CN118146146A - Hapten for detecting flunixin meglumine content and application thereof - Google Patents
Hapten for detecting flunixin meglumine content and application thereof Download PDFInfo
- Publication number
- CN118146146A CN118146146A CN202410578715.9A CN202410578715A CN118146146A CN 118146146 A CN118146146 A CN 118146146A CN 202410578715 A CN202410578715 A CN 202410578715A CN 118146146 A CN118146146 A CN 118146146A
- Authority
- CN
- China
- Prior art keywords
- flunixin meglumine
- content
- detecting
- hapten
- solution
- Prior art date
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- Granted
Links
- 229960000469 flunixin meglumine Drugs 0.000 title claims abstract description 123
- MGCCHNLNRBULBU-WZTVWXICSA-N flunixin meglumine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.C1=CC=C(C(F)(F)F)C(C)=C1NC1=NC=CC=C1C(O)=O MGCCHNLNRBULBU-WZTVWXICSA-N 0.000 title claims abstract description 123
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- 238000006243 chemical reaction Methods 0.000 claims description 22
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 21
- 239000000126 substance Substances 0.000 claims description 14
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- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
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- 108010078791 Carrier Proteins Proteins 0.000 claims description 8
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
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- 150000001875 compounds Chemical class 0.000 claims description 4
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- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 3
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Classifications
-
- 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
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/80—Acids; Esters in position 3
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/803—Processes of preparation
-
- 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
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Abstract
The application discloses a hapten for detecting the content of flunixin meglumine and application thereof, in particular relates to a hapten for detecting the content of flunixin meglumine and a preparation method thereof, and also discloses application of the hapten in the aspects of antibodies and kits. The ELISA kit established by hapten for detecting the content of the flunixin meglumine can realize rapid detection of a large number of samples to be detected, is convenient to use, low in detection cost, high in efficiency, accurate and rapid in detection method, can detect a large number of samples simultaneously, and is suitable for on-site monitoring of the flunixin meglumine in liquid milk and screening of a large number of samples.
Description
Technical Field
The application relates to the technical field of veterinary drug residue detection, in particular to a hapten for flunixin meglumine content detection and application thereof.
Background
Flunixin meglumine (Flunixin meglumine, FM) is the only animal-specific non-steroidal anti-inflammatory drug, and is formed by combining the solubilizing agent meglumine and flunixin in a 1:1 form. FM is a powerful cyclooxygenase inhibitor, and selectively inhibits cyclooxygenase to prevent thromboxane and inflammatory substances such as prostate, thereby having the effects of diminishing inflammation, relieving fever and easing pain. Is used for relieving inflammation and angina caused by viscera, muscle, skeleton, breast, uterus disorder of pig, horse and cattle, controlling various acute inflammation caused by infectious diseases, treating canine endotoxemia, etc. The medicine has the characteristics of small dosage, rapid absorption, long duration, obvious curative effect, light adverse reaction and the like, is deeply favored by veterinarians, and is the non-steroidal anti-inflammatory medicine with the largest clinical consumption of the veterinarians at home and abroad at present. FM is easily metabolized and eliminated in vivo, and about 90% of the original drug is excreted from the body through urine and feces 24 hours after administration, and its main metabolites are 4-hydroxyflunixin, 5-hydroxyflunixin and 2-methylhydroxyl, wherein 5-hydroxyflunixin is identified as a labeled residue in cow's milk.
Over the last two decades, many studies have demonstrated that FM and other non-steroidal anti-inflammatory drugs remain in foods of animal origin that can constitute a potential risk to human health, such as gastrointestinal damage, hematopoietic and renal system toxicity, hepatotoxicity, and aseptic meningitis. In order to reduce these toxic effects, european Union, the United states, canada, japan and China determined that the Maximum Residual Limit (MRLs) of flunixin meglumine and its metabolite 5-hydroxyflunixin in animal tissues and milk is 6-60 μg/kg. Therefore, the establishment of a rapid and effective detection method is important for the detection of the residues of FM and metabolites thereof in animal-derived foods.
The method for detecting the flunixin meglumine is mainly an instrument method such as a liquid chromatography method, a liquid chromatography mass spectrometry method and the like. The instrument analysis method has the advantages of high detection sensitivity, strong specificity and the like, but the pretreatment of the detection sample is tedious and time-consuming, the sample also needs to be extracted and purified, and meanwhile, the instrument detection method needs expensive large-scale instruments and equipment, is provided with professional detection technicians for operation and management, cannot perform on-site large-scale detection, has poor timeliness and is difficult to popularize.
The immunological detection analysis technology has been widely used in the field of drug residue detection by virtue of its advantages of high sensitivity, high specificity, rapidness, simple operation, etc., and has many advantages compared with the instrument detection method. However, the lack of detection reagents with specific reactions to flunixin meglumine in the prior art prevents the application of immunoassay detection methods in flunixin meglumine detection.
When an immunological detection method is established and the detection method is applied to detect the flunixin meglumine, the key technology is that the antibody with strong specificity and high sensitivity can be obtained, and the aim is to be fulfilled, provided that the proper flunixin meglumine hapten needs to be synthesized and prepared.
Disclosure of Invention
The application provides a hapten for detecting the content of flunixin meglumine and application thereof, which are used for solving the problems that the sample pretreatment is complicated, the detection efficiency is low and the rapid mass detection cannot be realized in the flunixin meglumine detection method in the prior art; the technical problem of the ELISA hapten with specificity aiming at flunixin meglumine is lacking.
The application provides a hapten for detecting the content of flunixin meglumine, which has the chemical structural formula:
Formula (1);
The preparation method of the hapten for detecting the content of the flunixin meglumine comprises the following steps:
2-amino-6- (trifluoromethyl) phenylpropionic acid is used as a raw material to carry out nucleophilic substitution reaction with 2-chloronicotinic acid, so as to obtain the hapten for detecting the content of the flunixin meglumine.
The hapten with the structural formula can be treated according to a common method in the prior art to prepare an antibody, and the antibody can generate a specific reaction on flunixin meglumine contained in the pretreated liquid milk. According to the characteristics, the flunixin meglumine standard substance can be subjected to gradient dilution to obtain flunixin meglumine standard substance solutions with different concentrations, an enzyme-labeled instrument is adopted to measure the OD value of each standard substance solution at 450nm, then pretreatment is carried out on a sample to be measured, the OD value at the same wavelength is measured, and the corresponding concentration is obtained through the standard curve, so that the quantitative effective and rapid detection of the content of the flunixin meglumine in the sample is realized.
The hapten with higher purity can be obtained by adopting the reaction. The reagents and reaction conditions used in the preparation method can be selected according to the reagents and conditions required by the existing nucleophilic substitution reaction.
The sensitivity of the kit prepared from the hapten for detecting the flunixin meglumine contained in the liquid milk is 0.1 mug/L, which indicates that the kit prepared from the hapten has higher detection sensitivity for the flunixin meglumine; after the kit is prepared according to the prior method, the detection limit of the flunixin meglumine in the liquid milk is 0.1 mug/L, and the kit can be used for effectively detecting the content of the flunixin meglumine in the liquid milk; after the hapten is prepared into a kit, the recovery rate of the flunixin meglumine is 100% +/-20%, the intra-batch variation coefficient is less than 10%, the inter-batch variation coefficient is less than 15%, the accuracy is good, and the detection requirement can be met.
After the hapten is prepared into a kit, the cross reactivity of the hapten to other nonsteroidal anti-inflammatory drugs is less than 1%, the cross reactivity of the hapten to flunixin meglumine can reach 100%, and the cross reactivity of the hapten to the metabolite 5-hydroxyflunixin is 80%, so that the hapten can specifically detect the flunixin meglumine and the metabolite thereof, avoid the interference of other structural analogues to detection results, and well fill the shortage of the hapten with obvious specificity to the flunixin meglumine in the prior art.
Preferably, the preparation method of the hapten for detecting the content of the flunixin meglumine comprises the following steps:
1) Adding N, N-dimethylformamide to dissolve 2-amino-6- (trifluoromethyl) phenylpropionic acid, adding 2-chloronicotinic acid, and fully stirring;
2) Adding anhydrous potassium hydroxide, installing a reflux condenser, heating to 70 ℃ for reaction for 6 hours, and stopping the reaction to obtain a reaction solution;
3) And separating and purifying the obtained reaction liquid to obtain the hapten for detecting the content of the flunixin meglumine.
The reaction equation of the hapten preparation method is shown as a formula (2):
formula (2).
From the above, it is clear that this reaction can normally occur, and hapten having the above structure can be produced.
Preferably, the separation and purification operation in step 3) of the preparation method comprises the following steps: and cooling the obtained reaction liquid to room temperature, adding 200mL of water, regulating the pH value to 6 by using 6mol/L of hydrochloric acid, adding 100mL of ethyl acetate for extraction, separating the water phase, evaporating the organic phase by rotary evaporation, loading the organic phase on a silica gel column, and eluting and separating by using a methylene dichloride-methanol mixed solution with the volume ratio of 10:1 to obtain the hapten for detecting the content of the flunixin meglumine.
To achieve effective application of hapten, the following applications can be performed:
The application also provides an antigen for detecting the content of flunixin meglumine, which is a conjugate obtained by coupling the hapten and carrier protein.
Preferably, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin. The carrier protein of the above kind can effectively exert the detection function of hapten.
The coupling method of hapten and carrier protein used in the invention can be the existing method for preparing antigen from hapten, for example, the coupling method using bovine serum albumin as carrier protein can be as follows:
Taking 13.2mg of hapten, adding 1mL of N, N-dimethylformamide for dissolution, adding 12.87mg of N-hydroxysuccinimide and 21.44mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, fully dissolving and uniformly mixing, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 4mL of 0.1mol/L CB buffer solution with pH of 9.5 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 6 hours, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times a day, centrifuging and subpackaging to obtain the flunixin meglumine antigen coupled with bovine serum albumin, and preserving at-20 ℃.
The coupling method using ovalbumin as carrier protein comprises the following steps:
Taking 15.73mg of hapten, adding 1mL of N, N-dimethylformamide for dissolution, adding 12.87mg of N-hydroxysuccinimide and 21.44mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, fully dissolving and uniformly mixing, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 100mg of Ovalbumin (OVA), and adding 4mL of 0.1mol/L CB buffer solution with pH of 9.5 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 6 hours, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times a day, centrifuging and subpackaging to obtain the flunixin meglumine antigen coupled with ovalbumin, and preserving at-20 ℃.
The invention also provides an antibody for detecting the content of flunixin meglumine, which is obtained by animal immunization of the antigen; the antibody reacts with flunixin meglumine specifically.
Preferably, the antibody is a flunixin meglumine monoclonal antibody.
The method for preparing antibodies from the above antigens used in this aspect may be animal immunization methods commonly used in the art, for example, comprising the steps of:
(1) Animal immunization: the flunixin meglumine antigen conjugated to bovine serum albumin was injected into Balb/c mice at an immunizing dose of 150 μg/mouse to generate polyclonal antibody serum.
(2) Cell fusion and cloning: after the serum determination result of the mice is higher, spleen cells are taken and fused with SP2/0 myeloma cells according to the ratio of 8:1, cell supernatant is determined by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain secreting monoclonal antibody.
(3) Cell cryopreservation and resuscitation: the monoclonal hybridoma cell strain of flunixin meglumine is prepared into a cell suspension of 1X 10 9 cells/mL by using a frozen stock solution, and the cell suspension is stored in liquid nitrogen for a long time. 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.
(4) Production and purification of monoclonal antibodies: balb/c mice were intraperitoneally injected with sterilized paraffin oil 0.5 mL/mouse, and after 7 days, the ascites was collected after 7 days by intraperitoneally injecting 6X 10 5 monoclonal hybridoma cell lines of flunixin meglumine. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and preserving at-20deg.C.
(5) Determination of monoclonal antibody titers: the titer of the antibodies was determined to be 1:270000 using indirect competition ELISA.
The antibody is put into use in the form of monoclonal antibody, and has higher potency.
The invention also provides an ELISA kit for detecting the content of flunixin meglumine, which comprises: the ELISA plate is coated with the antigen for detecting the content of the flunixin meglumine; the antibody is the antibody for detecting the content of the flunixin meglumine.
The kit is prepared according to the existing method, and a specific method can be prepared by a person skilled in the art by searching the existing production method of the ELISA kit.
Preferably, the enzyme-linked immunosorbent assay kit for detecting the content of the flunixin meglumine further comprises: the kit comprises flunixin meglumine standard substance solution with gradient concentration, enzyme-labeled secondary antibody, substrate solution A, substrate solution B, stop solution, washing solution and compound solution.
Preferably, the gradient concentration of the flunixin meglumine standard solution 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;
the enzyme-labeled secondary antibody is a goat anti-mouse antibody marked by horseradish peroxidase;
the substrate solution A is carbamide peroxide, and the substrate solution B is tetramethyl benzidine;
the stop solution is 2mol/L sulfuric acid solution;
The washing liquid is pH 7.2 and contains 0.8% -1.2% of Tween-20, 0.01% -0.03% of thimerosal preservative and 0.1% -0.3 mol/L carbonate buffer solution;
the compound solution is phosphate buffer solution with pH of 7.6 and containing 8% -12% casein and 0.1% -0.3 mol/L.
The kit prepared by the components has high detection efficiency and good accuracy, and is suitable for detecting mass samples on site.
The room temperature range of the application is 20-25 ℃, and the reading of the thermometer is the standard in operation.
The invention has the beneficial effects that:
1) The hapten for detecting the content of the flunixin meglumine provided by the application has the sensitivity of 0.1 mug/L when the hapten is used for detecting the flunixin meglumine contained in liquid milk after being prepared into a kit, which indicates that the hapten is higher in detection sensitivity of the flunixin meglumine after being prepared into the kit; after the kit is prepared according to the prior method, the detection limit of the flunixin meglumine in the liquid milk is 0.1 mug/L, and the kit can be used for effectively detecting the content of the flunixin meglumine in the liquid milk; after the hapten is prepared into a kit, the recovery rate of the flunixin meglumine is 100% +/-20%, the intra-batch variation coefficient is less than 10%, the inter-batch variation coefficient is less than 15%, the accuracy is good, and the detection requirement can be met. The hapten can keep the characteristic structure of the flunixin meglumine to the greatest extent, and after the hapten is used for preparing an artificial antibody, the antibody has the characteristics of high sensitivity and strong specificity for detecting the flunixin meglumine.
2) The hapten for detecting the content of the flunixin meglumine provided by the invention adopts the hapten as a raw material, and the antibody prepared by adopting the hapten as the raw material shows stronger immunogenicity aiming at the residue detection of the flunixin meglumine; the hapten is used as a raw material to prepare an artificial antigen system suitable for animal immunization to immunize animals, the titer, the specificity and the affinity of the obtained antibody are relatively good, and the detection sensitivity of the antibody against flunixin meglumine can reach 0.1 mug/L.
3) The antibody and the kit for detecting the content of the flunixin meglumine provided by the invention have the advantages that the detection operation is simple, large-scale instrument analysis equipment is not required to be purchased, the detection of a sample can be completed only by the kit, and the kit is suitable for on-site mass detection.
4) The ELISA kit provided by the invention is prepared from the obtained antibody, is convenient to use, has low detection cost, and can detect a large number of samples simultaneously, and the detection method is efficient, accurate and quick, and is suitable for on-site monitoring of flunixin meglumine in liquid milk and screening of a large number of samples.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of hapten obtained in the example of the present invention.
FIG. 2 is a standard curve obtained after detection of a flunixin meglumine standard solution with gradient concentration by using the antibody provided by the invention in the application of liquid milk detection in the ELISA kit obtained in the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings. The hapten-producing antigen, the antigen-producing antibody and the antibody-producing kit of the present invention are not described in detail in the methods disclosed in the prior art, and are not described here.
The materials and instruments used in the examples below are commercially available unless otherwise specified.
Example 1 preparation of hapten for detecting the content of flunixin meglumine
The hapten for detecting the content of the flunixin meglumine is prepared according to the following steps:
1) 2.33g of 2-amino-6- (trifluoromethyl) phenylpropionic acid (CAS number: 1806406-46-1) was dissolved in 100mL of N, N-dimethylformamide, and 1.88g of 2-chloronicotinic acid (CAS number: 2942-59-8), and stirring thoroughly;
2) Adding 0.78g of anhydrous potassium hydroxide, installing a reflux condenser, heating to 70 ℃ for reaction for 6 hours, and stopping the reaction to obtain a reaction solution;
3) And cooling the obtained reaction liquid to room temperature, adding 200mL of water, regulating the pH value to 6 by using 6mol/L of hydrochloric acid, adding 100mL of ethyl acetate for extraction, separating the water phase, evaporating the organic phase by rotary evaporation, loading the organic phase on a silica gel column, and eluting and separating by using a methylene dichloride-methanol mixed solution with the volume ratio of 10:1 to obtain the hapten for detecting the content of the flunixin meglumine.
The yield of hapten for detecting the content of flunixin meglumine is 48.58%. The hapten was subjected to hydrogen nuclear magnetic resonance (1 H-NMR) measurement, and the result was shown as ：1H NMR（500 MHz, Chloroform-d）：δ (ppm) 10.11 (s, 2H),9.77 (dd, J=5.0, 2.3 Hz, 2H),9.69 (s, 2H), 9.15 (dd, J=8.8, 2.2 Hz, 2H),7.75 (dd, J=8.7, 5.0 Hz, 2H),7.68 (dd, J=10.4, 1.6 Hz, 2H),7.64 (dd, J=10.4, 7.5 Hz, 2H),7.26 (dd, J=7.5, 1.6 Hz, 2H),3.50–3.33 (m, 4H),2.93 (d, J=16.8 Hz, 2H), in FIG. 1, which shows that the hapten structure was correct.
Example 2 preparation of antigen for detection of the content of flunixin meglumine coupled to bovine serum Albumin
The method comprises the following steps:
Taking 13.2mg of hapten for detecting the content of flunixin meglumine prepared in the example 1, adding 1mL of N, N-dimethylformamide for dissolution, adding 12.87mg of N-hydroxysuccinimide and 21.44mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 4mL of 0.1mol/L CB buffer solution with pH of 9.5 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 6 hours, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times a day, centrifuging and subpackaging to obtain the flunixin meglumine antigen coupled with bovine serum albumin, and preserving at-20 ℃.
Example 3 preparation of antigen for detection of content of flunixin meglumine coupled with ovalbumin
The method comprises the following steps:
Taking 15.73mg of hapten for detecting the content of flunixin meglumine prepared in the example 1, adding 1mL of N, N-dimethylformamide for dissolution, adding 12.87mg of N-hydroxysuccinimide and 21.44mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain hapten activating solution A; taking 100mg of Ovalbumin (OVA), and adding 4mL of 0.1mol/L CB buffer solution with pH of 9.5 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 6 hours, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times a day, centrifuging and subpackaging to obtain the flunixin meglumine antigen coupled with ovalbumin, and preserving at-20 ℃.
Example 4 preparation of monoclonal antibody for detecting Flunixin meglumine content
The method comprises the following steps:
(1) Animal immunization: the flunixin meglumine antigen conjugated to bovine serum albumin prepared in example 2 was injected into Balb/c mice at an immunizing dose of 150 μg/mouse to generate polyclonal antibody serum.
(2) Cell fusion and cloning: after the serum determination result of the mice is higher, spleen cells are taken and fused with SP2/0 myeloma cells according to the ratio of 8:1, cell supernatant is determined by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain secreting monoclonal antibody.
(3) Cell cryopreservation and resuscitation: the monoclonal hybridoma cell strain of flunixin meglumine is prepared into a cell suspension of 1X 10 9 cells/mL by using a frozen stock solution, and the cell suspension is stored in liquid nitrogen for a long time. 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.
(4) Production and purification of monoclonal antibodies: balb/c mice were intraperitoneally injected with sterilized paraffin oil 0.5 mL/mouse, and after 7 days, the ascites was collected after 7 days by intraperitoneally injecting 6X 10 5 monoclonal hybridoma cell lines of flunixin meglumine. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and preserving at-20deg.C.
(5) Determination of monoclonal antibody titers: the titer of the antibodies was determined to be 1:270000 using indirect competition ELISA.
Indirect competition ELISA method: coating an ELISA plate with the flunixin meglumine antigen coupled with ovalbumin prepared in the example 3, adding a flunixin meglumine standard substance solution and a monoclonal antibody working solution, reacting for 30min at 4 ℃, pouring out the liquid in the hole, washing for 3-5 times with a PBST washing solution, and drying by taking out with absorbent paper; adding goat anti-mouse antibody marked by horseradish peroxidase, reacting for 30min at 25 ℃, taking out, and repeating the step of washing the plate; adding a substrate solution, reacting for 15min at 25 ℃, and adding a stopping solution to stop the reaction; the absorbance value of each well was measured at a wavelength of 450nm by setting a microplate reader.
Example 5 preparation of enzyme-linked immunosorbent assay kit for detecting the content of flunixin meglumine
The resulting kit comprises:
(1) An ELISA plate coated with flunixin meglumine antigen coupled with ovalbumin;
(2) Flunixin meglumine monoclonal antibody working solution;
(3) Horseradish peroxidase-labeled goat anti-mouse antibody;
(4) 6 bottles of flunixin meglumine standard solution with the concentration of 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;
(5) The substrate solution consists of solution A and solution B, wherein the solution A is carbamide peroxide and the solution B is tetramethyl benzidine;
(6) The stop solution is 2mol/L sulfuric acid solution;
(7) The washing liquid is pH 7.2 and contains 1.0% Tween-20, 0.02%o thiomersal preservative and 0.2mol/L carbonate buffer solution;
(8) The complex solution is phosphate buffer solution with pH 7.6 and 10% casein and 0.2 mol/L.
The main reagent of the kit is provided in the form of working solution, and the detection method is convenient and feasible, has the characteristics of high specificity, high sensitivity, high accuracy and the like, and is convenient for rapidly detecting a large number of liquid milk samples in batches.
Application of enzyme-linked immunosorbent assay kit for detecting content of flunixin meglumine obtained in example 6 in liquid milk detection and performance detection thereof
1. Test method of the kit obtained in example 5
1. Sample pretreatment
The sample is recovered to room temperature and then is directly detected, and the sample to be detected needs to be uniform liquid and cannot be agglomerated, soured or precipitated.
2. The procedure for detection of the kit obtained in example 5
Adding 50 mu L/hole of a solution to be detected into a micropore of an ELISA plate (coated with the flunixin meglumine antigen coupled with ovalbumin obtained in the example 3), then adding 50 mu L/hole of the flunixin meglumine monoclonal antibody working solution prepared in the example 4, covering the plate by using a cover plate film, and performing light-shielding reaction at 25 ℃ for 30min; pouring out the liquid in the holes, adding 250 mu L of washing liquid into each hole, pouring out the liquid in the holes after 30s, repeating the operation of washing the plate for 5 times, and beating with absorbent paper; adding 100 mu L/hole of goat anti-mouse antibody marked by horseradish peroxidase, gently shaking and uniformly mixing, covering with a cover plate film, performing light-shielding reaction at 25 ℃ for 30min, and repeating the plate washing step;
Adding 50 mu L of substrate solution A urea peroxide and 50 mu L of substrate solution B Tetramethylbenzidine (TMB) into each hole, gently shaking and mixing, placing the cover film and the cover plate at 25 ℃ and developing for 15min in a dark place, adding 50 mu L of stop solution 2mol/L sulfuric acid solution into each hole, gently shaking and mixing, setting the wavelength at 450nm by using an enzyme-labeling instrument, and measuring the absorbance value (OD value) of each hole.
3. Detection and analysis of results thereof
According to the operation steps, respectively taking a flunixin meglumine standard solution with gradient concentration and a pretreated sample as a solution to be detected for detection;
The absorbance average (B) of each concentration of the obtained standard solution was divided by the absorbance average (B 0) of the first standard solution (0 standard) and multiplied by 100% to obtain a percent absorbance value.
The standard graph is plotted with the logarithmic value of the flunixin meglumine standard concentration (μg/L) as X-axis and the percent absorbance value as Y-axis, as shown in fig. 2.
And calculating the percentage absorbance value of the pretreated sample solution obtained by detection by the same method, and reading the concentration corresponding to the pretreated sample from the standard curve of the flunixin meglumine standard substance, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the flunixin meglumine in the pretreated sample.
2. Sensitivity determination of the kit obtained in example 5
Sensitivity in the application: the sensitivity of the quantitative kit can be generally understood as the concentration value of the standard substance with the minimum concentration or the concentration of the lowest standard substance corresponding to the establishment of a standard curve except for the 0 standard substance in the standard substance of the kit. The sensitivity of the kit provided by the application to flunixin meglumine is calculated to be 0.1 mug/L according to the prior method.
3. The detection limit of the kit obtained in example 5
Limit of detection: the minimum detection amount of the actual sample is measured by a kit product, and the theory is defined as follows: and (3) measuring 20 negative (blank) samples according to a reasonable pretreatment method, calculating an average value X and a Standard Deviation (SD), and obtaining a result according to a formula X+3SD, namely the detection (lower) limit of the samples. Therefore, the detection limit of the kit to flunixin meglumine in liquid milk is 0.1 mug/L according to the prior method.
4. Determination of the accuracy and precision of the kit obtained in example 5
Accuracy and precision: the accuracy of the ELISA assay is expressed as recovery, and the precision is expressed as coefficient of variation. Taking blank raw milk, pasteurized milk, UHT sterilized milk and goat milk samples, and carrying out addition recovery test on the blank raw milk, pasteurized milk, UHT sterilized milk and goat milk samples by using flunixin meglumine with three concentrations of 0.2, 0.4 and 0.8 mug/L according to the prior method, wherein the experimental results are as follows: the recovery rate of the kit prepared by the hapten to a sample is 100% +/-20%, the intra-batch variation coefficient is less than 10%, and the inter-batch variation coefficient is less than 15%.
The kit prepared by the hapten has good accuracy and precision, and can accurately detect the flunixin meglumine contained in the sample.
5. Kit antibody Cross-reactivity assay obtained in example 5
A structure similar to flunixin meglumine was used: the 5-hydroxyflunixin, aspirin, indomethacin, naproxen, diclofenac, ibuprofen, nimesulide, rofecoxib and celecoxib are respectively measured by adopting the existing indirect competition ELISA method, and the results show that except flunixin meglumine is 100%, 5-hydroxyflunixin is 80%, and the rest is less than 1%.
The antibody prepared by the hapten has better specificity on the flunixin meglumine and the metabolite thereof, can accurately detect the flunixin meglumine and the metabolite thereof contained in a sample, and is not influenced by other structural analogues.
6. Shelf life condition test of the kit obtained in example 5
And (3) after the kit is stored at the temperature of 2-8 ℃ for 12 months, detecting the maximum absorbance value (zero standard), 50% inhibition concentration and flunixin meglumine actual measured value, wherein each value is in a normal range.
The kit provided by the application can be stored for at least 12 months.
The detection result shows that the antibody prepared from the hapten has a more accurate detection effect in the detection of the residual quantity of flunixin meglumine in liquid milk after the antibody is prepared into a kit, and the kit provided by the invention can be used for effectively detecting various pretreated liquid milk.
Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.
Claims (10)
1. The hapten for detecting the content of the flunixin meglumine is characterized by comprising the following chemical structural formula:
Formula (1);
The preparation method of the hapten for detecting the content of the flunixin meglumine comprises the following steps:
2-amino-6- (trifluoromethyl) phenylpropionic acid is used as a raw material to carry out nucleophilic substitution reaction with 2-chloronicotinic acid, so as to obtain the hapten for detecting the content of the flunixin meglumine.
2. The hapten for flunixin meglumine content detection according to claim 1, wherein the preparation method of the hapten for flunixin meglumine content detection comprises the following steps:
1) Adding N, N-dimethylformamide to dissolve 2-amino-6- (trifluoromethyl) phenylpropionic acid, adding 2-chloronicotinic acid, and fully stirring;
2) Adding anhydrous potassium hydroxide, installing a reflux condenser, heating to 70 ℃ for reaction for 6 hours, and stopping the reaction to obtain a reaction solution;
3) And separating and purifying the obtained reaction liquid to obtain the hapten for detecting the content of the flunixin meglumine.
3. The hapten for flunixin meglumine content detection according to claim 2, wherein the separation and purification operation in step 3) comprises the steps of: and cooling the obtained reaction liquid to room temperature, adding 200mL of water, regulating the pH value to 6 by using 6mol/L of hydrochloric acid, adding 100mL of ethyl acetate for extraction, separating the water phase, evaporating the organic phase by rotary evaporation, loading the organic phase on a silica gel column, and eluting and separating by using a methylene dichloride-methanol mixed solution with the volume ratio of 10:1 to obtain the hapten for detecting the content of the flunixin meglumine.
4. An antigen for detecting the content of flunixin meglumine, which is a conjugate obtained by coupling the hapten according to any one of claims 1 to 3 with a carrier protein.
5. The antigen for detecting the content of flunixin meglumine according to claim 4, wherein the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
6. An antibody for detecting the content of flunixin meglumine, which is obtained by immunizing an animal with the antigen according to claim 4 or 5; the antibody reacts with flunixin meglumine specifically.
7. The antibody for detecting the content of flunixin meglumine according to claim 6, wherein the antibody is a flunixin meglumine monoclonal antibody.
8. An enzyme-linked immunosorbent assay kit for detecting the content of flunixin meglumine, which is characterized by comprising the following components: an ELISA plate coated with the antigen for detecting the content of flunixin meglumine according to claim 4 or 5; the antibody for detecting the content of flunixin meglumine according to claim 6 or 7.
9. The enzyme-linked immunosorbent assay kit for detecting the content of flunixin meglumine according to claim 8, further comprising: the kit comprises flunixin meglumine standard substance solution with gradient concentration, enzyme-labeled secondary antibody, substrate solution A, substrate solution B, stop solution, washing solution and compound solution.
10. The enzyme-linked immunosorbent assay kit for detecting the content of flunixin meglumine according to claim 9, wherein the gradient concentration of the flunixin meglumine standard solution is 0 μg/L, 0.1 μg/L, 0.3 μg/L, 0.9 μg/L, 2.7 μg/L, 8.1 μg/L, respectively;
The enzyme-labeled secondary antibody is a goat anti-mouse antibody marked by horseradish peroxidase;
the substrate solution A is carbamide peroxide, and the substrate solution B is tetramethyl benzidine;
the stop solution is 2mol/L sulfuric acid solution;
The washing liquid is pH 7.2 and contains 0.8% -1.2% of Tween-20, 0.01% -0.03% of thimerosal preservative and 0.1% -0.3 mol/L carbonate buffer solution;
The compound solution is phosphate buffer solution with pH of 7.6 and containing 8% -12% casein and 0.1% -0.3 mol/L.
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| CN118373916A (en) * | 2024-06-26 | 2024-07-23 | 北京纳百生物科技有限公司 | Flunixin meglumine monoclonal antibody, detection test strip and application thereof |
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