CN108558718B - Florfenicol, florfenicol amine antigen and antibody and simultaneous detection enzyme-linked immunoassay method thereof - Google Patents
Florfenicol, florfenicol amine antigen and antibody and simultaneous detection enzyme-linked immunoassay method thereof Download PDFInfo
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- CN108558718B CN108558718B CN201711489726.6A CN201711489726A CN108558718B CN 108558718 B CN108558718 B CN 108558718B CN 201711489726 A CN201711489726 A CN 201711489726A CN 108558718 B CN108558718 B CN 108558718B
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- AYIRNRDRBQJXIF-NXEZZACHSA-N (-)-Florfenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CF)NC(=O)C(Cl)Cl)C=C1 AYIRNRDRBQJXIF-NXEZZACHSA-N 0.000 title claims abstract description 107
- 229960003760 florfenicol Drugs 0.000 title claims abstract description 105
- 239000000427 antigen Substances 0.000 title claims abstract description 48
- 102000036639 antigens Human genes 0.000 title claims abstract description 48
- 108091007433 antigens Proteins 0.000 title claims abstract description 48
- XLSYLQDVLAXIKK-NXEZZACHSA-N (1r,2s)-2-amino-3-fluoro-1-(4-methylsulfonylphenyl)propan-1-ol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@H](N)CF)C=C1 XLSYLQDVLAXIKK-NXEZZACHSA-N 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000002965 ELISA Methods 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 title abstract description 18
- 239000011248 coating agent Substances 0.000 claims abstract description 33
- 238000000576 coating method Methods 0.000 claims abstract description 33
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 26
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 26
- 230000002163 immunogen Effects 0.000 claims abstract description 23
- 230000008878 coupling Effects 0.000 claims abstract description 10
- 238000010168 coupling process Methods 0.000 claims abstract description 10
- 238000005859 coupling reaction Methods 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 44
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 26
- 238000005406 washing Methods 0.000 claims description 26
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
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- 239000006228 supernatant Substances 0.000 claims description 8
- 230000005764 inhibitory process Effects 0.000 claims description 7
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 6
- 238000011161 development Methods 0.000 claims description 5
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- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 108010058846 Ovalbumin Proteins 0.000 claims description 3
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- 239000003814 drug Substances 0.000 claims description 3
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- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 230000031700 light absorption Effects 0.000 claims description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 abstract description 4
- -1 benzyl hydroxyl group Chemical group 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000001384 succinic acid Substances 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- 238000002649 immunization Methods 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 229960005091 chloramphenicol Drugs 0.000 description 6
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 6
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 5
- 230000002860 competitive effect Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
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- OTVAEFIXJLOWRX-NXEZZACHSA-N thiamphenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 OTVAEFIXJLOWRX-NXEZZACHSA-N 0.000 description 5
- 229960003053 thiamphenicol Drugs 0.000 description 5
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 208000027954 Poultry disease Diseases 0.000 description 1
- 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
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
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- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C317/00—Sulfones; Sulfoxides
- C07C317/26—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
- C07C317/32—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
- C07C317/34—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having sulfone or sulfoxide groups and amino groups bound to carbon atoms of six-membered aromatic rings being part of the same non-condensed ring or of a condensed ring system containing that ring
- C07C317/38—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having sulfone or sulfoxide groups and amino groups bound to carbon atoms of six-membered aromatic rings being part of the same non-condensed ring or of a condensed ring system containing that ring with the nitrogen atom of at least one amino group being part of any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylaminosulfones
- C07C317/40—Y being a hydrogen or a 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
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/94—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
Abstract
The invention discloses florfenicol, florfenicol amine antigen and antibody and a simultaneous detection enzyme-linked immunoassay method thereof. The florfenicol is used as a hapten to be coupled with carrier protein to obtain an artificial antigen which is used as a coating antigen; meanwhile, a succinic acid half-ester coupling arm is derived from the benzyl hydroxyl group of the florfenicol molecule to obtain a florfenicol hapten, and the florfenicol hapten is coupled with carrier protein to obtain a florfenicol artificial antigen which is used as an immunogen; preparing detection antibody, and establishing enzyme-linked immunoassay method for IC of florfenicol50=37.10 ng/mL, linear detection range 12.03-114.49 ng/mL, for florfenicol amine IC50The kit has the advantages of =45.3 ng/mL, linear detection range of 10.76-190.69 ng/mL, high sensitivity, good specificity and the like, can be used for quickly detecting residual florfenicol and florfenicol amine in food, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of florfenicol and florfenicol amine detection, and more particularly relates to a florfenicol and florfenicol amine antigen and antibody and a simultaneous detection enzyme-linked immunoassay method thereof.
Background
Florfenicol (Florfenicol, C)12H14Cl2FNO4S) belongs to broad-spectrum antibiotics of chloramphenicol, and is widely applied in the fields of aquaculture, livestock raising, treatment of poultry diseases and the like. Compared with the condition that chloramphenicol can cause serious harm to human health and is listed as a forbidden drug in many countries, thiamphenicol can cause animal aplastic anemia and the like, florfenicol has similar antibacterial activity with chloramphenicol and thiamphenicol and is safer to use. Florfenicol has been reported to have some immunotoxicity and embryotoxicity, and its maximum residual limit in animal tissues has been currently defined in many countries. Improper use and residues of florfenicol can be detrimental to human health, and therefore it is important to enhance the monitoring of the detection of florfenicol in animal products.
The final metabolite of florfenicol in animals is mainly florfenicol amine, which is specified to be detected simultaneously as a residual marker when detecting florfenicol residues. At present, the florfenicol and florfenicol amine analysis method is mainly an instrument detection method, is accurate and reliable, is often used as a confirmation method, and has the defects of expensive equipment, professional personnel requirement, low sample flux and the like. The immunoassay technology is an analysis method based on antigen-antibody specific reaction, has the characteristics of sensitivity, rapidness, high throughput and the like, can be matched with an instrument confirmation technology to meet the requirement of rapid screening of food safety, and has become a research hotspot in recent years. The research on the florfenicol and florfenicol amine enzyme-linked immunosorbent assay method has very important economic and social significance for the fast screening and detection of florfenicol and florfenicol amine large-scale samples.
Disclosure of Invention
It is a first object of the present invention to provide the use of florfenicol as a hapten for florfenicol and/or florfenicol amine.
The second purpose of the invention is to provide the application of florfenicol in preparing artificial antigens of florfenicol and/or florfenicol amine.
The third purpose of the invention is to provide an artificial antigen of florfenicol and/or florfenicol amine.
The fourth purpose of the invention is to provide the application of florfenicol or the artificial antigen in detecting florfenicol and/or florfenicol amine or in preparing a kit for detecting florfenicol and/or florfenicol amine. A fifth object of the present invention is to provide an immunogen and coating antigen combination for detecting florfenicol and/or florfenicol amine.
The sixth purpose of the invention is to provide an enzyme-linked immunoassay method for detecting florfenicol and/or florfenicol amine
The seventh purpose of the invention is an enzyme-linked immunoassay kit for florfenicol and/or florfenicol amine.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the florfenicol (FF) is used as a hapten of florfenicol and/or florfenicol amine, and the structure of the florfenicol is as follows:
an artificial antigen of florfenicol and/or florfenicol amine, which is a conjugate of the florfenicol and a carrier protein and has the following structure:
preferably, the carrier protein is keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin.
The florfenicol is applied to the preparation of artificial antigens of florfenicol and/or florfenicol amine.
The florfenicol or the artificial antigen is applied to the detection of florfenicol and/or florfenicol amine, or the preparation of a kit for detecting florfenicol and/or florfenicol amine.
The other hapten FFD of the florfenicol and/or the florfenicol amine is a carboxyl hapten of a coupling arm of succinic acid half ester derived from benzyl hydroxyl of a florfenicol molecule, and has the following structure:
FFD and carrier protein are coupled to obtain the florfenicol and/or florfenicol amine artificial antigen, and the structure of the florfenicol and/or florfenicol amine artificial antigen is as follows:
the combination of an immunogen and a coating antigen for detecting florfenicol and/or florfenicol amine is characterized in that the coating antigen is an artificial antigen obtained by coupling florfenicol and carrier protein, and the immunogen is a conjugate of florfenicol hapten FFD and keyhole limpet hemocyanin.
An enzyme-linked immunoassay kit for florfenicol and/or florfenicol amine comprises the immunogen and coating antigen combination.
Preferably, the coating antigen is a conjugate of florfenicol and ovalbumin.
In addition, according to the preparation method of the hapten FFD, florfenicol FF and succinic anhydride are placed in an organic solvent to be stirred and react.
Preferably, the method comprises the following steps:
s1, adding florfenicol and succinic anhydride into a proper amount of dichloromethane according to a molar ratio of 1: 1.5-2, wherein the molar ratio of the florfenicol to the succinic anhydride is preferably 1: 2;
s2, dropwise adding a proper amount of triethylamine into the mixed solution, heating in a water bath for reacting for 2-4 hours, wherein the molar ratio of the florfenicol to the succinic anhydride to the triethylamine is preferably 1:2:4, the water bath temperature is preferably 50 ℃, and the reaction time is preferably 3 hours;
s3, after the reaction is finished, extracting the product mixed liquor for 3 times by using ethyl acetate, wherein the volume ratio of the extracted mixed liquor to the ethyl acetate is preferably 1: 2;
s4, spin-drying the extract liquid in the S3, purifying the extract liquid through a silica gel column, and evaporating the extract liquid to dryness to obtain light yellow powder, namely the hapten FFD.
The preparation method of the artificial antigen prepared by FFD comprises the steps of dissolving florfenicol in N, N-dimethylformamide, adding N, N-carbonyl diimidazole by stirring, then adding a PBS solution dissolved with carrier protein, and reacting for 72 hours by stirring at 4 ℃; dialyzing the coupling mixture at 4 ℃ for 3 days by PBS to obtain a target product; the molar ratio of the hapten to the carrier protein is 25-100: 1.
Dissolving the hapten in N, N-dimethylformamide, stirring and adding N, N-Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), then adding a PBS solution dissolved with carrier protein, and stirring and reacting at 4 ℃ overnight; and dialyzing the coupling mixture at 4 ℃ by PBS for 3 days to obtain a target product, wherein the molar ratio of the hapten to the carrier protein is 25-100: 1.
An enzyme-linked immunoassay method for detecting florfenicol, which comprises the following steps:
s1, taking a conjugate of a florfenicol hapten FFD and a carrier protein as an artificial antigen immune animal to prepare an antibody for detecting the florfenicol;
s2, coating the artificial antigen obtained by coupling FF and carrier protein on a microporous plate as a coating antigen, and adding the antibody for detecting florfenicol, which is prepared in the step S1, into the microporous plate;
s3, adding a sample to be detected, and determining the content of the florfenicol and/or florfenicol amine in the sample to be detected by adopting an indirect competitive enzyme-linked immunosorbent assay.
Preferably, the enzyme-linked immunoassay method comprises the following steps:
s1, coating: diluting the coating antigen by a certain multiple with a coating buffer solution, preparing a blank control group, incubating at 37 ℃ overnight with 100 mu L of each well;
s2, washing: pouring out liquid in the holes, washing for 2 times by using a washing solution, and spin-drying;
s3, sealing: adding 120 μ L of sealing liquid into each hole, sealing at 37 deg.C for 3 hr, drying, and placing in oven at 37 deg.C for 1 hr;
s4, sample adding and incubation: the standard florfenicol is dissolved in DMF to prepare a solution of 1.0 mg/mL. Diluting the solution into a standard solution with a certain concentration by using 0.01mol/L PBS buffer solution, and then diluting the solution in a 5-time gradient manner to obtain 7 concentration gradients in total; adding 50 mu L of standard solution diluted in gradient and 50 mu L of antiserum with specific concentration into each hole of the ELISA plate, uniformly mixing by shaking, reacting at 37 ℃ for 40min, washing for 5 times, and spin-drying;
s5, adding a secondary antibody: adding 100 μ L of HRP-goat anti-rabbit IgG diluted 5000 times with PBS into each well, reacting at 37 deg.C for 30min, and washing for 5 times;
s6, color development: adding 100 μ L of developing solution into each well, developing at 37 deg.C for 10min, and adding 50 μ L of 10% H into each well2SO4Terminating the reaction;
s7, reading measurement: measuring the light absorption value at the wavelength of 450 nm; selecting antiserum dilution times with absorbance values within the range of 1.0-1.5 as antiserum titer, wherein the antiserum effect is obtained by the inhibition rate;
s8, calculating: IC of inhibition curves calculated using the four parameter fitting Module of origin8.650The value is obtained.
A high-sensitivity antibody against florfenicol and florfenicol amine is obtained by immunizing animals with an artificial antigen FFD-KLH obtained by coupling the hapten FFD with Keyhole Limpet Hemocyanin (KLH) as an immunogen.
Preferably, the immunogen is prepared by the following steps:
s1, dissolving the hapten FFD in mL of N, N-dimethylformamide;
s2, respectively adding N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide which are the same as the hapten in molar mass into the solution obtained in the step S1, stirring and reacting overnight at the temperature of 4 ℃, centrifuging and taking the supernatant as solution A;
s3, dissolving the carrier protein KLH in 0.01mol/L PBS (phosphate buffer solution), marking as solution B, dripping the solution A into the solution B under stirring, reacting for 12 hours at 4 ℃, and preferably controlling the dripping time to be about 30 minutes;
s4, after the reaction is finished, centrifuging and taking the supernatant, dialyzing the supernatant with PBS solution at 4 ℃ for 3 days to obtain the florfenicol artificial immunogen, subpackaging the florfenicol artificial immunogen with the concentration of 1mg/mL into 1mL centrifuge tubes, and freezing and storing the florfenicol artificial immunogen in a refrigerator at-20 ℃ for later use.
Wherein, the mole ratio of the hapten FFD, the DCC and the NHS is 1: 1.5: 1.5.
the antibody prepared by the artificial antigen comprises a monoclonal antibody, a polyclonal antibody or a genetic engineering antibody.
Preparing antibodies for resisting florfenicol and florfenicol amine by immunizing animals with the florfenicol artificial immune antigen FFD-KLH.
Preferably, the coated pro-FF-OVA is prepared by the following steps:
s1, dissolving the FF hapten in N, N-dimethylformamide;
s2, adding N' N-carbonyl diimidazole into the solution obtained in the step S1, stirring for 2 hours at 37 ℃, and marking as solution A;
s3, dissolving the carrier protein OVA in 0.01mol/L PBS (phosphate buffer solution), marking as solution B, slowly dripping the solution A into the solution B while stirring, stirring and reacting for 72 hours at 4 ℃, and preferably controlling the dripping time to be about 10 min;
and S, dialyzing the solution with PBS at 4.4 ℃ for 3 days to obtain the florfenicol artificial coating antigen, subpackaging the florfenicol artificial coating antigen in 1mL centrifuge tubes, and freezing and storing the florfenicol artificial coating antigen in a refrigerator at the temperature of-20 ℃ for later use.
Wherein, the mole ratio of the hapten FF, the DCC and the NHS is 1: 1.5: 1.5.
compared with the prior art, the invention has the following beneficial effects:
(1) aiming at florfenicol and florfenicol amine, carboxyl hapten FFD and technical hapten FF which are derived from benzyl hydroxyl of florfenicol molecules and coupled with arms are coupled with different carrier proteins to be respectively used as artificial immunogen and coating antigen.
(2) The polyclonal antibody for resisting florfenicol and florfenicol amine prepared by the invention has no obvious cross reaction on thiamphenicol and chloramphenicol which are structural analogues, the established enzyme-linked immunoassay method can realize direct detection, is convenient and quick, and provides a quick and efficient detection means for detecting the content of florfenicol and florfenicol amine in food.
(3) The antibody and the immunodetection method can be used for the specific detection of florfenicol and florfenicol amine residues, and the IC of the method for the florfenicol50The linear detection range is 12.03-114.49 ng/mL when the concentration is 37.10ng/mL, and the concentration is used for florfenicol amine IC50The linear detection range is 10.76-190.69 ng/mL when the concentration is 45.3 ng/mL. The antibody has the remarkable advantages of high sensitivity, good accuracy and the like, so that the antigen and the antibody provided by the invention can be used for establishing an enzyme-linked immunoassay method for florfenicol and florfenicol amine, thereby being used for rapidly detecting residual florfenicol and florfenicol amine in food and having wide application prospects.
Drawings
FIG. 1 is a diagram of identification of hapten FFD mass spectra.
FIG. 2 is a graph of the UV absorption spectra of carrier protein KLH, hapten FFD and immunogen FFD-KLH.
FIG. 3 is the ultraviolet absorption spectrum of carrier protein OVA, hapten FF and coating antigen FF-OVA.
Figure 4 is a plot of florfenicol and florfenicol amine inhibition curves.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 preparation of haptens
The hapten FFD is prepared according to the following method:
adding 580mg of florfenicol and 240mg of succinic anhydride into 5ml of dichloromethane, then dropwise adding 680 mu L of triethylamine into the mixed solution, heating in a water bath at 50 ℃ for reaction for 3 hours, and after the reaction is finished, extracting the product mixed solution with ethyl acetate for 3 times, wherein the volume ratio of the extracted mixed solution to the ethyl acetate is 1: 2; and (3) drying the extract liquid in a spinning way, purifying the extract liquid through a silica gel column, and evaporating the extract liquid to dryness to obtain light yellow powder, namely hapten FFD, wherein figure 1 is a mass spectrum identification chart of the FFD.
EXAMPLE 2 preparation of Artificial antigen
1, preparation of immunogen FFD-KLH
Active lipid method: 5mg of FFD hapten is dissolved in 0.2mL of N, N-dimethylformamide, 4.78mg of N, N-dicyclohexylcarbodiimide and 2.65mg of N-hydroxysuccinimide are added, the mixture is stirred at 4 ℃ for reaction overnight, and after centrifugation, the supernatant is taken and recorded as solution A. Dissolving carrier protein (14.6mg KLH) in 4mL of 0.01mol/LPBS, marking as solution B, dripping solution A into solution B under stirring, reacting for 12h at 4 ℃, centrifuging after the reaction is finished, taking supernatant, dialyzing by PBS solution at 4 ℃ for 3 days to obtain the florfenicol artificial immunogen, subpackaging in 1mL of centrifuge tubes at the concentration of 1mg/mL, and freezing and storing in a refrigerator at-20 ℃ for later use.
2, preparation of coated ProFF-OVA
N, N-carbonyldiimidazole method: dissolving 10.67mg of FF hapten in 0.25mL of N, N-dimethylformamide, adding 8.11mg of N' N-carbonyldiimidazole, and stirring at 37 ℃ for 2 hours to obtain solution A; dissolving carrier protein (13.41mg OVA) in 3mL of 0.01mol/L PBS (PBS), marking as solution B, slowly dripping the solution A into the solution B under stirring, controlling the dripping time to be about 10min, and stirring and reacting at 4 ℃ for 72 h; dialyzing with PBS solution at 4 ℃ for 3 days to obtain florfenicol artificial coating antigen, subpackaging in 1mL centrifuge tubes, and freezing in a refrigerator at-20 ℃ for later use.
3. Characterization of florfenicol immunogens and coatgens
(1) And (3) identifying the immunogen and the coating antigen by adopting an ultraviolet scanning method, measuring ultraviolet absorption spectra of the immunogen and the coating antigen within the wavelength range of 200-400 nm, comparing scanning curves of different substances, and identifying whether the coupling of the hapten and the carrier protein is successful. Because both the carrier protein and the hapten have maximum absorption peaks under the ultraviolet spectrum condition, if the coupling is successful, the characteristic absorption peaks are mutually superposed, thereby causing the blue shift of the maximum absorption peak.
(2) As shown in figures 2 and 3, the characteristic absorption peak of carrier protein at about 280nm shows obvious blue shift, so that the result of ultraviolet spectrum scanning can prove that the florfenicol immunogen and the coating antigen are successfully coupled.
Example 3 antibody preparation and characterization
When 2.5kg of female New Zealand white rabbits are immunized for the first time, 0.5mL of immunogen is injected, 0.5mL of 1mg/mL of immunogen is taken and added with equal volume of Freund complete adjuvant, and after full emulsification, subcutaneous multiple injection is carried out on the backs of the rabbits, wherein each injection is about 200 mul; after four weeks, carrying out 2 nd immunization, wherein the amount of the injected immunogen is 0.5 mL/mouse, and emulsifying by using equivalent volume of Freund incomplete adjuvant; three weeks later, the 3 rd immunization was performed, and the 4 th immunization and the 3 rd immunization were also performed at intervals of 3 weeks for a total of 4 immunizations. Blood is collected from the heart one week after 4-immunization, antibody serum is taken, the antiserum is purified by adopting an octanoic acid-ammonium sulfate precipitation method to obtain a polyclonal antibody, and the polyclonal antibody is stored at the temperature of minus 20 ℃ for later use.
Seventh day after 3 times of booster immunization, 200 μ L of blood was collected from the marginal vein of ear, incubated at 37 ℃ for 30min, centrifuged at 4000r/min for 10min, and the supernatant was collected and stored at-20 ℃.
The titer and specificity of antiserum were determined by indirect ELISA, which was carried out as follows:
s1, coating: 1mg/mL of the coated antigen was diluted 1000-fold with the coating buffer and used as a blank control, 100. mu.L/well was added to a 96-well microplate, and the microplate was incubated overnight in a 37 ℃ water bath.
S2, washing: and (3) pouring out liquid in the holes, setting parameters of the plate washing machine, adding 300 mu L of washing liquid into each hole, washing the plate for 2 times, and then drying the washing liquid.
S3, sealing: adding 120 μ L of sealing liquid into each hole, sealing at 37 deg.C for 3 hr, spin-drying the liquid in the hole, and oven-drying in an oven at 37 deg.C for 1 hr.
S4, sample adding and incubation: adding 50 mu L of anti-serum diluted in a gradient manner and 50 mu L of diluent into the titer column, shaking and uniformly mixing, reacting in a 37 ℃ water bath tank for 40min, washing for 5 times at 300 mu L/hole, and drying the liquid in the hole.
S5, adding a secondary antibody: adding 100 μ L HRP-goat anti-rabbit IgG (diluted 5000 times with PBS) into each well, reacting in a 37 deg.C constant temperature water bath for 30min, washing 5 times at 300 μ L/well, and draining off the liquid in the well.
S6, color development: adding 100 μ L of color developing solution into each well, placing in 37 deg.C water bath box for developing for 10min, adding 50 μ L of stop solution (10% H) into each well2SO4)。
S7, reading measurement: the absorbance (OD) was read with a microplate reader at a wavelength of 450 nm.
(3) And selecting the antiserum with the absorbance value within the range of 1.0-1.5 as the antiserum titer, wherein the result shows that the antiserum titer is 1: 8000.
Example 4 establishment of method for Indirect competitive enzyme-linked immunoassay of florfenicol and florfenicol amine
1. The indirect competitive enzyme-linked immunosorbent (iclelisa) reaction specifically comprises the following steps:
s1, coating: the coating source was diluted to the appropriate concentration with the coating solution, added to the wells of the microplate, 100. mu.L/well, and incubated overnight in a 37 ℃ water bath.
S2, washing: and (4) pouring out liquid in the holes, washing the plate for 2 times, adding 300 mu L of washing liquid into each hole, and drying by spin.
S3, sealing: adding 120 μ L of sealing solution into each well, sealing at 37 deg.C for 3 hr, drying, and placing in oven at 37 deg.C for 1 hr.
S4, sample adding and incubation: adding 50 mul of antibody diluted by a certain time and 50 mul of medicine diluent into each hole; shaking and mixing, reacting in a 37 ℃ water bath box for 40min, washing the plate 5 times by a plate washing machine, adding 300 mu L of washing liquid into each hole, and drying by spin drying.
S5, adding a secondary antibody: adding 100 μ L of HRP-goat anti-rabbit IgG diluted 5000 times into each well, reacting in a 37 deg.C water bath for 30min, washing the plate for 5 times, and spin-drying.
S6, color development: adding 100 μ L of color developing solution into each well, placing in 37 deg.C water bath box for developing for 10min, adding 50 μ L of stop solution (10% H) into each well2SO4)。
S7, determination: each well A was assayed by an enzyme-linked immunosorbent assay450nmThe absorbance of (a).
S8, calculating: IC of inhibition curves calculated using the four parameter fitting Module of origin8.650The value is obtained.
2. Preparing a florfenicol/florfenicol amine standard solution with the concentration of 3000ng/mL, 750ng/mL, 187.5ng/mL, 46.88ng/mL, 11.72ng/mL, 2.93ng/mL and 0.73ng/mL, and establishing a standard curve of an indirect enzyme-linked immunoassay method.
Figure 4 shows the inhibition curves for florfenicol and florfenicol amine. The method is directed to florfenicol IC50The linear detection range is 12.03-114.49 ng/mL, and the concentration is 37.10 ng/mL. For florfenicol amine IC50The linear detection range is 10.76-190.69 ng/mL when the concentration is 45.3 ng/mL.
EXAMPLE 5 determination of the Cross-reactivity ratio of antibodies
1. According to the optimal concentration of the coating antigen and the optimal dilution multiple of the antiserum obtained in the example 4, the structural analogs of florfenicol, florfenicol amine, thiamphenicol, chloramphenicol and the like are taken as competitive standards to carry out an indirect competitive ELISA experiment to detect the specificity of the florfenicol polyclonal antibody and the half Inhibition Concentration (IC)50) And the values of the cross-reactivity (CR) are given in Table 1.
TABLE 1
Experimental results show that 82% of florfenicol polyclonal antibody and florfenicol amine are crossed, and almost no cross is generated between the florfenicol polyclonal antibody and structural analogues thiamphenicol and chloramphenicol, which shows that the florfenicol polyclonal antibody has good specificity and the established indirect enzyme-linked immunoassay method has good specificity.
Claims (1)
1. An enzyme-linked immunoassay method for florfenicol and florfenicol amine is characterized by comprising the following steps:
s1, coating: diluting 1mg/mL coating antigen by 1000 times with a coating buffer solution, adding the diluted coating antigen into a hole of an enzyme-labeled plate, keeping the diluted coating antigen at 100 mu L/hole, and standing overnight in a 37 ℃ water bath box;
the bagIs a conjugate of florfenicol and carrier protein, and has the following structure:
the carrier protein is ovalbumin, and the preparation method of the coating antigen comprises the following steps: dissolving 10.67mg of florfenicol hapten in 0.25mL of N, N-dimethylformamide, adding 8.11mg of N, N' -carbonyldiimidazole, and stirring at 37 ℃ for 2 hours to obtain solution A; dissolving 13.41mg of carrier protein OVA in 3mL of 0.01mol/L PBS (phosphate buffer solution), marking as solution B, slowly dripping the solution A into the solution B under stirring, controlling the dripping time to be about 10min, and stirring and reacting at 4 ℃ for 72 h; dialyzing with PBS solution at 4 ℃ for 3 days to obtain florfenicol artificial coating antigen, subpackaging in 1mL centrifuge tubes, and freezing in a refrigerator at-20 ℃ for later use;
s2, washing: pouring out liquid in the holes, washing the plate for 2 times, adding 300 mu L of washing liquid into each hole, and spin-drying;
s3, sealing: adding 120 μ L of sealing liquid into each hole, sealing at 37 deg.C for 3 hr, spin-drying, and placing in oven at 37 deg.C for 1 hr;
s4, sample adding and incubation: adding 50 mul of antibody diluted by a certain time and 50 mul of medicine diluent into each hole; shaking and mixing, reacting in a 37 ℃ water bath tank for 40min, washing the plate for 5 times by a plate washing machine, adding 300 mu L of washing liquid into each hole, and drying; the dilution ratio of the antibody is 1: 8000;
the antibody is obtained by taking an artificial antigen FFD-KLH obtained by coupling hapten FFD to keyhole limpet hemocyanin as an immunogen and immunizing animals, wherein the hapten FFD has the following structure:
the preparation method of the immunogen comprises the following steps: dissolving 5mg of FFD hapten in 0.2mL of N, N-dimethylformamide, adding 4.78mg of N, N-dicyclohexylcarbodiimide and 2.65mg of N-hydroxysuccinimide, stirring at 4 ℃ for reacting overnight, centrifuging, taking supernatant as solution A, dissolving 14.6mg of carrier protein KLH in 4mL of 0.01mol/L PBS as solution B, dropping the solution A into the solution B under stirring, reacting at 4 ℃ for 12 hours, centrifuging after the reaction is finished, taking supernatant, dialyzing by using PBS solution at 4 ℃ for 3 days to obtain the florfenicol artificial immunogen, subpackaging at a concentration of 1mg/mL for 3 daysFreezing in a 1mL centrifuge tube in a refrigerator at-20 ℃ for later use;
s5, adding a secondary antibody: adding 100 μ L of HRP-goat anti-rabbit IgG diluted by 5000 times into each hole, reacting in a 37 ℃ water bath for 30min, washing the plate for 5 times, and spin-drying;
s6, color development: adding 100 μ L of color development solution into each well, placing in 37 deg.C water bath box for developing for 10min, adding 50 μ L of stop solution 10% H into each well2SO4；
S7, determination: measuring the light absorption value of the A450nm of each well by using an enzyme-linked immunosorbent assay (ELISA) detector;
s8, calculating: IC50 values for the inhibition curves were calculated using a four parameter fitting module from origin8.6.
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