CN116041211A - Rapid detection device for malachite green in aquatic product, and preparation and application thereof - Google Patents
Rapid detection device for malachite green in aquatic product, and preparation and application thereof Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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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
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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
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- 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
- G01N33/577—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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- 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
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
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- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a rapid detection device for malachite green in an aquatic product and preparation and application thereof, and relates to malachite green hapten, antigen, antibody, rapid detection device and preparation and application thereof in detection of malachite green in the aquatic product. The preparation method of the malachite green hapten provided by the invention has the advantages of easiness in obtaining the used chemical reagent, simple operation process, concise and effective synthesis steps, higher reaction yield and lower cost. According to the invention, the artificial antigen is prepared by using the malachite green hapten coupled carrier protein, and after an experimental animal is immunized, a monoclonal antibody aiming at the malachite green with high specificity is generated by the organism of the experimental animal, the malachite green is specifically detected, cross reaction with malachite green structural analogues can not occur, false positive results are avoided, whether the malachite green remains in the aquatic product is qualitatively detected through the ELISA detection kit, and the malachite green is specifically detected, so that the method has the advantages of good specificity, high sensitivity, simplicity and convenience in operation, capability of rapidly detecting on site and the like.
Description
Technical Field
The invention relates to the technical field of immunological detection of food safety, in particular to a malachite green hapten, an antigen, an antibody, a detection device and preparation and application thereof, wherein the detection device is particularly suitable for rapid detection of malachite green residues in aquatic products.
Background
Malachite Green (MG): the green crystal is also called alkaline green and salt block green, and is a green crystal with metallic luster. Because of its very effective effect in the treatment of saprolegniasis, it has been used in the fishery over a large area. However, research shows that malachite green and its metabolite, leucomalachite green, have high toxicity, high residue, high carcinogenicity, high teratogenicity, mutation and other side effects, so the use of malachite green has increased the supervision force worldwide. However, because malachite green is cheap and has no good substitute in the aspect of saprolegniasis prevention and treatment, some illegal vendors still use the malachite green, so that malachite green events still occur, and serious influence is caused on national economy and people health.
Patent publication numbers CN103012193A, CN108640850A and CN108640850A both disclose a synthesis method of malachite green hapten, namely, active arms with different electron cloud densities, different carbon chain lengths and suitable for macromolecular carrier coupling are derived at different chemical sites of malachite green structure. The leucomalachite green hapten is used for preparing malachite green artificial antigen and antibody, and is applied to detecting residual amount of leucomalachite green and malachite green, but the antibody has partial cross reaction on malachite green structural analogues, has low specificity, and is easy to generate false positive in actual rapid detection, so that the detection result is inaccurate. Therefore, there is an urgent need to develop a new and simple method for synthesizing malachite green hapten, and to prepare an artificial antibody which is highly specific to malachite green and specifically detects malachite green by using the plate antigen.
Disclosure of Invention
The invention aims to provide a rapid detection device for malachite green in an aquatic product, and preparation and application thereof, and aims to detect the residual malachite green in the aquatic product.
According to one aspect of the present invention there is provided a malachite green hapten of formula (I):
according to another aspect of the present invention there is provided a method of preparing malachite green hapten comprising the steps of:
s1, generating an intermediate 1 with a fatty chain structure by N-methylaniline and methyl acrylate under alkaline conditions, wherein the structural formula of the intermediate 1 is shown as a formula (II):
s2, the intermediate 1, benzaldehyde and N, N-dimethylaniline are catalyzed by zinc chloride to obtain an intermediate 2, and the structural formula of the intermediate 2 is shown as a formula (III):
s3, hydrolyzing the intermediate 2 under a strong alkaline condition to obtain a hidden malachite green hapten with an arm, namely an intermediate 3, wherein the structural formula of the intermediate 3 is shown as a formula (IV):
s4, the intermediate 3 is rapidly converted into malachite green hapten under the action of strong oxidant 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone, and the malachite green hapten is obtained through column chromatography purification, wherein the structural formula of the malachite green hapten is shown as a formula (I).
In some embodiments, the molar ratio of N-methylaniline to methyl acrylate in step S1 is 1 (1-5), the basic conditions in step S1 are provided by one or more of triethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate, and the molar ratio of intermediate 1 to benzaldehyde, N-dimethylaniline in step S2 is 1 (1-1.1): 1-1.1.
In some embodiments, the strongly basic conditions in step S3 are provided by a solution of a strongly basic compound, which is a sodium hydroxide solution, lithium hydroxide solution, or potassium hydroxide solution, the molar ratio of intermediate 2 to the strongly basic compound in step S3 is 1 (1.1-5), and the molar ratio of intermediate 3 to 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone in step S4 is 1 (1-2).
According to a further aspect of the invention, the malachite green antigen is a conjugate of malachite green hapten and a carrier protein, the carrier protein being one of bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin.
According to a fourth aspect of the invention, the malachite green antibody is prepared from malachite green antigen by animal immunization, and the malachite green antibody is an anti-malachite green monoclonal antibody.
According to a fifth aspect of the present invention there is provided the use of malachite green hapten or malachite green antigen in immunological detection of malachite green.
According to a sixth aspect of the invention there is provided a malachite green enzyme-linked immunosorbent assay kit comprising an enzyme-labeled plate coated with malachite green antigen, a malachite green antibody, an enzyme-labeled secondary antibody, an antibody diluent, malachite green standard solutions of different concentrations and a wash solution.
According to a seventh aspect of the present invention there is provided a method of detecting malachite green residue in a sample, the method comprising detecting malachite green in the sample using a malachite green enzyme-linked immunosorbent assay kit, the sample being an aquatic product.
The invention has the beneficial effects that: the preparation method of the malachite green hapten provided by the invention has the advantages of easiness in obtaining the used chemical reagent, simple operation process, concise and effective synthesis steps, higher reaction yield and lower cost. According to the invention, the artificial antigen is prepared by using the malachite green hapten coupled carrier protein, and after an experimental animal is immunized, a body of the animal generates a monoclonal antibody aiming at malachite green with high specificity, so that the malachite green is specifically detected, cross reaction with malachite green structural analogues can not occur, and false positive results are avoided. The invention uses the ELISA kit to qualitatively detect whether the malachite green is remained in the aquatic products, and the invention can achieve the purpose of rapid detection without using large-scale instruments such as liquid chromatography or mass spectrum, and the like, and can specifically detect the residual malachite green in the aquatic products, and has the advantages of high specificity, high sensitivity, accurate detection result, simple and convenient operation, and the like, and can realize on-site rapid detection.
Drawings
FIG. 1 is a mass spectrum of malachite green hapten according to one embodiment of the present invention.
FIG. 2 is a synthetic route pattern for malachite green hapten according to one embodiment of the present invention.
FIG. 3 is a graph of an indirect competition ELISA standard established based on malachite green monoclonal antibodies according to one embodiment of the invention.
Detailed Description
The invention will be described in further detail with reference to specific embodiments thereof, it being understood that these embodiments are for purposes of illustration only and not for purposes of limiting the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the invention, and are defined in the claims appended hereto. Unless otherwise specified, all materials and reagents of the invention are those commercially available in the conventional market.
Example 1 method for preparing malachite green hapten comprising the steps of:
s1, weighing 7g (1 eq) of N-methylaniline, placing the N-methylaniline into a 250ml single-mouth bottle, adding 100ml of absolute methanol, uniformly stirring, adding 17g (3 eq) of methyl acrylate and 3ml of triethylamine, carrying out oil bath at 85 ℃, reacting overnight, cooling the reaction solution to room temperature, concentrating, directly stirring, and purifying by column (PE: EA=50:1; EA) to obtain an intermediate 1;
s2, placing 1eq of intermediate 1 into a 250ml single-mouth bottle, adding 100ml of absolute ethyl alcohol, 4.6ml (1 eq) of benzaldehyde, 5.7ml (1 eq) of N, N-dimethylaniline, 20g (about 3 eq) of zinc chloride, carrying out oil bath at 100 ℃, reacting overnight, cooling the reaction solution to room temperature, concentrating, adding a proper amount of distilled water for dilution, adjusting the pH to about 8 by using a 10% sodium hydroxide aqueous solution, adding a proper amount of ethyl acetate for full stirring, separating liquid after suction filtration, washing an organic phase once by using water, concentrating the organic phase, purifying by a column, and finally obtaining 2g of intermediate 2;
s3, placing 2g of the intermediate 2 into a 100ml single-port bottle, adding 10ml of methanol for dissolution, adding 10ml of 10% sodium hydroxide aqueous solution, carrying out oil bath reaction at 70 ℃ for overnight, cooling the reaction liquid to room temperature, concentrating away part of solvent, adding a proper amount of distilled water for dilution, adjusting the pH to about 8 with dilute hydrochloric acid, extracting twice with ethyl acetate, merging organic phases, and purifying to obtain an intermediate 3;
s4, placing all the intermediate 3 into a 100ml single-mouth bottle, adding 20ml acetonitrile for dissolution, slowly adding 10ml acetonitrile solution dissolved with 1.1eq of 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) while stirring, enabling the color of the reaction solution to be deepened immediately, stirring for reaction for 0.5H, and directly stirring a sample and passing through a column to obtain a target product MG-H1, namely malachite green hapten.
The malachite green hapten is identified by mass spectrometry, and the obtained mass spectrum is shown in figure 1 of the specification. As can be seen from the mass spectrum, the molecular ion peak of malachite green hapten is M/z 386.4 [ M-H ]] - And is the highest peak, which is consistent with the molecular weight 387.49 of the malachite green hapten, indicating that the malachite green hapten represented by formula (I) was successfully synthesized.
EXAMPLE 2 preparation of antigen for malachite Green immunization and antigen for coating
Preparation of antigen for malachite green immunization: 30mg of malachite green hapten prepared in example 1 is weighed, dissolved in 2ml of DMF, added with 20mg of NHS and 22mg of EDC.Cl, and reacted overnight at room temperature to prepare an activated liquid; weighing 120MG of Bovine Lactoferrin (BLF), dissolving in 6ml of 0.1M boric acid buffer solution (pH 9.0), adding 1ml of DMF and 1.34ml of the activating solution, reacting for 5 hours, dialyzing for 2 to 3 days by PBS (0.01 mol/L, pH=7.4 phosphate buffer solution), changing the solution for 1 time every 4H, centrifuging for 5 minutes at 4000 revolutions per minute after dialysis, taking the supernatant, and preparing to obtain the malachite green antigen (MG-H1-BLF), namely the malachite green hapten-BLF conjugate, subpackaging, and storing at the temperature of minus 20 ℃.
Preparation of antigen for malachite green coating: dissolving 60mg of bovine serum albumin BSA in 1ml of water, adding 40ul of 1N NaOH solution, adding 30ul of 1, 4-butanediol diglycidyl ether, reacting for 12 hours at room temperature, adding 20ul of 1N NaOH solution, reacting for 12 hours again, dialyzing by using normal saline, and changing the solution once in the middle to prepare a reaction solution; 12mg of basic fuchsin was dissolved in 1ml of water, and 0.8ml of 0.4M Na was added 2 Adding HPO4 solution into the reaction solution, reacting at room temperature for 3 days, dialyzing with PBS (0.01 mol/L, phosphate buffer solution with pH=7.4) for 2-3 days, centrifuging to obtain malachite green coated antigen (MG-BSA), namely malachite green hapten-BSA conjugate, subpackaging, and storing at-20deg.C.
EXAMPLE 3 preparation and purification of Malachite Green monoclonal antibodies
3.1 immunization of animals
Healthy BALB/c mice with the age of 6-8 weeks are selected for immunization, and after the antigen for malachite green immunization prepared in the example 2 is mixed and emulsified in equal quantity with Freund's complete adjuvant, the BALB/c mice are subjected to subcutaneous multipoint injection immunization (except sprint immunization) at the back of the neck. The first immunization is carried out by using complete Freund's adjuvant, and the dosage is 150 mug/dose; boosting is carried out after 4 weeks at a dose of 75 mug/dose, the mixture is mixed and emulsified by incomplete Freund's adjuvant, and then the boosting is carried out for a plurality of times for 3 weeks; the dosage is halved again during the sprint immunization, which is 37.5 mug/dose, the complete antigen is diluted by normal saline, the intraperitoneal injection is carried out on the mice, the tail breaking blood sampling detection can be carried out after the third immunization of the mice, and the titer and the IC of the serum of the mice are detected by an indirect competition enzyme-linked immunosorbent assay (IC-ELISA) 50 High selective potency, IC 50 Low mice were fused.
3.2 cell fusion and cloning
Spleen cells of immunized BALB/c mice were taken at a ratio of 9:1 are fused with SP2/0 myeloma cells, and malachite green monoclonal hybridoma cell strains which stably secrete malachite green monoclonal antibodies are obtained through screening.
3.3 cryopreservation and resuscitation of cells
Freeze-stored solution for preparing malachite green monoclonal hybridoma cells into 1X 10 6 Cell suspensions of individual/mL were stored in liquid nitrogen for long periods. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.
3.4 preparation and purification of monoclonal antibodies
Incremental culture method: placing the malachite green monoclonal hybridoma cells in a cell culture medium, culturing at 37 ℃, purifying the obtained culture solution by an octanoic acid-saturated ammonium sulfate method to obtain the malachite green monoclonal antibody, and preserving at-20 ℃. Wherein, the cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI-1640 culture medium, the weight percentage of the calf serum in the cell culture medium is 15-20%, the weight percentage of the sodium bicarbonate in the cell culture medium is 0.1-0.2%, and the pH of the cell culture medium is 7.4. The sensitivity of detection of the malachite green antibody by means of iceelisa was 0.19ng/ml.
Example 4 experiments specific for malachite green antibodies and Cross-reactions
The test uses the iceelisa procedure to select several analogues with structural similarity to malachite green under the optimized iceelisa conditions and examine the specificity of the malachite green monoclonal antibodies prepared in example 3. The competition inhibition curves of the analogues of malachite green were sequentially made, the standard mass concentration was calculated at 50% inhibition, and then converted to molar concentration, and the cross-reactivity of each structural analogue with the antibody was calculated using the formula, see table 1 below, with 3 replicates per treatment set.
TABLE 1 Cross-reactivity results of malachite antibodies and malachite green structural analogues
From Table 1, it can be seen that the malachite green monoclonal antibody does not cross react with malachite green structural analogues, which indicates that the antibody has high specificity to malachite green and can specifically detect malachite green.
Example 5 construction of malachite green ELISA kit
5.1 Preparation of enzyme-labeled secondary antibody: goat is used as an immunized animal, and the pathogen-free goat is immunized by using a mouse protein, so that the goat is an anti-mouse antibody. And (3) coupling the goat anti-mouse antibody with horseradish peroxidase (HRP) to obtain the enzyme-labeled secondary antibody.
5.2 Preparing an ELISA plate: the malachite green coating antigen (MG-BSA) prepared in example 2 was diluted to 2. Mu.g/mL with a coating buffer (pH 9.6 carbonate solution), 100. Mu.l of each well was added, incubated at 4℃for 16h in the dark, the liquid in the well was decanted, washed 1 time with the washing solution, left to stand for 30s, and then dried, 200. Mu.l of a blocking solution was added to each well, incubated at 37℃for 2h in the dark, the liquid in the well was decanted, dried and stored in a vacuum sealed with an aluminum film.
5.3 The malachite green ELISA kit comprises the following components:
a. an ELISA plate coated with malachite green coating antigen MG-BSA;
b. 6 bottles of malachite green standard substance concentrated solution (acetonitrile is used as a solvent of the concentrated solution), wherein the concentration of malachite green is 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L respectively, and when the malachite green standard substance concentrated solution is used, the malachite green standard substance concentrated solution is diluted into a standard substance solution by 0.02mol phosphate buffer solution (pH 7.2) according to the volume ratio of 9:1, and the concentration after dilution is 0 mug/L, 0.01 mug/L, 0.03 mug/L, 0.09 mug/L, 0.27 mug/L and 0.81 mug/L respectively;
c. malachite green antibodies prepared in example 3;
d. the substrate color development liquid consists of liquid A and liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
e. the stop solution is 2mol/L sulfuric acid;
f. the washing solution is phosphate buffer solution with pH=7.2 and containing 0.01% (weight volume percentage) Tween-20, 3g/L sodium azide preservative and 0.01 mol/L;
g. the complex solution was phosphate buffer solution with ph=7.0 and 0.02 mol/L.
h. Goat anti-mouse enzyme-labeled secondary antibody
Example 6 method for detecting malachite green residue
6.1 the sample is fish or shrimp
1) Taking 1g of homogeneous boneless fish or shrimp meat sample, adding the sample into a 50ml centrifuge tube, adding 0.3ml of acetonitrile and 6ml of ethyl acetate, and sufficiently shaking (not less than 5 minutes to ensure that the sample does not agglomerate);
2) Centrifuging at room temperature at 4000r/min for 10min, collecting supernatant 3ml, adding into 10ml glass test tube, adding oxidant DDQ 50ul, and shaking for 2min;
3) Adding 50ul of cosolvent acetonitrile into each tube, drying with nitrogen in a water bath environment at 50 ℃ without oscillation (a drop of liquid substance should be arranged at the bottom of the tube after the blowing is finished), and when the grease content of a sample is too high, yellow viscous liquid drops remain at the bottom of the test tube;
4) Adding 1ml of sample complex solution, dissolving and mixing uniformly, taking 50ul of the mixed solution as a liquid to be detected, and analyzing and detecting the liquid to be detected by using the malachite green ELISA kit prepared in the example 5.
6.2 the sample is a water sample:
taking 1ml of water sample, adding 50ul of oxidant, and vibrating for 2min to obtain a liquid to be detected, and analyzing and detecting the liquid to be detected by using the malachite green ELISA kit prepared in the example 5.
The foregoing is merely illustrative of some embodiments of the invention, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concept.
Claims (9)
1. The malachite green hapten is characterized in that the malachite green hapten has a structure as shown in a formula (I):
2. a method of preparing the malachite green hapten of claim 1, comprising the steps of:
s1, generating an intermediate 1 with a fatty chain structure by N-methylaniline and methyl acrylate under alkaline conditions;
s2, reacting the intermediate 1 with benzaldehyde and N, N-dimethylaniline under the catalysis of zinc chloride to obtain an intermediate 2;
s3, hydrolyzing the intermediate 2 under a strong alkaline condition to obtain a hidden malachite green hapten with an arm, namely an intermediate 3;
s4, the intermediate 3 is rapidly converted into malachite green hapten under the action of strong oxidant 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone, and the malachite green hapten is obtained through column chromatography purification, wherein the structural formula of the malachite green hapten is shown as a formula (I).
3. The method according to claim 2, wherein the molar ratio of N-methylaniline to methyl acrylate in the step S1 is 1 (1-5), the alkaline condition in the step S1 is provided by one or more of triethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate, and the molar ratio of the intermediate 1 to benzaldehyde and N, N-dimethylaniline in the step S2 is 1 (1-1.1): 1-1.1.
4. The method according to claim 2, wherein the strongly basic conditions in step S3 are provided by a solution of a strongly basic compound, which is a sodium hydroxide solution, a lithium hydroxide solution or a potassium hydroxide solution, the molar ratio of intermediate 2 to the strongly basic compound in step S3 is 1 (1.1-5), and the molar ratio of intermediate 3 to 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone in step S4 is 1 (1-2).
5. The malachite green antigen is characterized in that the malachite green antigen is a conjugate of the malachite green hapten and a carrier protein, wherein the carrier protein is one of bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin.
6. The malachite green antibody is prepared from the malachite green antigen according to claim 5 through animal immunization, and the malachite green antibody is a malachite green monoclonal antibody.
7. Use of the malachite green hapten of claim 1 or the malachite green antigen of claim 5 in immunological detection of malachite green.
8. The malachite green enzyme-linked immunosorbent assay kit is characterized by comprising an ELISA plate coated with the malachite green antigen as claimed in claim 5, a malachite green antibody, an ELISA secondary antibody, an antibody diluent, malachite green standard substance solutions with different concentrations and washing solutions as claimed in claim 7.
9. A method for detecting malachite green residue in a sample, which is characterized in that the malachite green in the sample is detected by using the malachite green enzyme-linked immunosorbent assay kit as claimed in claim 8.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060211792A1 (en) * | 2003-08-01 | 2006-09-21 | Laursen Bo W | Triangulenium fluorescent dyes and polymers comprising such dyes |
| US20070072242A1 (en) * | 2005-09-22 | 2007-03-29 | Randox Laboratories Limited | Immunoassay method and kit to leucomalachite green and malachite green |
| CN103288661A (en) * | 2012-03-03 | 2013-09-11 | 北京勤邦生物技术有限公司 | Preparation method and application of malachite green hapten |
| CN105503632A (en) * | 2016-01-08 | 2016-04-20 | 广州润坤生物科技有限公司 | Preparation method and application of hapten and artificial antigen capable of being used for detecting crystal violet and malachite green together |
| CN108640850A (en) * | 2018-05-14 | 2018-10-12 | 广东达元绿洲食品安全科技股份有限公司 | A kind of malachite green hapten, artificial antigen and its application in fluorescent quantitation immunochromatography |
| CN113896651A (en) * | 2021-12-06 | 2022-01-07 | 信达安检测技术(天津)有限公司 | Synthesis method of dominant malachite green hapten and application of hapten |
-
2023
- 2023-04-03 CN CN202310345334.1A patent/CN116041211B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060211792A1 (en) * | 2003-08-01 | 2006-09-21 | Laursen Bo W | Triangulenium fluorescent dyes and polymers comprising such dyes |
| US20070072242A1 (en) * | 2005-09-22 | 2007-03-29 | Randox Laboratories Limited | Immunoassay method and kit to leucomalachite green and malachite green |
| CN103288661A (en) * | 2012-03-03 | 2013-09-11 | 北京勤邦生物技术有限公司 | Preparation method and application of malachite green hapten |
| CN105503632A (en) * | 2016-01-08 | 2016-04-20 | 广州润坤生物科技有限公司 | Preparation method and application of hapten and artificial antigen capable of being used for detecting crystal violet and malachite green together |
| CN108640850A (en) * | 2018-05-14 | 2018-10-12 | 广东达元绿洲食品安全科技股份有限公司 | A kind of malachite green hapten, artificial antigen and its application in fluorescent quantitation immunochromatography |
| CN113896651A (en) * | 2021-12-06 | 2022-01-07 | 信达安检测技术(天津)有限公司 | Synthesis method of dominant malachite green hapten and application of hapten |
Non-Patent Citations (1)
| Title |
|---|
| 许春向等: "《现代卫生化学》", vol. 1, 人民卫生出版社, pages: 612 - 614 * |
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