阅读说明：本技术 一种识别氟苯尼考、氯霉素和甲砜霉素的单克隆抗体及其应用 (Monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol and application thereof ) 是由 武玉香 沈志强 张莎莎 于 2019-09-04 设计创作，主要内容包括：本发明实施例公开了一种识别氟苯尼考、氯霉素和甲砜霉素的单克隆抗体及其应用,所述单克隆抗体由保藏编号为CCTCC NO:C2019152的氟苯尼考FF 3C11细胞株分泌得到；本发明上述单克隆抗体能够同时与氟苯尼考、氯霉素和甲砜霉素特异性结合,能够用于氟苯尼考、氯霉素和甲砜霉素中任意一种或多种的同时检测,且上述单克隆抗体特异性强,检测灵敏度较高。(The embodiment of the invention discloses a monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol and application thereof, wherein the monoclonal antibody is secreted by a florfenicol FF 3C11 cell strain with the preservation number of CCTCC NO: C2019152; the monoclonal antibody can be specifically combined with florfenicol, chloramphenicol and thiamphenicol at the same time, can be used for simultaneously detecting any one or more of florfenicol, chloramphenicol and thiamphenicol, and has strong specificity and high detection sensitivity.)

1. The monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol is characterized by being secreted by a florfenicol FF 3C11 cell strain with the preservation number of CCTCC NO: C2019152.

2. The monoclonal antibody of claim 1 for use in the detection of florfenicol, chloramphenicol, and thiamphenicol.

3. The detection card for detecting florfenicol, chloramphenicol and thiamphenicol residues in meat is characterized by comprising a bottom plate, a sample pad, a combination pad, a base membrane and a water absorption pad, wherein the combination pad contains a quantum dot labeled monoclonal antibody, the monoclonal antibody in the quantum dot labeled monoclonal antibody is the monoclonal antibody in claim 1, the base membrane is respectively provided with a detection line and a control line, the control line is coated with an antibody of goat anti-mouse IgG, and the detection line is coated with a chloramphenicol holoantigen.

4. The detection card of claim 3, wherein the quantum dots are ZnS core/CdSe composite quantum dots.

5. The detection card according to claim 3 or 4, wherein the quantum dot-labeled monoclonal antibody is prepared by the following method:

(a) cleaning and activating the quantum dots;

(b) mixing the activated quantum dot microspheres and the monoclonal antibody uniformly, and incubating;

(c) adding a sealing solution into the incubated mixture, incubating again, centrifuging, collecting the precipitate, and resuspending to obtain the quantum dot labeled monoclonal antibody.

6. The detection card according to claim 5, characterized in that said activation comprises in particular the steps of:

NHS and DCC were dissolved in pH 6.050mM MES buffer to give NHS solution and DCC solution, respectively; and then adding an NHS solution into the washed quantum dots, uniformly mixing, adding a DCC solution, uniformly mixing, incubating at room temperature for 20-30min, centrifuging to remove a supernatant, and resuspending.

7. The detection card of claim 6, wherein the concentration of the solution of NHS and DCC is 20 mg/ml; the volume ratio of the NHS solution to the DCC solution to the quantum dots is 1: 2.

8. The test card of claim 3, wherein the conjugate pad is made by:

the extract contains Triton X-100 0.5%, alanine 0.5%, and NaN 0.05%₃The PBS buffer solution with the pH value of 9.00.01M is sprayed on the bonding pad and dried; and then, spraying the quantum dot marked monoclonal antibody on the dried bonding pad, and drying.

9. The test card of claim 3, wherein the sample pad is made by:

spraying PBS buffer solution (pH9.00.01M) containing 0.5% Triton X-100 and 0.5% BSA onto the sample pad, and oven drying.

10. A method for detecting florfenicol, chloramphenicol and thiamphenicol residues in meat, which is characterized in that the detection is carried out by using the detection card of any one of claims 3-9;

the detection method comprises the steps of putting a sample into an ethyl acetate aqueous solution for extraction, taking supernatant, drying the supernatant by using nitrogen, re-dissolving the supernatant by using a sample diluent to obtain a mixed solution, and using the mixed solution for detection; the sample diluent comprises the following components in parts by weight: 0.6g of potassium dihydrogen phosphate, 0.3g of potassium chloride, 7.5g of disodium hydrogen phosphate dodecahydrate, 4.5g of sodium chloride and 0.5L of distilled water.

Technical Field

The embodiment of the invention relates to the technical field of detection, and particularly relates to a monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol and application thereof.

Background

Florfenicol is an antibiotic drug special for 3 rd generation animals of amidol, has the characteristics of broad spectrum, high efficiency, good absorption, wide in-vivo distribution, no aplastic anemia caused action and the like, and is widely used for treating bacterial diseases of livestock and poultry as a substitute drug of chloramphenicol. Research shows that florfenicol, chloramphenicol and thiamphenicol have embryotoxicity and drug resistance, and a large amount of applications in livestock and poultry production also cause residues in livestock and poultry products, thus harming public health. Standards for the maximum residual amounts of florfenicol, chloramphenicol and thiamphenicol in animal food are established in countries such as the European Union and Japan, and the No. 235 bulletin of the Ministry of agriculture in China also strictly regulates the residual amount, the maximum residual amount of florfenicol (FF) in poultry muscle is 100 mug/kg, chloramphenicol Chloramphenico (CAP) and salts and esters thereof are not detected in edible tissues of all food animals, and the maximum residual amount of Thiamphenicol (TAP) in poultry muscle is 50 mug/kg.

At present, the detection methods of antibiotics are various, and the instrument methods mainly comprise Thin Layer Chromatography (TLC), Gas Chromatography (GC), High Pressure Liquid Chromatography (HPLC) and various combined technologies such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (HPLC-MS) and the like; the rapid method is mainly based on immune analysis method based on immunochemistry, such as immune affinity column-fluorescence detection (IAC-FLD), enzyme linked immunosorbent assay (ELlSA), colloidal gold immune chromatography and the like.

The determination method has accurate determination result, but the price of instruments and equipment is high, the pretreatment process is complex, the operation is complicated, the efficiency is low, and the cost is extremely high, so the method is not suitable for large-scale sample screening and daily internal control detection. The rapid detection method is a rapid detection method commonly used by enterprises, and the rapid detection card adopts an enzyme-linked immunosorbent assay (ELISA) method and colloidal gold, but has the advantages and disadvantages: the ELISA method has relatively high sensitivity and can carry out quantitative detection, but the operation process is relatively complex, the steps are more, the detection time is longer, the requirements on environment and personnel are high, the interference on the environment and a matrix is serious, and a standard substance is required to be used, so that the harm to the operators is relatively large; the colloidal gold detection card can meet the requirement of rapid field, can rapidly detect within 5-10min, but can only determine qualitatively, has narrow quantitative range and low sensitivity, and has strong subjectivity and poor repeatability when poultry is observed by naked eyes.

Disclosure of Invention

Therefore, the embodiment of the invention provides a monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol, which can be specifically combined with florfenicol, chloramphenicol and thiamphenicol at the same time, so that the monoclonal antibody can be used for detecting any one or more of florfenicol, chloramphenicol and thiamphenicol, and has high detection sensitivity.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

according to the first aspect of the embodiment of the invention, the monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol is obtained by secreting a florfenicol FF 3C11 cell strain with the preservation number of CCTCC NO: C2019152.

The monoclonal antibody can be specifically combined with florfenicol, chloramphenicol and thiamphenicol at the same time, and can detect any one or more of the florfenicol, the chloramphenicol and the thiamphenicol.

According to a second aspect of the embodiment of the invention, the application of the monoclonal antibody in the detection of florfenicol, chloramphenicol and thiamphenicol is provided.

According to a third aspect of the embodiment of the invention, the detection card for detecting florfenicol, chloramphenicol and thiamphenicol residues in meat comprises a bottom plate, a sample pad, a combination pad, a base membrane and a water absorption pad, wherein the combination pad contains a quantum dot labeled monoclonal antibody, the monoclonal antibody in the quantum dot labeled monoclonal antibody is secreted by a florfenicol FF 3C11 cell strain with the preservation number of CCTCC NO: C2019152, the base membrane is respectively provided with a detection line and a control line, the control line is coated with an antibody of goat anti-mouse IgG, and the detection line is coated with a chloramphenicol holoantigen.

The detection card can rapidly, accurately and quantitatively detect the residues of florfenicol, chloramphenicol and thiamphenicol in meat by selecting a specific monoclonal antibody and adopting quantum dots for marking.

Further, the quantum dots are ZnS core/CdSe composite quantum dots. The ZnS core/CdSe composite quantum dot is selected, the fluorescence intensity is high, the efficiency series of the quantum dot is 0.5, the stability is good, and the ZnS core/CdSe composite quantum dot can endure multiple excitations; the absorption spectrum of the quantum dots is wide and continuous, unitary excitation and multivariate emission can be realized, the quantum dots are suitable for multicolor marks, can emit red light, have the effect of long tolerance time, and can better improve the detection effect of the detection card.

Further, the quantum dot labeled monoclonal antibody is prepared by the following method:

(a) cleaning and activating the quantum dots;

(b) mixing the activated quantum dot microspheres and the monoclonal antibody uniformly, and incubating;

(c) adding a sealing solution into the incubated mixture, incubating again, centrifuging, collecting the precipitate, and resuspending to obtain the quantum dot labeled monoclonal antibody.

Further, the activation specifically comprises the following steps:

respectively dissolving N-hydroxysuccinimide (NHS) and N, N' -Dicyclohexylcarbodiimide (DCC) in a pH 6.050mM MES buffer solution to respectively obtain an NHS solution and a DCC solution; and then adding an NHS solution into the washed quantum dots, uniformly mixing, adding a DCC solution, uniformly mixing, incubating at room temperature for 20-30min, centrifuging to remove a supernatant, and resuspending.

Further, the concentration of the solution of NHS and DCC is 20 mg/ml; the volume ratio of the NHS solution to the DCC solution to the quantum dots is 1: 2.

Further, the conjugate pad is prepared by the following method:

spraying PBS buffer solution (pH9.00.01M) containing 0.5% Triton X-100, 0.5% alanine, and 0.05% NaN3 onto the bonding pad, and oven drying; and then, spraying the quantum dot marked monoclonal antibody on the dried bonding pad, and drying.

Further, the sample pad is prepared by the following method:

spraying PBS buffer solution (pH9.00.01M) containing 0.5% Triton X-100 and 0.5% BSA onto the sample pad, and oven drying.

Further, the whole antigen is prepared by the following method:

dissolving chloramphenicol and succinic anhydride in anhydrous pyridine, refluxing at 60 deg.C for 24h, removing pyridine by nitrogen blowing, redissolving with ethyl acetate, standing for layering, and washing to obtain derivative solution; then carrying out reduced pressure concentration and light-shielding vacuum drying on the derivative solution to obtain hapten; dissolving hapten by adopting N, N-dimethylformamide, then adding N-hydroxysuccinimide and N-N-dicyclohexylcarbodiimide, and stirring at room temperature in a dark place for reaction to obtain an activated intermediate product; and then, dropwise adding the activated intermediate product into 1ml of PBS buffer solution containing 10mg of carrier protein under stirring, reacting and dialyzing to obtain the complete antigen.

Further, the carrier protein is bovine serum albumin or a tooth hole hemocyanin.

According to a fourth aspect of the embodiments of the present invention, there is provided a method for detecting florfenicol, chloramphenicol, and thiamphenicol residues in meat, wherein the method employs the above-mentioned detection card for detection;

the detection method comprises the steps of putting a sample into an ethyl acetate aqueous solution for extraction, taking supernatant, drying the supernatant by using nitrogen, re-dissolving the supernatant by using a sample diluent to obtain a mixed solution, and using the mixed solution for detection; the sample diluent comprises the following components in parts by weight: 0.6g of potassium dihydrogen phosphate, 0.3g of potassium chloride, 7.5g of disodium hydrogen phosphate dodecahydrate, 4.5g of sodium chloride and 0.5L of distilled water.

The embodiment of the invention has the following advantages:

(1) the invention adopts the cross cooperation of several universities such as immunology, chemistry, biotechnology, computer software, food science and the like, and the detection card which is prepared by the invention and is based on quantum dot labeling and simultaneously quantitatively detects the residues of florfenicol, chloramphenicol and thiamphenicol in meat can simultaneously, rapidly, accurately and quantitatively detect the residues of florfenicol, chloramphenicol and thiamphenicol in poultry meat, thereby solving the bottleneck that three antibiotics can be simultaneously and quantitatively detected by using one antibody and one antigen in the prior art.

(2) The detection card adopts the quantum dots to mark the monoclonal antibody, coats the chloramphenicol holoantigen of the original coupling carrier protein by adopting a succinic anhydride method, and meets the simultaneous quantitative detection of three substances of florfenicol, chloramphenicol and thiamphenicol.

(3) The detection card can rapidly, sensitively and accurately simultaneously and quantitatively detect the residues of the florfenicol, the chloramphenicol and the thiamphenicol in the poultry meat, greatly shortens the detection time, reduces the operation steps, has simple preparation of all raw materials, reduces the cost and provides convenience for field detection; in addition, the detection card has good stability and long storage time.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other implementation drawings can be derived from the drawings provided by those of ordinary skill in the art without any creative effort.

FIG. 1 is a diagram showing the purity identification of monoclonal antibodies recognizing florfenicol, chloramphenicol and thiamphenicol provided in example 2 of the present invention;

FIG. 2 is a standard curve of chloramphenicol detection using the detection card of Experimental example 1 of the present invention;

FIG. 3 is a standard curve for detecting florfenicol heavy metal cadmium by the detection card provided in Experimental example 1 of the present invention;

FIG. 4 is a standard curve of thiamphenicol detection using the detection card provided in Experimental example 1.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.