CN112174838A - 2,4, 5-trichlorophenoxyacetic acid hapten, artificial antigen and application thereof in immunodetection - Google Patents
2,4, 5-trichlorophenoxyacetic acid hapten, artificial antigen and application thereof in immunodetection Download PDFInfo
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- CN112174838A CN112174838A CN202011088212.1A CN202011088212A CN112174838A CN 112174838 A CN112174838 A CN 112174838A CN 202011088212 A CN202011088212 A CN 202011088212A CN 112174838 A CN112174838 A CN 112174838A
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- artificial antigen
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- trichlorophenoxyacetic acid
- hapten
- acid
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- SMYMJHWAQXWPDB-UHFFFAOYSA-N (2,4,5-trichlorophenoxy)acetic acid Chemical compound OC(=O)COC1=CC(Cl)=C(Cl)C=C1Cl SMYMJHWAQXWPDB-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 239000003559 2,4,5-trichlorophenoxyacetic acid Substances 0.000 title claims abstract description 75
- 239000000427 antigen Substances 0.000 title claims abstract description 50
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- 238000000034 method Methods 0.000 claims abstract description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 48
- 238000012360 testing method Methods 0.000 claims description 37
- 150000001875 compounds Chemical class 0.000 claims description 30
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical group O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 4
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- 238000012286 ELISA Assay Methods 0.000 claims 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
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- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- -1 2,4, 5-trichlorophenoxyacetic acid (2, 4, 5-trichlorophenoxyacetic acid) 2,4, 5-trichlorophenoxyacetic acid Chemical compound 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
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- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
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- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
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- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
- C07C217/78—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
- C07C217/80—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings
- C07C217/82—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings of the same non-condensed six-membered aromatic ring
- C07C217/84—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings of the same non-condensed six-membered aromatic ring the oxygen atom of at least one of the etherified hydroxy groups being further bound to an acyclic carbon atom
-
- 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
-
- 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
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- 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
-
- 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
-
- 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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- Immunology (AREA)
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- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
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- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Gastroenterology & Hepatology (AREA)
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- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a 2,4, 5-trichlorophenoxyacetic acid hapten, an artificial antigen and application thereof in immunodetection. The arm introduced by the designed 2,4, 5-trichlorophenoxy acetic acid hapten (2- (2-amido-3, 4, 6-trichlorophenoxy) acetic acid) not only has an active group, but also can completely reserve a carboxyl group of a target object to be detected, so that the electron cloud density of the hapten and the electron cloud density of the target object to be detected are kept consistent, and the coupling is simple. The 2,4, 5-trichlorophenoxyacetic acid artificial antigen and the monoclonal antibody constructed based on the method are strong in ELISA detection specificity, and the IC50 value is 4.9 mu g/L; when the fluorescent quantitative immunochromatography reagent is used for a fluorescent quantitative immunochromatography technology, the sensitivity is 3 mu g/L, and the stability is excellent.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to a 2,4, 5-trichlorophenoxyacetic acid hapten, an artificial antigen and application thereof in immunodetection
Background
2,4, 5-trichlorophenoxyacetic acid (6-benzylpenining, 2,4, 5-trichlorophenoxyacetic acid) is the first artificially synthesized cytokinin, is white or white-like crystal, is insoluble in water, slightly soluble in ethanol, stable in acid and alkali, and has the effects of inhibiting the decomposition of chlorophyll, nucleic acid and protein in plant leaves, keeping green and preventing aging; the amino acid, the auxin, the inorganic salt and the like are transported to the treatment part, and the like, and are widely used in various stages from germination to harvest of agricultural, fruit trees and horticultural crops. Because the 2,4, 5-trichlorophenoxyacetic acid has good drug effect and low price, the 2,4, 5-trichlorophenoxyacetic acid is taken as a growth regulator and is widely applied to the planting of the rootless cultured bean sprouts. If a human body ingests too much 2,4, 5-trichlorophenoxyacetic acid, skin mucosa can be stimulated, esophagus and gastric mucosa are damaged, and symptoms such as nausea and vomiting occur. The original national food and drug administration, agricultural department, national health and family planning committee have jointly issued bulletins (No. 11 in 2015) to prohibit the use of substances such as 2,4, 5-trichlorophenoxyacetic acid and the like in the bean sprout production process, so that the enhancement of the monitoring of the residual amount of the 2,4, 5-trichlorophenoxyacetic acid is very important for ensuring the food quality and safety.
The research has been carried out to determine whether the chemical herbicide of 2,4, 5-trichlorophenoxyacetic acid can induce human peripheral blood lymphocytes, and in the experiment, it is found that the SCE value of normal human lymphocytes has a certain range when the chemical herbicide of 2,4, 5-trichlorophenoxyacetic acid is not added, when the concentration of the chemical herbicide is added to 20 picograms, the mutagenesis effect is strong, when the concentration is increased to 80 picograms, the mutagenesis effect is stronger, which indicates that the mutagenesis effect is related to the concentration of the added chemical herbicide.
The existing methods for detecting 2,4, 5-trichlorophenoxyacetic acid mainly comprise traditional detection methods, such as spectrophotometry, high performance liquid chromatography and combined technology thereof, and the methods can accurately quantify, but cannot realize real on-site rapid detection due to expensive equipment and instruments, long detection time and operation of professional staff; the other detection method is an immunological detection technology, which has the characteristics of high specificity and high selectivity, is very suitable for separating or detecting trace components of complex matrixes, and along with the mutual permeation among disciplines, the detection method is endless, the application range is increasingly expanded, and compared with the traditional detection method, the immunological detection technology has the characteristics of economy, rapidness, low technical key points, simplicity and convenience in operation and the like. However, the key of the immunoassay detection technology is the performance determination of the antigen and the antibody, and the key of the antigen and the antibody is the hapten of the corresponding medicine.
Therefore, designing haptens with higher recognition degree and corresponding artificial antigens become a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a 2,4, 5-trichlorophenoxyacetic acid hapten;
another object of the present invention is to provide a method for preparing the above hapten;
it is another object of the present invention to provide an artificial antigen;
another object of the present invention is to provide a method for preparing the above artificial antigen;
the invention also aims to provide the application of the artificial antigen in preparing the 2,4, 5-trichlorophenoxyacetic acid monoclonal antibody;
the invention also aims to provide the application of the artificial antigen in ELISA detection.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a hapten of 2,4, 5-trichlorophenoxyacetic acid, which has a structure shown in formula I:
compared with the existing 2,4, 5-trichlorophenoxyacetic acid hapten, the artificial antigen prepared by the hapten can keep carboxyl characteristic groups, so that the consistency of the electron cloud density and the structural chemical property of the artificial antigen and a target object to be detected is not influenced, and the artificial antigen has excellent specificity and accuracy.
In a second aspect of the present invention, there is provided:
the preparation method of the hapten comprises the following steps:
(1) mixing the compound a, the compound b, a solvent and an acid-binding agent, removing the solvent, adding water for mixing, sequentially extracting with petroleum ether and ethyl acetate, combining ethyl acetate layers, and removing the petroleum ether and the ethyl acetate to obtain a compound c;
(2) mixing the compound c, a solvent, zinc and hydrochloric acid, adding water, extracting with ethyl acetate, adjusting the pH to 8-9 with a pH regulator, extracting with ethyl acetate again, combining organic phases, and removing ethyl acetate to obtain a compound d;
(3) mixing the compound d, a solvent, lithium hydroxide and water, adding sodium chloride, extracting with dichloromethane, and adjusting the pH to 4-5 with a pH regulator to obtain a 2,4, 5-trichlorophenoxyacetic acid hapten;
wherein the structural formula of the compound a is shown as formula II:
the structural formula of the compound b is shown as the formula III:
the structural formula of the compound c is shown as a formula IV:
the structural formula of the compound d is shown as the formula V:
further, the solvent is selected from acetone and ethanol; the acid-binding agent is selected from potassium carbonate.
Further, the volume ratio of the ethyl acetate to the petroleum ether in the step (1) is 1: 5.
further, the mixing condition in the step (1) is that the reaction is carried out for more than 5 hours at the temperature of 60-70 ℃; the mixing condition in the step (2) is room temperature reaction for more than 2 hours; the mixing condition in the step (3) is room temperature reaction for more than 16 h.
In a third aspect of the present invention, there is provided:
an artificial antigen is obtained by coupling the hapten and a protein carrier, and the structural formula of the artificial antigen is as follows:
wherein the protein is a protein carrier.
Further, the protein carrier is selected from any one of bovine serum albumin, ovalbumin, human serum albumin and hemocyanin.
In a fourth aspect of the present invention, there is provided:
the preparation method of the artificial antigen comprises the following steps:
(1) reacting the hapten with an activator and a protein carrier for 16-24 hours;
(2) stirring for 16-24 h, and dialyzing and separating to obtain the artificial antigen;
wherein the activating agent is glutaraldehyde.
In a fifth aspect of the present invention, there is provided:
the artificial antigen is applied to the preparation of the 2,4, 5-trichlorophenoxyacetic acid monoclonal antibody.
The preparation method of the 2,4, 5-trichlorophenoxyacetic acid monoclonal antibody comprises the preparation method of the monoclonal antibody which is conventional in the field.
In a sixth aspect of the present invention, there is provided:
the artificial antigen is applied to ELISA detection.
In a seventh aspect of the present invention, there is provided:
the fluorescent quantitative immunochromatographic test paper for detecting the 2,4, 5-trichlorophenoxyacetic acid is coated with the artificial antigen.
The invention has the beneficial effects that:
1. the arm introduced by the designed 2,4, 5-trichlorophenoxyacetic acid hapten not only has an amino group, but also completely retains the carboxyl of a target object to be detected, so that the electron cloud density of the hapten and the electron cloud density of the target object to be detected are consistent.
2. The 2,4, 5-trichlorophenoxyacetic acid artificial antigen and the monoclonal antibody have strong detection specificity when used for ELISA, and the IC50 value is 4.9 mu g/L.
3. The 2,4, 5-trichlorophenoxyacetic acid artificial antigen and the monoclonal antibody are used for a fluorescence quantitative immunochromatography technology, the detection of the 2,4, 5-trichlorophenoxyacetic acid can be rapidly and conveniently realized, and the fluorescence quantitative immunochromatography test strip prepared in the invention has the detection sensitivity of 3 mu g/L for the 2,4, 5-trichlorophenoxyacetic acid.
Drawings
FIG. 1 is a scheme showing the synthesis of 2,4, 5-trichlorophenoxyacetic acid hapten;
FIG. 2 is a circuit diagram of the synthesis of an artificial antigen of 2,4, 5-trichlorophenoxyacetic acid;
FIG. 3 is a standard curve diagram of the combination of 2,4, 5-trichlorophenoxyacetic acid artificial antigen and monoclonal antibody against 2,4, 5-trichlorophenoxyacetic acid;
FIG. 4 is a standard curve chart of the fluorescent quantitative test paper of the present invention for 2,4, 5-trichlorophenoxyacetic acid.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
EXAMPLE 12 preparation of 4, 5-Trichlorophenoxyacetic acid hapten
A hapten of 2,4, 5-trichlorophenoxyacetic acid, which has a structure shown in formula I:
as shown in FIG. 1, the hapten of 2,4, 5-trichlorophenoxyacetic acid is prepared by the following method:
(1) 1.0g (4.1mmol) of Compound a in a 100ml round-bottomed flask, followed by addition of 10ml of acetone, 1.3g (8.2mmol) of Compound b and 1.1g (8.2mmol) of K2CO3And reacting for more than 5 hours at the temperature of 60-70 ℃. After the reaction is finished, decompressing and removing the solvent, adding 30ml of purified water, extracting twice with petroleum ether, extracting the water phase twice with ethyl acetate (the volume ratio of the ethyl acetate to the petroleum ether is 1: 5), combining the ethyl acetate layers, decompressing and removing the solvent to obtain a compound c.
(2) Dissolving 1.2g (3.7mmol) of the compound c in 10ml of ethanol, adding 1.0g (14.6mmol) of reduced zinc powder, slowly dropwise adding 7.5ml of 2M hydrochloric acid while stirring, reacting at room temperature for more than 2h, adding 40ml of purified water, extracting twice with ethyl acetate, adjusting the pH value of the water phase to 8-9 by 1M potassium carbonate aqueous solution, extracting twice with ethyl acetate, combining ethyl acetate layers, and removing the solvent under reduced pressure to obtain a compound d.
(3) Dissolving 0.8g (2.7mmol) of compound d in 5ml of ethanol, adding 0.7g (16.7mmol) of LiOH and 5ml of purified water, reacting at room temperature for more than 16h, adding 20ml of saturated sodium chloride aqueous solution, extracting twice with dichloromethane, adjusting the pH value of the aqueous phase to 4-5 by 2M dilute hydrochloric acid, filtering, and drying to obtain the 2,4, 5-trichlorophenoxyacetic acid hapten.
Wherein the structural formula of the compound a is shown as formula II:
the structural formula of the compound b is shown as the formula III:
the structural formula of the compound c is shown as a formula IV:
the structural formula of the compound d is shown as the formula V:
example 22 preparation of artificial antigen of 4, 5-trichlorophenoxyacetic acid
The structural formula of the 2,4, 5-trichlorophenoxyacetic acid artificial antigen is as follows:
wherein the protein is a protein carrier, and the protein carrier is selected from any one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
The synthetic route of the artificial antigen is shown in FIG. 2.
1. The carrier protein is 2,4, 5-trichlorophenoxyacetic acid artificial antigen of bovine serum albumin, and the synthesis method is as follows:
(1) weighing 40mg of Bovine Serum Albumin (BSA), fully dissolving the BSA in 4mL of MES buffer solution with the pH value of 6.0, adding 40 mu L of 50% glutaraldehyde, and stirring for 16-24 h at room temperature in a dark place to obtain bovine serum albumin buffer solution;
(2) carrying out ultrafiltration treatment on the bovine serum albumin buffer solution prepared in the step (1) by using an ultrafiltration tube with the aperture of 6kD, carrying out ultrafiltration for 6 times, and redissolving by using 4mL of MES buffer solution every time so as to remove unreacted small molecular substances;
(3) taking 10mg of the 2,4, 5-trichlorophenoxyacetic acid hapten prepared in the example 1, fully dissolving the hapten in 0.4mL of Dimethylformamide (DMF), dropwise adding the mixture into the bovine serum albumin buffer solution subjected to ultrafiltration in the step (2) under stirring, and stirring the mixture at room temperature in a dark place for 16-24 hours to obtain a carrier protein solution;
(4) dialyzing the carrier protein solution with 0.01mol/L PBS at room temperature for 3 days, and changing the dialyzate 3 times per day to remove unreacted small molecular substances;
(5) subpackaging and storing at 4 ℃ for later use.
2. The carrier protein is 2,4, 5-trichlorophenoxyacetic acid artificial antigen of hemocyanin, and the synthesis method is as follows:
(1) weighing 20mg of hemocyanin (KLH), fully dissolving the hemocyanin (KLH) in 2mL of MES buffer solution with the pH value of 6.0, adding 40 mu L of glutaraldehyde with the content of 50%, and stirring for 16-24 h at room temperature in a dark place to obtain a hemocyanin buffer solution;
(2) carrying out ultrafiltration treatment on the hemocyanin buffer solution prepared in the step (1) by using an ultrafiltration tube with the aperture of 6kD, carrying out ultrafiltration for 6 times, and redissolving by using 2mL of MES buffer solution every time to remove unreacted small molecular substances;
(3) taking 5mg of the 2,4, 5-trichlorophenoxyacetic acid hapten prepared in the example 1, fully dissolving the hapten in 0.2mL of Dimethylformamide (DMF), dropwise adding the mixture into the hemocyanin buffer solution subjected to ultrafiltration in the step (2) under stirring, and stirring the mixture at room temperature in a dark place for 16-24 hours to obtain a carrier protein solution;
(4) dialyzing the carrier protein solution with 0.01mol/L PBS at room temperature for 3 days, and changing the dialyzate 3 times per day to remove unreacted small molecular substances;
(5) subpackaging and storing at 4 ℃ for later use.
EXAMPLE 32 preparation of 4, 5-Trichlorophenoxyacetic acid monoclonal antibody
The preparation method of the monoclonal antibody for resisting 2,4, 5-trichlorophenoxyacetic acid comprises the following steps:
the BALB/C mice were immunized by emulsifying the 2,4, 5-trichlorophenoxyacetic acid artificial antigen, the carrier protein of which was hemocyanin in example 2, with an equal volume of Freund's adjuvant. The immunization dose of each mouse is 100 mu g, the immunization interval is 2 weeks, and after 3 times of immunization, tail venous blood of the mouse is taken to detect the serum titer. In the case of an antibody titer that did not meet the requirements, the booster immunization was carried out, and after the antibody titer did not increase any more, the subcutaneous booster immunization was carried out with 100. mu.g of whole antigen, and 5 days later, the mouse spleen cells were taken and fused with SP20 cells (mouse myeloma cells). The fused cells were selected in HAT medium, and after 5 days, the HAT medium was replaced with complete medium. And (3) detecting the cell supernatant by using ELISA, carrying out limited dilution method cloning culture on the cells in the holes with strong positive detection results, and detecting through 3 times of cloning culture, wherein the positive cells in the holes are hybridoma cells secreting the monoclonal antibody. After the hybridoma cells are subjected to amplification culture, the hybridoma cells are inoculated to the abdominal cavity of a mouse to generate ascites containing the antibody. The ascites is purified by an octanoic acid-ammonium sulfate precipitation method, and the monoclonal antibody with high purity and high specificity can be obtained after freeze drying.
Example 42 application and Effect evaluation of 4, 5-Trichlorophenoxyacetic acid Artificial antigen in ELISA
Using carbonate buffer solution with pH9.6 as coating diluent, diluting 2,4, 5-trichlorophenoxyacetic acid artificial antigen of which the carrier protein is bovine serum albumin in the embodiment 2 to 0.5 mu g/mL, adding the diluted artificial antigen into a polystyrene micropore plate according to 100 mu L/hole, coating overnight at 4 ℃, drying, adding phosphate buffer solution containing 1% BSA according to 250 mu L/hole, sealing for 1h at 37 ℃, drying, and then packaging and storing in vacuum.
Adding 100 mu L/well of 2,4, 5-trichlorophenoxyacetic acid standard solution (gradient diluted by phosphate buffer solution with pH 7.4) into a micropore ELISA plate coated with 2,4, 5-trichlorophenoxyacetic acid artificial antigen, correspondingly adding 50 mu L/well of the 2,4, 5-trichlorophenoxyacetic acid monoclonal antibody solution in the above example 3 (diluting the monoclonal antibody to 0.3 mu g/mL by phosphate buffer solution containing 0.05% sodium azide and pH7.4, and reacting for 0.5h at 37 ℃; after spin-drying, adding 250 mu L/hole of washing liquid, washing for 3 times and then drying by beating; then adding 100 mu L of enzyme-labeled secondary antibody per hole, and reacting for 0.5h at 37 ℃; washing for 3 times again, drying, adding 100 μ L/hole color development solution, and reacting at 37 deg.C for 15 min; the addition of 50. mu.L/well of 0.5M sulfuric acid was terminated and the OD per well was determined at a wavelength of 450nm using a microplate reader. The results are shown in table 1 below:
TABLE 1 ELISA test OD values of standard solutions of 2,4, 5-trichlorophenoxyacetic acid at different concentrations
By using the data in table 1, a standard curve is drawn by performing four-parameter Logistic curve fitting by using ELISA Calc software, the obtained standard curve is shown in fig. 3, and the linear equation of 2,4, 5-trichlorophenoxyacetic acid is as follows:
r2the concentration of the substance to be detected is defined as 0.999, A is 1.93124, B is 0.92268, C is 4.49748, D is 0.07451, x is the concentration of the substance to be detected, y is the OD value, and the IC50 value is calculated to be 4.9 mug/L and is in a linear relation of 1-81 mug/L.
From the above results, it was found that the IC50 value of 4.9. mu.g/L indicates that the 2,4, 5-trichlorophenoxyacetic acid artificial antigen and monoclonal antibody of the present invention have strong specificity in ELISA detection.
Example 52 preparation of fluorescence quantitative immunochromatography test paper for 4, 5-trichlorophenoxyacetic acid and evaluation of Performance
Preparation of 1.2,4, 5-trichlorophenoxyacetic acid fluorescence quantitative immunochromatographic test paper
The preparation method of the 2,4, 5-trichlorophenoxyacetic acid fluorescence quantitative immunochromatographic test paper comprises the following steps:
(1) preparation of a reaction membrane coated with artificial antigen and rabbit IgG:
adjusting the antigen of example 2 to 3 concentrations (0.2 mg/mL, 0.4mg/mL and 0.8mg/mL respectively) by using a nitrocellulose membrane (NC membrane) as a reaction membrane, adjusting the concentration of rabbit IgG to 0.25mg/mL by using a coating buffer solution, spraying the antigen and rabbit IgG of example 2 to a detection area (T line) and a control area (C line) corresponding to the reaction membrane according to the membrane liquid amount of 1uL/cm, wherein the interval between the detection area and the control area is 5mm, placing the reaction membrane in an oven at 40-45 ℃ for treatment for 16-35 hours, and placing the reaction membrane in a constant-temperature and constant-humidity preservation box for standby, wherein the coating buffer solution contains 0.5% PEG20000, 1% sucrose, 0.5% BSA and 0.05% sodium azide (NaN)3) 0.01M Phosphate Buffered Saline (PBS).
(2) Preparation of sample pad
And soaking the cut blank sample pad of 30 x 30cm in the sample pad treatment solution for 5min, and taking out and drying at 37 ℃ for 16 h. After drying, the four sides of the sample pad need to be cut, the cutting width is 0.7cm, the sample pad is placed in a constant-temperature constant-humidity preservation box for standby after cutting, and the sample pad treatment solution is 0.1M phosphate buffer solution containing 0.3% of Tween 20, 1% of sucrose, 0.5% of BSA and 0.05% of sodium azide.
(3) Assembly of fluorescent quantitative test paper
And (3) sequentially sticking the sample pad prepared in the step (2), the reaction membrane prepared in the step (1) and the water absorption pad on a back lining of the PVC board to obtain a test paper board, cutting the test paper board into test paper strips, and loading the test paper strips into the test paper card to obtain the fluorescent quantitative immunochromatographic test paper.
The fluorescent quantitative detection test paper prepared by the invention is further detected.
Performance evaluation detection of 2,4, 5-trichlorophenoxyacetic acid fluorescence quantitative immunochromatographic test paper
(1) Preparing fluorescent microsphere labeled protein solution:
taking 1.5ml of fluorescent microsphere solution (the solid content of the fluorescent microspheres is 1 percent, namely 15mg of the fluorescent microspheres), and carrying out ultrasonic treatment; stirring, respectively adding 15mg of NHS and 15mg of EDC, controlling the temperature of the fluorescent microsphere solution to be 4-10 ℃, and activating for 20min at the temperature; after activation, the reaction solution is centrifuged at 10000rpm for 10min, the supernatant is removed, MES buffer solution is added, ultrasonic resuspension is carried out, and then centrifugation and supernatant removal are carried out, and the operation is repeated for 3 times; after the last supernatant removal, re-dissolution with MES buffer, ultrasonic re-suspension, 2ml of the fluorescent microsphere solution was divided into 4 tubes of 500. mu.L each, and the 2,4, 5-trichlorophenoxyacetic acid monoclonal antibody of example 3 and goat anti-rabbit IgG were added as shown in Table 2.
TABLE 2 component proportion of fluorescent microsphere-labeled protein solution
| Volume of fluorescent microsphere solution | 500μL | 500μL | |
| Tagging items | |||
| 2,4, 5-trichlorophenoxyacetic acid |
2,4, 5-trichlorophenoxyacetic acid monoclonal antibody | Goat anti-rabbit IgG | |
| Marker concentration | 0.5mg/mL | 0.25mg/mL | 0.5mg/mL |
After adding the protein, uniformly stirring, removing the ice bath, naturally heating to room temperature, and stirring at room temperature for 4-6 h; after coupling, the reaction solution is centrifuged at 10000rpm for 10min, the supernatant is removed, 1mL of fluorescent buffer solution is added, after ultrasonic resuspension, the supernatant is centrifuged and removed, the operation is repeated for 3 times, the last centrifugation is carried out, after the supernatant is removed, 0.5mL of fluorescent buffer solution is added, the fluorescent microspheres are ultrasonically resuspended, and the mixture is stored in a refrigerator at the temperature of 2-8 ℃ for later use, wherein the used fluorescent buffer solution is 0.01M phosphate buffer solution PBS containing 0.5% of PEG20000, 2% of sucrose, 0.1% of Tween 20, 0.5% of BSA and 0.05% of sodium azide.
(2) Preparing a fluorescent microsphere labeled protein detection solution:
and (2) taking out the fluorescent microsphere labeled protein liquid in the step (1), placing the fluorescent microsphere labeled protein liquid to room temperature, diluting the fluorescent microsphere labeled protein liquid by 1000 times with a fluorescent buffer solution as the fluorescent microsphere labeled protein detection liquid, wherein the fluorescent buffer solution is shown in the step (1), and then placing the fluorescent microsphere labeled protein liquid in a refrigerator at the temperature of 2-8 ℃ for storage and standby.
(3) Preparing a 2,4, 5-trichlorophenoxyacetic acid fluorescence detection solution:
taking 10mL of fluorescence buffer solution to a 20mL brown glass bottle, respectively adding 100 μ L of 2,4, 5-trichlorophenoxyacetic acid (2, 4, 5-trichlorophenoxyacetic acid) 2,4, 5-trichlorophenoxyacetic acid monoclonal antibody labeling solution with the concentration of 0.25mg/mL (2, 4, 5-trichlorophenoxyacetic acid fluorescence detection solution 1) and 0.5mg/mL (2, 4, 5-trichlorophenoxyacetic acid fluorescence detection solution 2), adding 20 μ L of goat anti-rabbit IgG labeling solution with the concentration of 0.5mg/mL, uniformly mixing, and placing in a refrigerator at 2-8 ℃ for storage.
(4) And (3) testing the signal value of the fluorescent quantitative detection test paper:
after 20. mu.L of each of the prepared fluorescent microsphere labeled protein detection solutions was mixed with 100. mu.L of 0.01M phosphate buffer solution for 1min, 80. mu.L of each of the prepared fluorescent microsphere labeled protein detection solutions was added dropwise to three types of fluorescent quantitative test strips (coating concentrations: 0.2mg/mL, 0.4mg/mL, and 0.8mg/mL) with different coating concentrations prepared in example 5, and after 15min, the T-line signal value, the C-line signal value, and the ratio of the T/C fluorescent signal values were read by a fluorescent quantitative detector.
TABLE 3 detection effect of fluorescent quantitative test paper with coating concentration of 0.2mg/mL
| Fluorescent liquid detection liquid | Signal value of T line | C line signal value | Ratio of fluorescence signal values of T/ |
| 2,4, 5-trichlorophenoxyacetic acid fluorescence detection liquid 1 | 3280 | 2276 | 1.44 |
| 2,4, 5-trichlorophenoxyacetic acid |
5485 | 2215 | 2.48 |
TABLE 4 detection effect of fluorescent quantitative test paper with coating concentration of 0.4mg/mL
| Fluorescent liquid detection liquid | Signal value of T line | C line signal value | Ratio of fluorescence signal values of T/ |
| 2,4, 5-trichlorophenoxyacetic acid fluorescence detection liquid 1 | 8766 | 2330 | 3.76 |
| 2,4, 5-trichlorophenoxyacetic acid |
15017 | 2314 | 6.49 |
TABLE 5 test effect of fluorescent quantitative test paper with coating concentration of 0.8mg/mL
| Fluorescent liquid detection liquid | Signal value of T line | C line signal value | Ratio of fluorescence signal values of T/ |
| 2,4, 5-trichlorophenoxyacetic acid fluorescence detection liquid 1 | 16492 | 2309 | 7.14 |
| 2,4, 5-trichlorophenoxyAcetic acid |
34922 | 2147 | 16.3 |
And (4) displaying and comparing the signal value test results, and selecting the moderate signal value condition to perform uniformity test.
(5) And (3) evaluating the uniformity of the fluorescent quantitative detection test paper:
respectively adding 20 mu L0.01M phosphate buffer solution into a 10-tube 1.5mL centrifuge tube, respectively adding 100 mu L2, 4, 5-trichlorophenoxyacetic acid fluorescence detection solution 2 into the centrifuge tube, mixing for 1 minute, then taking 80 mu L and dropwise adding onto fluorescence quantitative detection test paper with antigen concentration of 0.4mg/mL, reacting for 15 minutes, reading the ratio of the fluorescence signals of T/C by using a fluorescence quantitative detector, recording data, and calculating the coefficient of variation of 10 results.
TABLE 6 evaluation of homogeneity of the fluorescent quantitative test paper
(6) Evaluating the sensitivity detection effect of the fluorescent quantitative detection test paper:
preparing a series of standard solutions of 2,4, 5-trichlorophenoxyacetic acid with different concentrations (0, 3, 9, 27, 81 and 243 mu g/L) by using 0.01M phosphate buffer solution, then taking 20 mu L of the fluorescent microsphere labeled protein detection solution prepared in the step (2), mixing the fluorescent microsphere labeled protein detection solution with 100 mu L of the prepared series of standard solutions for 1min, dripping 80 mu L of the mixed solution onto the prepared fluorescent quantitative detection test paper, and reading the ratio of the fluorescent signal values of T/C by using a fluorescent quantitative detector after 15 min. And then establishing a four-parameter Logistic curve through T/C values corresponding to the concentrations of the series of standard products, and inputting four parameter values obtained by the curve into calibration software of the fluorescence quantitative detection instrument, so that the rapid quantitative detection of the fluorescence quantitative detection test paper on the fluorescence immunochromatographic analyzer can be realized.
The T/C values of different 2,4, 5-trichlorophenoxyacetic acid concentrations were measured, and B/B0 (the ratio of the T/C value of the standard test card at different concentrations to the T/C value of the standard test card at 0 content) was calculated, with the results shown in Table 7. As can be seen from the table, when the content of the 2,4, 5-trichlorophenoxyacetic acid is 3 mug/L, the B/B0 value is 74.31%, which indicates that the T/C value detected under the concentration is obviously different from the T/C value of the test card containing the concentration of 0 mug/L, so that the detection sensitivity of the fluorescence quantitative detection test paper on the 2,4, 5-trichlorophenoxyacetic acid can reach 3 mug/L.
TABLE 7 measurement results of 2,4, 5-trichlorophenoxyacetic acid with fluorescent quantitative test paper
By using the data in the table above, a standard curve (fig. 4) is drawn by performing four-parameter Logistic curve fitting by using ELISA Calc software, and the linear equation of the obtained curve is as follows:
r20.998, 6.55744 for A, 0.82884 for B, 9.25363 for C and 0.20595 for D, x in the linear equation represents the concentration of the substance to be measured, and y represents the value of T/C. In the quantitative curve setting interface of the fluorescence immunochromatographic analyzer, the values of A, B, C and D are respectively recorded into the calibration software of the fluorescence immunochromatographic analyzer, so that the rapid quantitative detection of the fluorescence quantitative detection test paper on the fluorescence immunochromatographic analyzer can be realized.
(7) Stability test of fluorescent microsphere labeling liquid and fluorescent quantitative detection test paper
And carrying out an aging experiment on the fluorescent quantitative detection test paper and the fluorescent microglobulin test solution. The fluorescence quantitative detection test paper is respectively sealed and placed in a thermostat with the room temperature and the 50 ℃ for comparison experiments. And respectively storing the fluorescent microsphere labeled protein detection solution in an environment with the temperature of 2-8 ℃ and 37 ℃.
TABLE 8 preservation effect of fluorescent microsphere labeled protein detection solution at 2-8 deg.C and 37 deg.C
TABLE 9 preservation effect of the test paper in a thermostat at room temperature and 50 deg.C
The test result shows that the fluorescent microsphere labeling solution and the fluorescent quantitative detection test paper in the embodiment of the invention have excellent stability and are not easily interfered by temperature.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A2, 4, 5-trichlorophenoxyacetic acid hapten, wherein the hapten has the structure shown in formula I:
2. the method of preparing a hapten for 2,4, 5-trichlorophenoxyacetic acid of claim 1, comprising the steps of:
(1) reacting the mixed compound a, the compound b, a solvent and an acid-binding agent, removing the solvent, adding water for mixing, and extracting by using petroleum ether and ethyl acetate in sequence to obtain a compound c;
(2) mixing the compound c, a solvent, zinc and hydrochloric acid for reaction, adding water, extracting with ethyl acetate, adjusting the pH to 8-9 with a pH regulator, and extracting with ethyl acetate again to obtain a compound d;
(3) mixing the compound d, a solvent, lithium hydroxide and water for reaction, then adding a sodium chloride solution, extracting with dichloromethane, taking a water phase, and adjusting the pH of the water phase to 4-5 by using a pH regulator to obtain the 2,4, 5-trichlorophenoxyacetic acid hapten;
wherein the structural formula of the compound a is shown as formula II:
the structural formula of the compound b is shown as the formula III:
the structural formula of the compound c is shown as a formula IV:
the structural formula of the compound d is shown as the formula V:
3. the method of claim 2, wherein the solvent is selected from the group consisting of acetone, ethanol; the acid-binding agent is selected from potassium carbonate.
4. The preparation method according to claim 2, wherein the reaction conditions in the step (1) are 60-70 ℃ for more than 5 hours; the reaction condition in the step (2) is room temperature reaction for more than 2 hours; the mixing condition in the step (3) is room temperature reaction for more than 16 h.
5. An artificial antigen, wherein the structural formula of the artificial antigen is shown in VI:
wherein the protein is a carrier protein.
6. The artificial antigen according to claim 5, wherein the carrier protein is selected from any one of bovine serum albumin, ovalbumin, human serum albumin, and hemocyanin.
7. The method for producing the artificial antigen according to claim 5 or 6, comprising the steps of:
(1) taking the hapten, the activator and the protein carrier of claim 1, and reacting for 16-24 hours;
(2) dialyzing and separating to obtain the artificial antigen;
wherein the activating agent is glutaraldehyde.
8. Use of the artificial antigen of claim 5 or 6 in the preparation of a 2,4, 5-trichlorophenoxyacetic acid monoclonal antibody.
9. Use of the artificial antigen of claim 5 or 6 in an ELISA assay.
10. A fluorescence quantitative immunochromatographic test paper for detecting 2,4, 5-trichlorophenoxyacetic acid, which is characterized in that the fluorescence quantitative immunochromatographic test paper is coated with the artificial antigen of claim 5 or 6.
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| CN108727204A (en) * | 2018-05-29 | 2018-11-02 | 上海交通大学 | A kind of preparation method and its usage of 2,4,4 '-tribromo Biphenyl Ether haptens |
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| CN112047837A (en) * | 2020-08-10 | 2020-12-08 | 广东达元绿洲食品安全科技股份有限公司 | 4-chlorophenoxyacetic acid hapten, artificial antigen and application thereof in immunoassay |
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