191,
c=16.94755, d=0.22014, x represents the concentration of the analyte, and y represents the OD value.
IC is obtained through calculation 50 The value is 21.29 mug/L, and the linear relation is between 5 mug/L and 405 mug/L.
Example 5 colloidal gold qualitative immunochromatography kit for detecting propamocarb and effect evaluation
1. Composition of colloidal gold qualitative immunochromatography kit
The kit comprises an immune layer test paper card, and the preparation method thereof is as follows:
1. preparation of coated reaction film
The propamocarb artificial antigen with the carrier protein prepared in example 2 as bovine serum albumin is used as a coating source, and the coating buffer (0.01M PBS buffer containing 1 wt percent of sucrose and 0.05 wt percent of sodium azide, pH=7.6) is used for diluting the concentration to 0.1 mg/mL-0.5 mg/mL; the concentration of the mouse IgG is regulated to be 0.1 mg/mL-0.5 mg/mL by using a coating buffer solution.
Taking a Nitrocellulose (NC) film as a reaction film, spraying the diluted coating raw material to a detection area (T line) on the reaction film according to the film liquid amount of 0.8 mu L/cm-1.2 mu L/cm, spraying the diluted mouse IgG to a control area (C line) on the reaction film, wherein the interval between the detection area and the control area is 2.5mm, and placing the reaction film in a 45 ℃ oven for 12-16 h to obtain the coating reaction film, and placing the coating reaction film in a constant temperature and humidity preservation box for standby.
2. Preparation of sample pad
The cut blank sample pad with the size of 30cm multiplied by 30cm is soaked in the sample treatment liquid (0.01M PBT buffer solution containing 0.5%wt polyethylene glycol (PEG), 0.1%wt BSA, 0.1%wt Triton X-100, 1%wt sucrose and 0.05%wt sodium azide) for 5min, taken out, dried at 37 ℃ for 16h, and the sample pad is obtained and placed in a constant temperature and humidity preservation box for standby.
3. Preparation of gold seed pad
(1) Preparation of colloidal gold solution
1g of chloroauric acid is taken, dissolved by pure water and ultrasound, and then fixed to 100mL, thus obtaining chloroauric acid solution, and the chloroauric acid solution is preserved at 4 ℃ in a dark place for standby. And (3) heating 1m chloroauric acid solution to 100mL of pure water, boiling, adding 0.5mL of 0.06 wt% sodium citrate solution, continuously heating for 10min, cooling to room temperature, adding pure water to complement to a volume of 100mL, and standing in a dark place at normal temperature for later use. All glassware is soaked in a mixed solution of potassium permanganate and sulfuric acid overnight, washed and dried.
(2) Labeling of propamocarb monoclonal antibodies
Taking 1mL of colloidal gold solution, adding a proper amount of 0.1mol/L K 2 CO 3 Adding 5 mu g of the propamocarb monoclonal antibody prepared in the example 3 into a colloidal gold solution, reacting for 5 minutes at room temperature, adding 10 mu L of 10%wt bovine serum albumin for sealing, fully mixing, centrifuging at 12,000rpm for 10 minutes, and discarding all clear liquid to obtain precipitate, namely the propamocarb monoclonal antibody marked by the colloidal gold.
(3) Preparation of gold seed pad
1mL of gold seed diluent (aqueous solution containing 2% wtTris, 5% wt of bovine serum albumin, 0.05% wt of merthiolate and 5% wt of sucrose) is used for redissolving the colloidal gold-labeled propamocarb monoclonal antibody obtained in the previous step, and the colloidal gold-labeled propamocarb monoclonal antibody is uniformly smeared on a glass fiber pad with the area of 5cm multiplied by 5cm, and is dried at 37 ℃ for 16 hours, so that the gold seed pad is obtained and is preserved for standby.
4. Assembly
And sticking a sample pad, a gold sub-pad, a coating reaction film and a water absorption pad at the middle part of the PVC board, and sequentially overlapping the sample pad, the gold sub-pad, the coating reaction film and the water absorption pad, wherein the water absorption pad is adjacent to a C line of the coating reaction film, and the gold sub-pad is adjacent to a T line of the coating reaction film, so that the test board is obtained. Cutting the test paper plate into test strips with the width of 3mm, loading the test strips into the test paper card, and obtaining the colloidal gold qualitative immunochromatography test paper card by arranging the sample pad adjacent to the sample adding hole.
2. Application method
(1) Sample processing
2g of fruit and vegetable sample (1 cm cut of leaf vegetable) 3 About fragments, tuber samples or epidermis thereof are taken out), the tuber samples or epidermis thereof are placed into a 50mL centrifuge tube, 6mL of 0.01M PBS is added, the mixture is mixed for 2min under intense shaking, the centrifuge tube is kept stand for 2min, and supernatant fluid is collected, thus obtaining the liquid to be measured. If the PBS is added, the mixture can be filtered or centrifuged at 4000 rpm for 3min, and then the supernatant is collected as the liquid to be tested.
(2) Detection of
And (3) placing the colloidal gold qualitative immunochromatographic test paper card horizontally, taking 100 mu L of liquid to be tested, and adding the liquid to be tested into the sample adding hole, wherein the liquid to be tested sequentially passes through the sample pad, the gold sub-pad, the T line of the reaction film, the C line of the reaction film and the absorbent paper. And (3) starting timing after sample addition, standing for 5-8 min, observing results, and judging that the reading is invalid for more than 8 min. The test set up was repeated for 3 groups.
(3) Interpretation of results
The interpretation method comprises the following steps:
when the line C does not develop color, the detection result is invalid, the operation process is incorrect or the test paper strip is invalid; when the color of the C line is developed, and the color of the T line is stronger than that of the C line or has no obvious difference with that of the C line, the detection result is negative (-), which indicates that the sample to be detected has no propamocarb; when the C line is developed and the T line is obviously weaker than the C line or the T line is not developed, the detection result is positive (+), which indicates that the propamocarb exists in the sample to be detected.
3. Evaluation of Performance
1. Sensitivity of
The propamocarb standard was diluted to a concentration of 0. Mu.g/L, 12.5. Mu.g/L, 25. Mu.g/L, 50. Mu.g/L and 100. Mu.g/L with 0.01M PBS buffer and tested using the colloidal gold qualitative immunochromatographic kit of the present example.
TABLE 2 sensitivity measurement results
As shown in Table 2, the colloidal gold qualitative immunochromatographic kit prepared by the invention has high sensitivity for detecting propamocarb, and the lower limit of detection is 25 mug/L.
2. Specificity (specificity)
The cross-reactivity of other bactericides (carbendazim, cymoxanil, pyraclostrobin, imidacloprid and procymidone) was determined with a propamocarb kit. The above detection targets were spotted in a solution of 2500. Mu.g/L in 0.01M PBS, and 3 detection cards were repeated for each target according to the detection procedure, and the results are shown in Table 3.
TABLE 3 specificity determination results
As shown in Table 3, the colloidal gold qualitative immunochromatographic kit prepared by the invention has negative detection results of carbendazim, cymoxanil, pyraclostrobin, imidacloprid and procymidone at the concentration of 2500 mug/L, and the cross reaction rate is less than 1%.
3. Stability of
The storage condition of the colloidal gold qualitative immunochromatographic test paper card of the present example is room temperature, in order to determine the stability of the test paper strip, an accelerated destructive test is performed on the test paper strip, the test paper strip is placed continuously at 45 ℃ for 60 days, and the test is set up to 3 groups of repetitions on days 0, 5, 10, 20, 30, 40, 50 and 60 by diluting the propamocarb standard substance with 0.01M PBS buffer solution to a concentration of 0 μg/L, 12.5 μg/L, 25 μg/L and 50 μg/L, respectively.
TABLE 4 stability measurement results
As shown in Table 4, after the colloidal gold qualitative immunochromatographic test paper card hermetically stored at 45 ℃ is stored for 60 days, the color depth interpretation results of the T line and the C line of the test paper card are not obviously changed, which indicates that the colloidal gold qualitative immunochromatographic test paper card can be stably stored for at least 60 days at 45 ℃ in an accelerated experiment. The acceleration of 37.5 days at 45 ℃ is equivalent to one year of storage at room temperature, so that the propamocarb colloidal gold qualitative immunochromatography test paper card prepared by the invention can be stably stored for more than one year at room temperature, and can completely meet the requirements of the market in the storage and transportation processes.
4. Fruit and vegetable sample marking detection limit
The cauliflower, eggplant, brussels sprouts, chicory, chinese cabbage, grape and plum are used as samples to be tested, and the sample liquid of each sample to be tested is obtained according to the sample treatment method in the embodiment. The sample solutions of the samples to be tested were subjected to gradient labeling detection with the propamocarb standard, and the labeling degree was 0. Mu.g/kg, 50. Mu.g/kg, 100. Mu.g/kg and 200. Mu.g/kg, using the colloidal gold qualitative immunochromatographic kit of the present example.
Table 5 fruit and vegetable sample labeled detection limit determination results
As shown in Table 5, the colloidal gold qualitative immunochromatographic kit prepared by the invention has good repeatability of detection results in 7 samples, and is negative when the propamocarb content in the samples is lower than 100 mug/kg; above 100. Mu.g/kg, all positive. Therefore, the detection limit of the propamocarb colloidal gold immunochromatographic reagent card prepared by the invention in the fruit and vegetable sample is 100 mug/kg.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (10)

1. A propamocarb hapten is characterized in that the structural formula is shown as a formula (I),
2. the preparation method of the compound with the structural formula shown as the formula (I) is characterized in that propamocarb and bromoacetic acid fully react, and the obtained reactant is purified to obtain the compound;
3. the application of a compound with a structural formula shown as a formula (I) in the preparation of propamocarb artificial antigen,
4. a propamocarb artificial antigen is characterized in that the propamocarb artificial antigen is obtained by coupling a propamocarb hapten with a carrier protein according to claim 1, the structural formula of the propamocarb artificial antigen is shown as a formula (II),
wherein the Protein is carrier Protein, and the carrier Protein is bovine serum albumin or hemocyanin.
5. The method for preparing the propamocarb artificial antigen according to claim 4, which is characterized in that the propamocarb hapten according to claim 1 is coupled with carrier protein by an active ester method.
6. The application of a compound with a structural formula shown as a formula (II) in the preparation of the propamocarb antibody,
wherein the Protein is carrier Protein, and the carrier Protein is bovine serum albumin or hemocyanin.
7. A combination of propamocarb artificial antigens, comprising a coating antigen and an immunogen, said coating antigen being derived from the propamocarb hapten coupled bovine serum albumin of claim 1; the immunogen is obtained by coupling propamocarb hapten with hemocyanin as claimed in claim 1.
8. Use of the propamocarb-artificial antigen combination of claim 7 for preparing a kit for detecting propamocarb.
9. A kit for detecting propamocarb, comprising the propamocarb artificial antigen combination of claim 7.
10. The kit according to claim 9, comprising an immunochromatographic test strip, wherein the immunochromatographic test strip comprises a bottom plate, and a gold pad, a sample pad, a coating reaction membrane and a water absorption pad which are sequentially overlapped are arranged on the bottom plate; the gold sub-pad is a glass fiber pad containing a propamocarb-detecting antibody, the propamocarb-detecting antibody is prepared from the immunogen immune animal of claim 7, and is marked with colloidal gold; the reaction membrane is a nitrocellulose membrane provided with a detection area and a quality control area, wherein the detection area is coated with the coating antigen in claim 7, and the quality control area is coated with IgG.
CN202311071967.4A 2023-08-23 2023-08-23 Propamocarb hapten, artificial antigen and application thereof Pending CN117263827A (en)

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