An antibody, a detection product and a method for detecting thiamphenicol and / or florfenicol

Abstract

The application discloses an antibody against thiamphenicol and / or florfenicol, a detection product and a method for detecting thiamphenicol and / or florfenicol, and relates to the technical field of thiamphenicol / florfenicol detection. The thiamphenicol or florfenicol monoclonal antibody provided by the application has high specificity for thiamphenicol or florfenicol, and has high detection sensitivity and a wide linear detection range for thiamphenicol or florfenicol. The application is helpful for rapidly detecting thiamphenicol and florfenicol antibiotic residues in livestock and poultry products and aquatic products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of thiamphenicol and / or florfenicol detection, in particular to an antibody against thiamphenicol and / or florfenicol, a detection product and a method for detecting thiamphenicol and / or florfenicol. BACKGROUND

[0002] Thiamphenicol (TAP) and florfenicol (FF) belong to the amphenicol class of broad-spectrum antibiotics, which are widely used to treat bacterial diseases in animals. With the large-scale and non-standard use of thiamphenicol and florfenicol in breeding, the problem of residues in animal food such as livestock and poultry, aquatic products, etc. has been increasingly valued. Both of them have hemotoxicity, immunotoxicity and embryotoxicity, etc. to the human body and animals, which threatens the health of consumers. Therefore, it is necessary to strengthen the monitoring and control of such drugs and the residues in animal food. GB-31650 stipulates that the maximum residue limit of thiamphenicol and florfenicol in eggs is 10 μg / kg, and the maximum residue limit in animal tissues such as cattle, sheep, pigs, poultry, fish, etc. is 50 μg / kg-3000 μg / kg.

[0003] At present, the analysis methods for thiamphenicol and florfenicol mainly include liquid chromatography, liquid chromatography-tandem mass spectrometry and other instrumental methods. Although instrumental methods are accurate and reliable, they have the disadvantages of high cost, complex operation, low throughput, difficulty in popularization, and large amount of organic solvent use. Immunoassay has the characteristics of rapidness, sensitivity, low cost, and easy on-site operation, and can be used as a preliminary screening method in combination with instrumental methods, which is expected to meet the rapid screening needs of large quantities of samples.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The present application aims to provide an antibody against thiamphenicol and / or florfenicol, a detection product and a method for detecting thiamphenicol and / or florfenicol to solve the above technical problems.

[0006] The present application is implemented as follows:

[0007] In a first aspect, the present application provides a hybridoma cell, which is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC NO. 46125.

[0008] In a second aspect, the present application provides a monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol, comprising: a heavy chain complementarity determining region comprising CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NOs: 1-3 in order, and a light chain complementarity determining region comprising CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NOs: 4-5 in order, and the amino acid sequence of CDR-L2 is APS.

[0009] In a third aspect, the present application provides an antibody conjugate formed by coupling the above-mentioned monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol and a label selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent and a nanoparticle label.

[0010] In a fourth aspect, the present application provides the use of the above-mentioned monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol or the above-mentioned antibody conjugate in any one of the following:

[0011] (1) detecting thiamphenicol and / or florfenicol;

[0012] and (2) preparing a thiamphenicol and / or florfenicol detection product;

[0013] The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

[0014] In a fifth aspect, the present application provides a thiamphenicol and / or florfenicol detection product, which is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; the detection product comprising the above-mentioned monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol, or the antibody secreted by the above-mentioned hybridoma cell.

[0015] In a sixth aspect, the present application provides a method for detecting thiamphenicol and / or florfenicol, comprising any one of the following methods:

[0016] (1) adding the diluent and the above-mentioned anti-thiamphenicol and / or florfenicol monoclonal antibody or antigen-binding fragment thereof into the control wells of the microwell plate coated with the florfenicol antigen, and incubating; adding the anti-thiamphenicol and / or florfenicol monoclonal antibody or antigen-binding fragment thereof and the thiamphenicol standard, florfenicol standard or sample to be tested into the inhibition wells of the microwell plate coated with the florfenicol antigen, and incubating; adding the enzyme-labeled secondary antibody into the control wells and the inhibition wells respectively, and incubating, and then detecting the absorbance of the control wells and the inhibition wells through color development, and then drawing a standard curve of the absorbance and the thiamphenicol concentration or the florfenicol concentration, and then obtaining the thiamphenicol and / or florfenicol concentration of the sample to be tested according to the standard curve;

[0017] (2) contacting the sample to be tested with the sample pad of the test strip, and judging whether the sample contains thiamphenicol and / or florfenicol according to the chromatographic result of the test strip, or drawing a standard curve of the ratio of the colorimetric value of the T line to the C line on the test strip and the thiamphenicol and / or florfenicol concentration, and then obtaining the thiamphenicol and / or florfenicol concentration of the sample to be tested according to the standard curve.

[0018] In a seventh aspect, the present application provides a nucleic acid molecule encoding the above-mentioned anti-thiamphenicol and / or florfenicol monoclonal antibody or antigen-binding fragment thereof.

[0019] The present application has the following beneficial effects:

[0020] The present application immunizes mice with the antigen of florfenicol, and then fuses hybridomas to screen hybridoma cell strains capable of stably secreting anti-thiamphenicol or florfenicol monoclonal antibodies, and then obtains specific and sensitive thiamphenicol or florfenicol monoclonal antibodies through screening. The immunodetection experiment proves that the thiamphenicol or florfenicol monoclonal antibodies provided by the present application have high specificity for thiamphenicol or florfenicol, high detection sensitivity for thiamphenicol or florfenicol, and wide linear detection range.

[0021] Therefore, the anti-thiamphenicol and / or florfenicol monoclonal antibodies provided by the present application can be used for developing thiamphenicol or florfenicol detection kits, test strips and other products, and the present application provides detection tools and means for rapid detection of thiamphenicol or florfenicol antibiotic residues in livestock and poultry products and aquatic products. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0023] Figure 1 Preparation process chart of fluorfenicol succinic anhydride hapten;

[0024] Figure 2 Standard curve chart for detecting thiamphenicol and florfenicol standard products;

[0025] Figure 3 Detection result chart for detecting fresh egg sample added with thiamphenicol standard product and fresh egg sample added with florfenicol standard product by using colloidal gold test paper strip respectively;

[0026] Figure 4 Detection result chart for detecting pork sample added with thiamphenicol standard product and pork sample added with florfenicol standard product by using colloidal gold test paper strip respectively;

[0027] Figure 5 Stability test result chart of 1ppb thiamphenicol standard product. DETAILED DESCRIPTION

[0028] Reference will now be made in detail to the embodiments of the present application, one or more examples of which are set forth below. Each example is provided as an explanation and not as a limitation of the present application. Indeed, it will be apparent to one of ordinary skill in the art that modifications and variations to the present application can be made without departing from the scope or spirit of the application. One of ordinary skill in the art will readily recognize a wide variety of alternatives for example methods from this disclosure and the functionality of the same. Additionally, the various instances of in this disclosure are not meant to be all inclusive or exclusive; rather, they are provided for illustrative purposes to aid in the understanding of the present application.

[0029] Laboratory Manual, 2nd Ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0030] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If no specific conditions are indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If no manufacturers of the reagents or instruments are indicated, the reagents or instruments are all conventional products which can be purchased in the market.

[0031] Nomenclature

[0032] The term "antigen binding fragment" is used in the broadest sense and includes all protein / protein fragments that comprise CDR regions, particularly antibodies or antibody functional fragments. "Antigen binding fragments" include antigen binding fragments of the above-mentioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, diabodies, multispecific antibodies, and minimal recognition units of antibodies, as well as single chain derivatives of these antibodies and fragments. The class of antibodies can be selected from IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0033] The term "antibody" herein is used in the broadest sense and specifically covers full length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments so long as they exhibit the desired biological activity, such as specific binding to florfenicol antigen or fragments thereof.

[0034] In the present application, the term "complementarity determining region or complementarity determining region", "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to the region comprising one or more or even all of the main amino acid residues that affect the binding affinity of the antibody or antigen binding fragment to the antigen or epitope it recognizes. In the detailed description of the present application, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0035] In the present application, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. The commonly used CDR labeling method in the art includes: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of immunoglobulin superfamily in 1997. Kabat et al. was the first to propose a standardized numbering scheme for immunoglobulin variable regions. In the past few decades, the accumulation of sequences led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be the standard widely adopted for numbering antibody residues. The present application adopts the standard labeling of CDR regions by Kabat annotation, but CDR regions labeled by other methods are also within the protection scope of the present application.

[0036] Generally, the variable region of the heavy chain of the antibody VH can be obtained by connecting the CDRs and FRs numbered as follows in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0037] The variable region of the light chain of the antibody VL can be obtained by connecting the CDRs and FRs numbered as follows in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0038] The term "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars, and internucleosidic (backbone) linkages. The term also encompasses modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of the present application can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and can contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases can also be included. Examples of these modified bases include nitrogenous and deazapurines, deazopyrimidines, and xanthine and hypoxanthine.

[0039] In a first aspect, the present application provides a hybridoma cell, which is deposited in China General Microbiological Culture Collection Center, and the deposit number is CGMCC NO. 46125. The hybridoma cell can stably secrete a monoclonal antibody against thiamphenicol and / or florfenicol. The deposit date is October 23, 2024, the name of the biological material submitted is JFMS-3B12H11, the classification name is mouse hybridoma cell strain, and the deposit address is No. 3, Beichen West Road, Beijing City, Chaoyang District. The identification result is survival.

[0040] In a second aspect, the present application provides a monoclonal antibody or antigen binding fragment thereof against thiamphenicol and / or florfenicol, which comprises: a heavy chain complementarity determining region and a light chain complementarity determining region, the heavy chain complementarity determining region comprises: CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 1-3 in turn, and the light chain complementarity determining region comprises: CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5 in turn, and the amino acid sequence of CDR-L2 is APS.

[0041] The anti-thiamphenicol and / or florfenicol monoclonal antibody provided by the application has high specificity to thiamphenicol or florfenicol, high detection sensitivity to thiamphenicol or florfenicol, and wide linear detection range. Therefore, the anti-thiamphenicol and / or florfenicol monoclonal antibody provided by the application can be used for developing a thiamphenicol or florfenicol detection kit, test strip and other products, and the application provides a detection tool and means for rapid detection of thiamphenicol or florfenicol antibiotic residues in livestock and poultry products.

[0042] In a preferred embodiment of the application, the antibody or antigen-binding fragment thereof further comprises a heavy chain framework region and a light chain framework region; the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4 having at least 80% homology to the amino acid sequences shown in SEQ ID NOs: 6-9 in order; for example, the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequences shown in SEQ ID NOs: 6-9 in order.

[0043] The light chain framework region comprises LFR1, LFR2, LFR3 and LFR4 having at least 80% homology to the amino acid sequences shown in SEQ ID NOs: 10-13 in order; for example, the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequences shown in SEQ ID NOs: 10-13 in order.

[0044] In an embodiment, the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 14, and the light chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 15.

[0045] In a preferred embodiment of the application, the antibody or antigen-binding fragment thereof further comprises a constant region, the constant region comprises a heavy chain constant region, and / or, a light chain constant region, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the kappa type or lambda type light chain constant region.

[0046] The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of an antibody.

[0047] The antigen-binding fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they are derived. It is readily understood by one skilled in the art in light of the teachings of the present application that functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by methods of chemical reduction to split disulfide bonds.

[0048] The antigen-binding fragments of the above-mentioned antibodies can also be obtained by recombinant genetic engineering techniques, which are also known to those skilled in the art, or by synthesis, for example, using an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems and the like.

[0049] In a third aspect, the present application provides an antibody conjugate formed by conjugating the above-mentioned monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol and a label selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle label.

[0050] The above-mentioned label refers to a type of substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by the naked eye or detected or probed by an instrument, and through which qualitative or quantitative detection of the corresponding target substance can be achieved. In actual use, one skilled in the art can select a suitable label according to the detection conditions or actual needs, and regardless of the label used, it falls within the scope of protection of the present application.

[0051] The fluorescent dyes include, but are not limited to, fluorescein dyes and derivatives thereof (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorin (FAM), tetrafluorin (TET), etc., or analogs thereof), rhodamine dyes and derivatives thereof (for example, including but not limited to red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc., or analogs thereof), Cy series dyes and derivatives thereof (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc., or analogs thereof), Alexa series dyes and derivatives thereof (for example, including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc., or analogs thereof), and protein dyes and derivatives thereof (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0052] In alternative embodiments, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0053] In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu, and 18 F.

[0054] In alternative embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, metachromatic animal fluorescein and its derivatives, ruthenium bispyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lucigenin and its derivatives, and peroxymalonic acid salt and its derivatives.

[0055] In preferred embodiments of the application, the nanoparticle-based label is selected from the group consisting of nanoparticles or colloids; in preferred embodiments of the application, the nanoparticle-based label is selected from the group consisting of nanoparticles or colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0056] In preferred embodiments of the application, the colloid is selected from the group consisting of colloidal gold, colloidal silver, or colloidal selenium.

[0057] In a fourth aspect, the present application provides the use of the monoclonal antibody or antigen binding fragment thereof against thiamphenicol and / or florfenicol or the antibody conjugate thereof as described above in any one of the following:

[0058] (1) detecting thiamphenicol and / or florfenicol;

[0059] (2) preparing a detection product of thiamphenicol and / or florfenicol;

[0060] The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

[0061] In order to improve the stability of the reagent and prolong the effective period of preservation, the skilled person in the art can add functional components such as stabilizers and protective agents to the reagent as needed, the protein stabilizer is selected from the group consisting of sucrose, trehalose, BSA, glycerol, mannitol, Triton X-100 and Tween-20. The protective agent is selected from the group consisting of cryoprotective agents such as polyols and sugars. The polyol is selected from the group consisting of sorbitol, mannitol or a mixture thereof. The form of the reagent includes but is not limited to solid, liquid and semi-solid.

[0062] The antibody chip refers to a chip formed by fixing the antibody or antigen binding fragment thereof against thiamphenicol described above on a carrier.

[0063] In a preferred embodiment of the application, the kit comprises a solid phase, and the antibody or antigen binding fragment thereof is coated on the solid phase; for example, the antibody or antigen binding fragment thereof is connected to the solid phase by chemical coupling.

[0064] In a preferred embodiment of the application, the solid phase is selected from the group consisting of microspheres, plates and membranes.

[0065] In a preferred embodiment of the application, the solid phase is selected from the group consisting of magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0066] In a preferred embodiment of the application, the sample to be tested is an animal food;

[0067] In a preferred embodiment of the application, the animal food is selected from the group consisting of livestock and poultry meat products, dairy products, egg products and egg-containing food products, and aquatic products.

[0068] The egg products and egg-containing food products include but are not limited to: chicken eggs, duck eggs, goose eggs, quail eggs, ostrich eggs, and egg yolk powder, egg liquid, dried egg of any of the above.

[0069] The aquatic products include but are not limited to: fish, shrimp, crab, and shellfish.

[0070] Fish is, for example, hairtail, squid, yellow croaker, cod, sardine, shark, whale, salmon, tuna, sea perch, flying fish, skate, sea eel, Spanish mackerel and yellow croaker, etc.

[0071] Livestock meat products include, but are not limited to, beef, mutton, horse meat, donkey meat, camel meat, pork, chicken, duck, goose, etc. Meat products include, but are not limited to, fresh meat, frozen meat, dried meat, meat paste, etc.

[0072] Milk products are, for example, cow's milk, goat's milk, horse milk, etc.

[0073] In a fifth aspect, the present application provides a thiamphenicol and / or florfenicol detection product, which is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; the detection product comprises the above-mentioned anti-thiamphenicol and / or florfenicol monoclonal antibody or antigen-binding fragment thereof, or the antibody secreted by the above-mentioned hybridoma cell.

[0074] In a preferred embodiment of the application, the detection product is an ELISA kit, which comprises a microplate and an anti-thiamphenicol and / or florfenicol monoclonal antibody or antigen-binding fragment thereof, and the microplate is coated with a florfenicol antigen; preferably, it is a competitive ELISA kit.

[0075] Alternatively, the detection product is a test strip, which comprises a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with an anti-thiamphenicol and / or florfenicol monoclonal antibody or antigen-binding fragment thereof. The test strip is assembled in the order of sample pad, conjugate pad, nitrocellulose membrane and absorbent paper, and then cut into a certain width by a cutting machine.

[0076] In a preferred embodiment of the application, the nitrocellulose membrane has a T line and a C line, the T line has a florfenicol antigen, and the C line has an X anti-mouse antibody; X is a goat, a rabbit, a horse, a monkey or a chicken;

[0077] In a preferred embodiment of the application, the coating concentration of the florfenicol antigen on the T line is 0.1-2 mg / mL, and the coating concentration of the X anti-mouse antibody on the C line is 0.2-1.0 mg / mL; under this coating concentration, the detection effect is good.

[0078] In a preferred embodiment of the application, the florfenicol antigen is a conjugate of a florfenicol succinic anhydride hapten and a carrier protein;

[0079] In a preferred embodiment of the application, the carrier protein is selected from BSA or OVA;

[0080] In a preferred embodiment of the application, the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol is coated on the binding pad with colloidal labels; and the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol is 5-20 μg per mL of colloid.

[0081] The colloid is, for example, colloidal gold, colloidal silver or colloidal selenium.

[0082] The chip can also be referred to as a suspension array or a liquid array. It comprises a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound on the surface of the carrier.

[0083] The carrier can be of various materials and forms, for example, it can be preferably selected from containers with flat bottoms. A more typical preferred example is a multe-well plate, a microplate, a microfluidic-based device (such as a microfluidic chip), a container similar to a petri dish, and the like, and is not limited thereto.

[0084] The microfluidic chip is selected from a PDMS chip or a metal droplet generator of a T-shaped chip, a flow focusing type chip or a coaxial flow type chip, or a PMMA microfluidic chip.

[0085] Further, the kit can further comprise at least one of a buffer, a detection reagent, a diluent, a washing solution, a thiamphenicol or florfenicol antigen, and a thiamphenicol or florfenicol standard, and is not limited thereto.

[0086] In a sixth aspect, the present application provides a method for detecting thiamphenicol and / or florfenicol, comprising any one of the following methods:

[0087] (1) adding a diluent and the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol to the control wells of the microplate coated with the florfenicol antigen, and incubating; adding the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol, and a thiamphenicol standard, a florfenicol standard or a sample to be tested to the inhibition wells of the microplate coated with the florfenicol antigen, and incubating; adding an enzyme-labeled secondary antibody to the control wells and the inhibition wells, respectively, and incubating, and then detecting the absorbance of the control wells and the inhibition wells through a color development reaction, and drawing a standard curve of the absorbance versus the thiamphenicol concentration or the florfenicol concentration, and obtaining the concentration of thiamphenicol and / or florfenicol in the sample to be tested according to the standard curve;

[0088] (2) make the sample to be tested contact with the sample pad of the test strip, and determine whether the sample contains thiamphenicol and / or florfenicol according to the chromatographic result of the test strip or draw the ratio of the colorimetric value of the T line to the C line on the test strip to the standard curve of thiamphenicol and / or florfenicol, and obtain the concentration of thiamphenicol and / or florfenicol in the sample to be tested according to the standard curve.

[0089] The enzyme-labeled secondary antibody includes but is not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate deoxyenzyme-labeled secondary antibody.

[0090] When drawing the standard curve of absorbance and thiamphenicol concentration, it includes but is not limited to drawing the standard curve of the ratio of the absorbance of the inhibition well to the control well to the concentration of the thiamphenicol standard, or drawing the standard curve of the ratio of the absorbance of (control well-inhibition well) to the control well to the concentration of the thiamphenicol standard.

[0091] When drawing the standard curve of absorbance and florfenicol concentration, it includes but is not limited to drawing the standard curve of the ratio of the absorbance of the inhibition well to the control well to the concentration of the florfenicol standard, or drawing the standard curve of the ratio of the absorbance of (control well-inhibition well) to the control well to the concentration of the florfenicol standard.

[0092] In a preferred embodiment of the application, the coating concentration of the florfenicol antigen on the microwell plate is 2.5 x 10 -5 -1 x 10 -4 mg / mL; and the addition concentration of the anti-thiamphenicol and / or florfenicol monoclonal antibody or its antigen-binding fragment is 2.5 x 10 -5 -1 x 10 -3 mg / mL (or dilution of 1:1000-4000). Under the above coating concentration and addition concentration, the inhibition effect of thiamphenicol or and / or florfenicol is the best, and the higher the inhibition rate is, the higher the binding activity of the antibody to thiamphenicol and / or florfenicol in the test sample is, and the more accurate the detection result is.

[0093] In a seventh aspect, the present application provides a nucleic acid molecule encoding the above-mentioned anti-thiamphenicol and / or florfenicol monoclonal antibody or its antigen-binding fragment.

[0094] In an embodiment, the coding sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 16, and the coding sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 17.

[0095] In an eighth aspect, the present application further provides a vector containing the above-mentioned nucleic acid molecule.

[0096] The term "vector" is used herein in its broadest meaning and includes any intermediate vehicle for a nucleic acid which is capable of, for example, introducing the nucleic acid into a prokaryotic and / or eukaryotic cell and, where appropriate, integrating into the genome. Vectors of this type are preferably replicated and / or expressed in the cell. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors which are well known in the art. The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, usually circular DNA double strands, which can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and be stable in the host.

[0097] In an alternative embodiment, the above-mentioned vector is an expression vector, an important feature of which is that it usually contains an origin of replication, a promoter, a marker gene and a translation control element.

[0098] In a ninth aspect, the present application also provides a recombinant cell comprising the above-mentioned vector.

[0099] The term "recombinant cell" refers to any cell which can be transformed or transfected with an exogenous nucleic acid. The term "recombinant cell" according to the present application comprises prokaryotic (e.g. E. coli) or eukaryotic cells (e.g. mammalian cells, in particular human cells, yeast cells and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats or primates. The cells can be derived from a plurality of tissue types and comprise primary cells and cell lines. The nucleic acid can be present in the host cell in a single copy or in two or more copies and, in one embodiment, is expressed in the recombinant cell.

[0100] In an alternative embodiment, the recombinant cell is a eukaryotic cell.

[0101] In an alternative embodiment, the recombinant cell is a mammalian cell.

[0102] The features and nature of the present application will appear still more clearly from the following detailed description of specific embodiments, given by way of example only.

[0103] Example 1

[0104] This example describes the production of monoclonal antibodies.

[0105] 1. Preparation of florfenicol hapten and complete antigen

[0106] The preparation of florfenicol hapten was carried out according to the method reported in the reference. Florfenicol (FF) 358 mg (1 mmol) and succinic anhydride (HS) 159 mg (1.5 mmol) were weighed into a 50 mL round-bottom flask, dichloromethane 15 mL was added as solvent, then 150 μL of triethylamine was added as catalyst, and the reaction was carried out under 60 °C oil bath condition for 8-10 h. The dichloromethane was evaporated, and the product was dissolved in ethyl acetate, and diluted with dilute hydrochloric acid to make it acidic. The organic layer was collected and washed repeatedly for 2-3 times. The organic phase was combined and dehydrated with anhydrous magnesium sulfate, and then evaporated to obtain white powder, which was the hapten florfenicol succinic anhydride (FF-HS) (the preparation process is shown in Figure 1

[0107] The florfenicol succinic anhydride 10 mg was weighed and dissolved in 0.5 mL of DMF, and then DCC 9.3 mg and N-hydroxysuccinimide (NHS) 5.2 mg were added. The reaction was carried out at room temperature for 8 h in the dark, and the supernatant was obtained by centrifugation, which was the hapten activation solution. 20 mg of BSA was weighed and added to 4 mL of 0.01 M PBS. The hapten activation solution 0.2 mL was slowly added to the BSA solution under ice bath condition, and the reaction was carried out at 4 °C for 8-10 h. The product was loaded into a dialysis bag and dialyzed with PBS at 4 °C for 3 days, and the dialysis solution was changed 3 times a day. The supernatant obtained by centrifugation of the dialysate was FF-HS-BSA. The preparation method of FF-HS-OVA was the same as above, and BSA was replaced by OVA, the volume of the hapten activation solution was 0.3 mL, and the other conditions were the same. The prepared complete antigen was stored at -20 °C.

[0108] 2. Preparation of monoclonal antibody:

[0109] (1) 3 BALB / C female mice aged 6-8 weeks were selected as experimental animals.

[0110] (2) Primary immunization: The diluted complete antigen FF-HS-BSA solution (concentration 1 mg / mL) was mixed with an equal volume of Freund's complete adjuvant, and then emulsified with a magnetic stirrer. The mice were immunized by subcutaneous injection at multiple points on the back, and the immunization dose was 0.1 mg of antigen per mouse.

[0111] (3) Booster immunization: Two weeks after the primary immunization, the diluted complete antigen FF-HS-BSA solution (concentration 1 mg / mL) was mixed with an equal volume of Freund's incomplete adjuvant, and then emulsified with a magnetic stirrer. The mice were immunized by subcutaneous injection at multiple points on the back, and the immunization dose was 0.1 mg of antigen per mouse. Booster immunization was carried out every 2 weeks, and the antibody titer and inhibition effect were detected by indirect competitive ELISA method on the 7th day after each immunization, and the coating antigen was the complete antigen FF-HS-OVA prepared in step 1. ​

[0112] (4) Boosting immunization: select mice with high titer and good inhibition, and perform boosting immunization on the 10th day after the 5th immunization, 1 mg / mL 100 uL intraperitoneal injection, without adjuvant.

[0113] (5) Cell fusion

[0114] On the 3rd day after the boosting immunization, hybridoma fusion was performed, and the mouse spleen cells were fused with mouse myeloma cells sp2 / 0 by PEG 1450, and selected and cultured by 2% HAT medium, and the cell wells with high titer and good inhibition were screened by indirect competitive ELISA method, and subcloned by limiting dilution, and finally the monoclonal antibody hybridoma cell strain JFMS-3B12H11 of thiamphenicol was obtained. The antibody secreted by the cell strain has good specificity to thiamphenicol and florfenicol, and the detection sensitivity can reach 1 ug / L. The cell strain is preserved in China General Microbial Culture Collection Management Center, and the preservation number is: 46125.

[0115] (6) Freezing and recovery of cells

[0116] After the obtained hybridoma cell strain JFMS-3B12H11 is expanded and cultured, it is resuspended by blowing with DMEM and centrifuged, and frozen in liquid nitrogen with a cell freezing solution at 1x10 9 / mL for long-term preservation. When recovering, the frozen tube is taken out from the liquid nitrogen tank and immediately placed in a 37°C water bath for thawing, and then transferred to preheated 10 mL DMEM with a pipette for centrifugation to remove the freezing solution, and then transferred to a culture plate for culture.

[0117] (7) Preparation and purification of monoclonal antibody.

[0118] Take 8 10-12 week old BALB / C female mice, intraperitoneally inject sterile paraffin oil, 0.3 mL each. One week later, each mouse was injected with monoclonal cell strain JFMS-3B12H11 intraperitoneally, and the number of cells injected per mouse was about 10 6 When the mouse abdomen swells, the mouse ascites is collected, and the ascites is purified by saturated ammonium sulfate method and stored at -20°C. Thus, the purified monoclonal antibody secreted by the hybridoma cell strain JFMS-3B12H11 is obtained.

[0119] Example 2

[0120] The monoclonal antibody obtained in Example 1 was detected for antibody effect.

[0121] The various buffers used in the following experiments are as follows:

[0122] 1) Coating buffer (pH 9.6 0.05M carbonate buffer): Na2CO31.5g; NaHCO32.94g, add pure water to 1000mL;

[0123] 2) Phosphate buffer PBS (0.01M pH 7.4): 0.2g KH2PO4; 8g NaCl; 2.92g NaH2PO4·12H2O, add pure water to 1000mL;

[0124] 3) Washing buffer (PBST): add 1mL Tween-20 to the prepared 1000mL PBS solution;

[0125] 4) Sample diluent (PBSTG): add 1mL Tween-20, 1g gelatin (microwave heating and melting) to the prepared PBS, and make up to 1L;

[0126] 5) Color developing solution: TMB storage solution (TMB solid 375mg + 30mL DMSO (prepared in proportion, avoid light and store at room temperature)), BUFFER (sorbitol potassium 0.1g + citric acid dihydrogen potassium hydrate 46.04g + pure water 1L), color developing solution prepared on site: TMB storage solution 200uL + 11mL BUFFER + 3.34uL 30% hydrogen peroxide solution;

[0127] 6) Stop solution (2M H2SO4): distilled water 445.6mL, add 54.4mL of concentrated sulfuric acid (98%) dropwise and stir.

[0128] The following is a chessboard experiment of antigen and antibody:

[0129] 1) Coating:

[0130] Dilute 1mg / mL complete antigen FF-HS-OVA with coating buffer at 1:2000, 1:4000 and 1:8000 to obtain coating solutions of different concentrations of complete antigen FF-HS-OVA, add 100uL of the prepared coating solution to each well of a 96-well enzyme-labeled plate, and incubate at 4°C overnight, then wash 3 times with PBST;

[0131] 2) Competition:

[0132] Dilute the 1ug / mL thiamphenicol standard purchased from the test tank to 10ng / mL with PBSTG, add 50uL of sample diluent to each well of the zero hole, and add 50uL of the diluted 10ng / mL thiamphenicol standard solution to each well of the inhibition hole.

[0133] The sulfomycin antibody prepared in Example 1 was diluted with PBSTG at a gradient of 1:2000, 1:4000, 1:8000, and 1:16000 to obtain a sulfomycin antibody diluent (50 uL / well), and was placed in a wet box at 37°C for 30 min, and the plate was washed 3 times.

[0134] 3) Add enzyme-labeled secondary antibody: dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson Company) 10000 times with PBSTG, add 100 uL per well, and place in a wet box at 37°C for 30 min, and wash the plate 3 times.

[0135] 4) Color development: color developing solution is prepared on site, and the prepared TMB solution is mixed with hydrogen peroxide according to the proportion, 100 uL per well, color development at room temperature for 10 min.

[0136] 5) Termination: add 50 uL of 2M H2SO4 per well, and measure the OD value of each well at 450 nm with an enzyme-labeled instrument.

[0137] The calculation formula of the inhibition rate is: inhibition rate = (B0-B) / B0x100%, B0 is the OD value of the control well; B is the OD value of the inhibition well.

[0138] The results are shown in the following table:

[0139]

[0140] Note: I represents the inhibition well in the enzyme-labeled plate, and C represents the control well in the enzyme-labeled plate.

[0141] The results show that when the coating antigen dilution is 1:4000 and the antibody dilution is 1:4000, the inhibition effect on sulfomycin is the best, with an inhibition rate of 82%, indicating that the antibody produced by the above hybridoma cell JFMS-3B12H11 can detect sulfomycin. The higher the inhibition rate, the higher the binding activity of the antibody to sulfomycin in the test sample, and the more accurate the detection result, indicating that the antibody produced by the above hybridoma cell can detect sulfomycin.

[0142] Example 3

[0143] This example provides a standard curve for detecting sulfomycin and florfenicol, and tests the sensitivity of the detection method.

[0144] The sulfathiazole and florfenicol standard solution was diluted with sample diluent to the following different concentrations: 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.3125 ng / mL. The standard curve experiment was performed according to the checkerboard experiment procedure, 3 parallel samples were prepared for each standard concentration, the coating antigen dilution factor was 1:4000, and the antibody dilution factor was 1:4000.

[0145] The standard curve was plotted: the sulfathiazole and florfenicol standard solution with different concentrations (ng / mL) was taken as the X axis, and the ratio of absorbance values (B / B0, wherein B is the average absorbance value of the sulfathiazole / florfenicol standard solution, and B0 is the average absorbance value of the control well) was taken as the Y axis. The standard curve was plotted according to the average value of three experimental results, as shown in Figure 2 .

[0146] The results show that the sensitivity (IC 50 ) of sulfathiazole is 1.14 ng / mL, and the linear range is 0.4 ng / mL-5 ng / mL. The sensitivity (IC 50 ) of florfenicol is 0.66 ng / mL, and the linear range is 0.4 ng / mL-5 ng / mL. The above method prepared the antibody with high sensitivity to sulfathiazole and florfenicol, and the detection effect was good.

[0147] Example 4

[0148] In this embodiment, the antibody prepared in Example 1 was subjected to specific detection.

[0149] According to the preparation method of the sulfathiazole standard sample in Example 3, chloramphenicol and florfenicol standard samples were prepared.

[0150] The above two kinds of analogues were diluted with sample diluent to the following concentrations: 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL.

[0151] The standard curve was established, and the inhibition concentration IC 50 (the standard sample concentration value at which the inhibition rate reached 50%) was determined. The establishment method of the standard curve was the same as the establishment method of the above sulfathiazole standard curve.

[0152] The cross-reactivity (%) = (sulfathiazole IC 50 ) / (analog IC 50 ) x 100%.

[0153]

[0154]

[0155] The results show that the antibody prepared in Example 1 has good specificity for sulfamethoxazole and florfenicol.

[0156] Example 5

[0157] The present embodiment provides a colloidal gold test strip.

[0158] 1) Preparation of gold-labeled antibody pad: Take 1 mL of colloidal gold solution (30 nm) in a 2 mL centrifuge tube (rinse with pure water), add 2 μL of 0.2M K2CO3 solution, then add 5 μL of 1 mg / mL antibody, mix well by inverting up and down, and then let stand for 20 min. Then add 50 μL of 3% BSA blocking solution, mix well by inverting up and down, and then let stand for 20 min. Then use a high-speed refrigerated centrifuge to centrifuge the mixture at 10000 r / min for 10 min (4°C). Discard the supernatant, and add 0.4 mL of resuspension solution (0.01M PBS containing 3% sucrose) to the precipitate for resuspension. Spread the solution on a 1 cm x 15 cm sample pad, and dry in a 37°C oven (1.5 h).

[0159] 2) The line concentration of the NC membrane is: the T-line antigen (FF-HS-BSA) concentration is 1.0 mg / mL, and the C-line secondary antibody concentration is 1.0 mg / mL.

[0160] 3) Chicken egg spiked detection: Crush the fresh eggs into a 100 mL beaker, mix well (stir the egg white and yolk evenly), weigh 1.00 ± 0.05 g of the well-mixed fresh egg sample into a 10 mL centrifuge tube, then according to the maximum residual limit in GB 31650.1-2022, add 100 μL of 10 ng / mL sulfamethoxazole and florfenicol standard respectively, mix well by vortexing for 1 min, then add 4 mL of sample diluent, vortex for 2 min to obtain the sample solution to be detected, which should be detected immediately. Take 100 μL of the sample solution to be detected and add it to the test paper card for detection. The detection result is shown in Figure 3 As shown in the figure, CK is repeated twice, and MRL refers to the maximum residual limit in GB 31650.1-2022. It can be clearly seen that the test strip developed by the antibody prepared by the present application can be used for rapid detection of sulfamethoxazole and florfenicol in fresh egg samples.

[0161] 4) Pork spiked detection: Take the fat-removed pork sample and homogenize it in a homogenizer, weigh 1.0 ± 0.05 g of the homogenized meat sample into a 10 mL centrifuge tube, then according to the maximum residue limit in GB 31650-2019, add 100 μL of 500 ng / mL thiamphenicol or 100 μL of 2000 ng / mL florfenicol standard, vortex for 1 min, then add 2 mL of sample diluent, vortex for 2 min, centrifuge at 3000 r / min or more at room temperature (20-25°C) for 5 min, the upper clear part is the sample solution, dilute it with sample diluent and take 100 μL to add to the test paper card for detection. The specific dilution method is as follows. The detection result is as follows Figure 4 As shown in the figure, MRL refers to the maximum residue limit in GB 31650-2019, 1 / 2 MRL is 1 / 2 of the maximum residue limit, and CK is the treatment group without adding standard (repeated 3 times). The national standard limit of thiamphenicol and florfenicol in pork is 50 μg / kg and 200 μg / kg respectively, and it can be obviously seen that the test strip developed by the antibody prepared by the application can be used for rapid detection of thiamphenicol and florfenicol in meat samples. It can be used for the development of thiamphenicol or florfenicol detection kit, test strip and other products. The application provides a detection tool and means for rapid detection of thiamphenicol, florfenicol and other antibiotic residues in livestock and poultry products and aquatic products.

[0162] Dilution method table:

[0163]

[0164] Example 6

[0165] Prepare 1 ppb thiamphenicol standard with 0.02 M PB, randomly take 10 test strips prepared in Example 5 to detect the standard solution, detect the color value of T line and C line on each test strip by test strip card reader, obtain the ratio of T line and C line, and mark on the test strip respectively, as shown in the figure. Figure 5 The calculation method of batch stability is as follows (STDEV.S (10 detection data) / AVERAGE (10 detection data)) * 100 (i.e. the ratio of standard deviation and average value of all detection data), the detection result shows that the batch stability is 2.9%, indicating that the colloidal gold test strip has good stability.

[0166] In summary, the immunodetection experiment proves that the thiamphenicol or florfenicol monoclonal antibody provided by the application has high specificity for thiamphenicol or florfenicol, high detection sensitivity for thiamphenicol or florfenicol, and wide linear detection range.

[0167] The sequence information involved in the application is as follows:

[0168]

[0169]

[0170] The above descriptions are only the preferred embodiments of the present application, not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the scope of the present application.

Claims

1. A hybridoma cell, characterized in that, It is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 46125.

2. A monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol, characterized in that, It comprises: The heavy chain complementarity determining region comprises CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are sequentially shown as SEQ ID NO: 1-3; the light chain complementarity determining region comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences of CDR-L1 and CDR-L3 are sequentially shown as SEQ ID NO: 4-5, and the amino acid sequence of CDR-L2 is APS.

3. The monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol according to claim 2, characterized in that, The antibody or the antigen binding fragment thereof further comprises a heavy chain framework region and a light chain framework region; the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4 which have at least 80% homology with the amino acid sequences shown as SEQ ID NO: 6-9 in sequence; and the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4 which have at least 80% homology with the amino acid sequences shown as SEQ ID NO: 10-13 in sequence.

4. The monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol according to claim 3, characterized in that, The antibody or the antigen binding fragment thereof further comprises a constant region, and the constant region comprises a heavy chain constant region and / or a light chain constant region; the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the kappa type or lambda type light chain constant region. The antigen binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

5. An antibody conjugate, characterized in that, The antibody conjugate is formed by coupling the monoclonal antibody or the antigen binding fragment thereof against thiamphenicol and / or florfenicol according to any one of claims 2-4 with a label selected from at least one of an enzyme which catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent and a nanoparticle label.

6. The antibody conjugate of claim 5, wherein, The nanoparticle label is selected from a nanoparticle or a colloid.

7. The antibody conjugate of claim 6, wherein, The colloid is selected from colloidal gold, colloidal silver or colloidal selenium.

8. The monoclonal antibody or the antigen binding fragment thereof against thiamphenicol and / or florfenicol according to any one of claims 2-4 or the antibody conjugate according to any one of claims 5-7 is applied in any one of the following: (1) detecting thiamphenicol and / or florfenicol; and (2) preparing a detection product of thiamphenicol and / or florfenicol. The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

9. Use according to claim 8, characterized in that, The sample to be detected is an animal food.

10. Use according to claim 9, characterized in that, The animal food is selected from livestock and poultry meat products, egg products, dairy products, egg-containing food products or aquatic products.

11. A thiamphenicol and / or florfenicol test product, characterized in that, The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; and the detection product comprises the monoclonal antibody or the antigen binding fragment thereof against thiamphenicol and / or florfenicol according to any one of claims 2-4 or the antibody secreted by the hybridoma cell according to claim 1.

12. The thiamphenicol and / or florfenicol detection product according to claim 11, characterized by, The detection product is an ELISA kit, which comprises a microplate and the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol coated on the microplate, and florfenicol antigen; Or, the detection product is a test strip, which comprises a sample pad, a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol.

13. The thiamphenicol and / or florfenicol detection product according to claim 12, characterized by, The conjugate pad is coated with the colloidal-labeled monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol; the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol is 5-20 μg per milliliter of colloid.

14. A method of detecting thiamphenicol and / or florfenicol, characterized in that, The method comprises any one of the following: (1) adding the diluent and the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol according to any one of claims 2-4 to the control well of the microplate coated with florfenicol antigen, and incubating; adding the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol according to any one of claims 2-4, and thiamphenicol standard, florfenicol standard or sample to be tested to the inhibition well of the microplate coated with florfenicol antigen, and incubating; adding the enzyme-labeled secondary antibody to the control well and the inhibition well respectively, and incubating, and then detecting the absorbance of the control well and the inhibition well through color development reaction, and obtaining the concentration of thiamphenicol and / or florfenicol in the sample to be tested according to the standard curve of the absorbance and the concentration of thiamphenicol or florfenicol; (2) contacting the sample to be tested with the sample pad of the test strip according to claim 12, and determining whether the sample contains thiamphenicol and / or florfenicol according to the chromatography result of the test strip.

15. The method for detecting thiamphenicol and / or florfenicol according to claim 14, wherein, The method for determining whether the sample contains thiamphenicol and / or florfenicol according to the chromatography result of the test strip in method (2) is as follows: drawing the standard curve of the ratio of the colorimetric value of T line to C line on the test strip and the concentration of thiamphenicol and / or florfenicol, and obtaining the concentration of thiamphenicol and / or florfenicol in the sample to be tested according to the standard curve.

16. The method for detecting thiamphenicol and / or florfenicol according to claim 14, wherein, The coating concentration of the florfenicol antigen on the microplate is 2.5 x 10 -5 -1 x 10 -4 mg / mL; and the addition concentration of the anti-thiamphenicol and / or florfenicol monoclonal antibody or antigen-binding fragment thereof is 2.5 x 10 -5 -1 x 10 -3 mg / mL.

17. A nucleic acid molecule, characterized in that, The nucleic acid encodes the monoclonal antibody or antigen-binding fragment thereof against thiamphenicol and / or florfenicol according to any one of claims 2-4.

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