Anti-thiamphenicol and / or florfenicol antibody, detection product and method for detecting thiamphenicol and / or florfenicol

Abstract

The invention discloses an antibody resisting 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. Immunodetection experiments prove that the thiamphenicol or florfenicol monoclonal antibody provided by the invention has high specificity on thiamphenicol or florfenicol, the detection sensitivity on thiamphenicol or florfenicol is high, and the linear detection range is wide. The rapid detection of thiamphenicol and florfenicol antibiotic residues in livestock and poultry products and aquatic products is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of thiamphenicol and / or florfenicol detection. Specifically, the present invention relates to an antibody against thiamphenicol and / or florfenicol, a detection product, and a method for detecting thiamphenicol and / or florfenicol. Background Art

[0002] Thiamphenicol (TAP) and florfenicol (FF) both belong to the broad-spectrum antibiotics of the amide alcohol class. Such drugs are widely used to treat bacterial diseases in animals. With the large-scale and non-standard application of thiamphenicol and florfenicol in breeding, the residue problems in animal-derived foods such as livestock, poultry, and aquatic products have been increasingly emphasized. Both of them have toxicities such as blood toxicity, immunotoxicity, and embryo toxicity to humans and animals, threatening the health of consumers. Therefore, it is necessary to strengthen the monitoring of such drugs and control their residues in animal-derived foods. 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, and fish is 50 μg / kg - 3000 μg / kg.

[0003] Currently, the main analytical methods for thiamphenicol and florfenicol are instrumental methods such as liquid chromatography and liquid chromatography-tandem mass spectrometry. Although instrumental methods are accurate and reliable, they have the disadvantages of high cost, complex and time-consuming operation, low throughput, difficulty in popularization, and large consumption of organic solvents. Immunoassay has the characteristics of rapidity, sensitivity, low cost, and easy on-site operation. As a primary screening method, it can be used in combination with instrumental methods and is expected to meet the rapid screening needs of a large number of samples.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is 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 invention is implemented as follows:

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

[0008] In a second aspect, the present invention provides a monoclonal antibody against thiamphenicol and / or florfenicol or an antigen-binding fragment thereof, which comprises: heavy chain complementarity-determining regions and light chain complementarity-determining regions. The heavy chain complementarity-determining regions include: CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light chain complementarity-determining regions include: CDR-L1, CDR-L2 and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is APS.

[0009] In a third aspect, the present invention provides an antibody conjugate, which is formed by conjugating the above-mentioned monoclonal antibody against thiamphenicol and / or florfenicol or an antigen-binding fragment thereof with a labeling agent, and the labeling agent is selected from at least one of fluorescent dyes, enzymes that catalyze substrate chromogenesis, radioisotopes, chemiluminescent reagents and nanoparticle-based labeling agents.

[0010] In a fourth aspect, the present invention provides the use of the monoclonal antibody against thiamphenicol and / or florfenicol or an antigen-binding fragment thereof 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 invention provides a thiamphenicol and / or florfenicol detection product. The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; the detection product comprises the above-mentioned monoclonal antibody against thiamphenicol and / or florfenicol or an antigen-binding fragment thereof, or an antibody secreted by the above-mentioned hybridoma cells.

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

[0016] (1) Add the dilution solution and the above-mentioned monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment to the control wells of the microplate coated with florfenicol antigen, and incubate; add the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment, as well as thiamphenicol standard, florfenicol standard or the sample to be tested to the inhibition wells of the microplate coated with florfenicol antigen, and incubate; by adding the enzyme-labeled secondary antibody to the control wells and inhibition wells respectively for incubation, after the color reaction, detect the absorbance of the control wells and inhibition wells, and obtain the concentration of thiamphenicol and / or florfenicol in the sample to be tested by plotting the standard curve of absorbance vs. thiamphenicol concentration or vs. florfenicol concentration;

[0017] (2) Make the sample to be tested contact with the sample pad of the test strip, and judge whether the sample contains thiamphenicol and / or florfenicol according to the chromatography result of the test strip, or plot the standard curve of the ratio of the chromaticity values of the T line and the C line on the test strip vs. the concentration 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.

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

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

[0020] In the present invention, mice are immunized with the antigen of florfenicol, and after hybridoma fusion, hybridoma cell lines that can stably secrete monoclonal antibodies against thiamphenicol or florfenicol are screened out, and specific and sensitive monoclonal antibodies against thiamphenicol or florfenicol are obtained through screening. The monoclonal antibodies against thiamphenicol or florfenicol provided by the present invention are proved to have high specificity for thiamphenicol or florfenicol through immunoassay experiments, and have high detection sensitivity for thiamphenicol or florfenicol, and a wide linear detection range.

[0021] Therefore, the monoclonal antibody against thiamphenicol and / or florfenicol provided by the present invention can be used to develop detection kits, test strips and other products for thiamphenicol or florfenicol. The present invention provides detection tools and means for the rapid detection of antibiotic residues such as thiamphenicol and florfenicol in livestock and poultry products and aquatic products. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 Process diagram for the preparation of florfenicol succinic anhydride hapten;

[0024] Figure 2 Standard curve diagram for detecting thiamphenicol and florfenicol standards;

[0025] Figure 3 Detection result diagram for detecting fresh egg samples added with thiamphenicol standard and fresh egg samples added with florfenicol standard using colloidal gold test strips respectively;

[0026] Figure 4 Detection result diagram for detecting pork samples added with thiamphenicol standard and pork samples added with florfenicol standard using colloidal gold test strips respectively;

[0027] Figure 5 Result diagram for the stability test of 1 ppb thiamphenicol standard; Detailed implementation manners

[0028] Reference to the embodiments of the present invention will now be provided in detail, with one or more examples described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0029] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (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 hereby expressly incorporated by reference.

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0031] Definition of Terms

[0032] The term "antigen-binding fragment" generally refers to any protein / protein fragment containing CDR regions, especially antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen compound-binding fragments of these antibodies, including Fab, F(ab’)2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies and the minimum antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, as well as related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

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

[0034] In the present invention, the term "complementary determining region or complementarity-determining region", "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins, and refers to the regions containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0035] In the present invention, the heavy chain complementary determining regions are denoted as HCDR and include HCDR1, HCDR2 and HCDR3; the light chain complementary determining regions are denoted as LCDR and include LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label the CDR regions, but CDR regions labeled by other methods also fall within the protection scope of the present invention.

[0036] Under normal circumstances, the variable region VH of the heavy chain of an antibody can be obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

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

[0038] As used herein, the term "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers consisting of natural bases, sugars, and sugar-sugar (backbone) linkages. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of the present invention 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 nitrogen-containing and de-nitrogenated adenine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine.

[0039] In a first aspect, the present invention provides a hybridoma cell that is deposited with the China General Microbiological Culture Collection Center under the deposit number CGMCC NO. 46125. This hybridoma cell can stably secrete monoclonal antibodies against thiamphenicol and / or florfenicol. The deposit date is October 23, 2024, the name of the biological material submitted is JFMS-3B12H11, the taxonomic designation is: mouse hybridoma cell line, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The identification result is viable.

[0040] In a second aspect, the present invention provides a monoclonal antibody against thiamphenicol and / or florfenicol or an antigen-binding fragment thereof, which includes: heavy chain complementary determining regions and light chain complementary determining regions. The heavy chain complementary determining regions include: CDR-H1, CDR-H2, and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light chain complementary determining regions include: CDR-L1, CDR-L2, and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is APS.

[0041] Through immunoassay experiments, it is proved that the monoclonal antibodies against thiamphenicol and / or florfenicol provided by the present invention have high specificity for thiamphenicol or florfenicol, high detection sensitivity for thiamphenicol or florfenicol, and a wide linear detection range. Therefore, the monoclonal antibodies against thiamphenicol and / or florfenicol provided by the present invention can be used to develop products such as detection kits and test strips for thiamphenicol or florfenicol. The present invention provides detection tools and means for the rapid detection of thiamphenicol and florfenicol residues in livestock and poultry products.

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

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

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

[0045] In a preferred embodiment of the application of the present invention, the antibody or its antigen-binding fragment 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 regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light-chain constant region is selected from the κ-type or λ-type light-chain constant regions.

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

[0047] The antigen-binding fragments of the above-mentioned antibodies generally have the same binding specificity as the antibodies from which they are derived. It is readily understood by those skilled in the art from the content described in the present invention that the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemically reducing and cleaving disulfide bonds.

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

[0049] In a third aspect, the present invention provides an antibody conjugate formed by conjugating the above-mentioned monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment with a label, and the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate chromogenesis, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0050] The above-mentioned label refers to a class of substances having characteristics such as luminescence, chromogenesis, radioactivity, etc. that can be directly observed by the naked eye or detected or probed by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through these characteristics. In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it falls within the protection scope of the present invention.

[0051] Fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (such as, for example, but not limited to fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, for example, but not limited to red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, for example, but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, for example, but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, for example, but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0052] In alternative embodiments, the enzymes that catalyze substrate chromogenesis include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0053] In alternative embodiments, the radioisotopes include, but are 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 reagents include, but are not limited to, luminol and its derivatives, lucigenin, copepod luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rhodamine and its derivatives, and peroxyoxalate and its derivatives.

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

[0056] In a preferred embodiment of the application of the present invention, the colloid is selected from colloidal gold, colloidal silver, or colloidal selenium.

[0057] Fourthly, the present invention provides the use of a monoclonal antibody against thiamphenicol and / or florfenicol, or an antigen-binding fragment thereof, or the above-mentioned antibody conjugate in any one of the following:

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

[0059] and (2) preparing a detection product for 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 extend the storage validity period, those skilled in the art can add functional components such as stabilizers and protectants to the reagent as needed. The protein stabilizer is selected from: sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100 and Tween-20. The protectant is selected from cryoprotectants, such as polyols and sugars. The polyols are selected from 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 immobilizing the above-mentioned antibody against thiamphenicol or an antigen-binding fragment thereof on a carrier.

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

[0064] In a preferred embodiment of the application of the present invention, the solid phase is selected from microspheres, plates and membranes;

[0065] In a preferred embodiment of the application of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic particles, latex microspheres, microtiter plates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0066] In a preferred embodiment of the application of the present invention, the sample to be tested is animal-derived food;

[0067] In a preferred embodiment of the application of the present invention, the animal-derived food is selected from livestock and poultry meat products, dairy products, eggs and egg-containing foods or aquatic products.

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

[0069] The aquatic products include, but are not limited to: fresh fish, shrimps, crabs and shellfish.

[0070] The fish are, for example, hairtail, squid, yellow croaker, cod, sardine, shark, whale, salmon, tuna, sea bass, saury, skate, conger eel, Spanish mackerel and yellow croaker, etc.

[0071] Livestock and poultry meat products include, but are not limited to: meat products of cattle, sheep, horses, donkeys, camels, pigs, chickens, ducks, geese, etc. The meat products include, but are not limited to, fresh meat, frozen meat, dried meat, meat mince, etc.

[0072] Dairy products are, for example: milk, goat milk, horse milk, etc.

[0073] In a fifth aspect, the present invention provides a thiamphenicol and / or florfenicol detection product, and the detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; the detection product includes the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment as described above, or the antibody secreted by the above-mentioned hybridoma cells.

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

[0075] Alternatively, the detection product is a test strip, and the test strip includes a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with a monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment. It is assembled in the order of sample pad, conjugate pad, nitrocellulose membrane and absorbent paper, and then cut into test strips of a certain width with a cutting machine.

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

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

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

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

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

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

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

[0083] The aforementioned carrier can be of various materials and forms. For example, it can be preferably selected from containers with a flat bottom. A more typical preferred example is the multi-well plates, microplates, microfluidics-based devices (such as microfluidic chips), petri-dish-like containers, etc. widely used in biochemical detection, and is not limited thereto.

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

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

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

[0087] (1) Add a diluent and the above-mentioned monoclonal antibody or its antigen-binding fragment against thiamphenicol and / or florfenicol to the control wells of a microplate coated with a florfenicol antigen, and incubate; add the monoclonal antibody or its antigen-binding fragment against thiamphenicol and / or florfenicol, as well as a thiamphenicol standard, a florfenicol standard or a sample to be tested to the inhibition wells of the microplate coated with a florfenicol antigen, and incubate; add an enzyme-labeled secondary antibody to the control wells and the inhibition wells respectively for incubation, and after a color reaction, detect the absorbance of the control wells and the inhibition wells, and by plotting a standard curve of absorbance versus the concentration of thiamphenicol or versus the concentration of florfenicol, obtain the concentration of thiamphenicol and / or florfenicol in the sample to be tested according to the standard curve;

[0088] (2) Bring the sample to be tested into contact with the sample pad of the above test strip, and determine whether thiamphenicol and / or florfenicol is contained in the sample according to the chromatography result of the test strip, or draw the ratio of the chromaticity values of the T line and 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 tested sample according to the standard curve.

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

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

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

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

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

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

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

[0096] The term "vector" is used herein in its most common sense and includes any intermediate agent for nucleic acids that can enable the nucleic acids to be introduced into, for example, prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector preferably replicates and / or expresses in cells. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually circular double-stranded DNA, 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 an expression vector is that it usually contains an origin of replication, a promoter, a marker gene and translation control elements.

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

[0099] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. The term "recombinant cell" according to the present invention includes prokaryotes (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells, especially 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 multiple tissue types and include primary cells and cell lines. The nucleic acid can exist in the host cell in single copy form or in two or more copy forms 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 properties of the present invention will be further described in detail below in conjunction with examples.

[0103] Example 1

[0104] This example is for the preparation of monoclonal antibodies.

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

[0106] The method reported in the references was used to prepare the hapten of florfenicol. Weigh 358 mg (1 mmol) of florfenicol (FF) and 159 mg (1.5 mmol) of succinic anhydride (HS), place them in a 50 mL round-bottom flask, add 15 mL of dichloromethane as the solvent, then add 150 μL of triethylamine as the catalyst, and reflux for 8 - 10 h under the condition of a 60 °C oil bath. Evaporate the dichloromethane, dissolve it with ethyl acetate, adjust to acidic with dilute hydrochloric acid water, collect the organic layer, wash it 2 - 3 times repeatedly, combine the organic phases, dehydrate with anhydrous magnesium sulfate, and evaporate to dryness to obtain a white powdery solid, that is, the hapten florfenicol succinic anhydride (FF-HS) (the preparation process route refers to Figure 1 as shown).

[0107] Weigh 10 mg of florfenicol succinic anhydride, dissolve it in 0.5 mL of DMF, add 9.3 mg of DCC and 5.2 mg of N-hydroxysuccinimide (NHS), and react in the dark at room temperature for 8 h. Centrifuge and take the supernatant as the hapten activation solution. Weigh 20 mg of BSA respectively, add 4 mL of 0.01 M PBS, and under the condition of an ice bath, slowly drip 0.2 mL of the hapten activation solution into the BSA solution and react at 4 °C for 8 - 10 h. Put the product into a dialysis bag and dialyze with PBS at 4 °C for 3 d, changing the dialysis solution 3 times a day. The supernatant obtained by centrifuging the collected dialysate is FF-HS-BSA. The preparation method of FF-HS-OVA is the same as the above, replace BSA with OVA, the added volume of the hapten activation solution is 0.3 mL, and other conditions are the same. Store the prepared complete antigen in aliquots at minus 20 degrees.

[0108] 2. Preparation of monoclonal antibodies:

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

[0110] (2) Primary immunization: The diluted complete antigen FF-HS-BSA solution (concentration 1 mg / mL) and an equal volume of Freund's complete adjuvant are fully emulsified with a magnetic stirrer and then injected subcutaneously at multiple points on the back of the mice for immunization. The immunization dose is 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) and an equal volume of Freund's incomplete adjuvant are fully emulsified with a magnetic stirrer and then injected subcutaneously at multiple points on the back of the mice for immunization. The immunization dose is 0.1 mg of antigen per mouse. The booster immunization is carried out once every 2 weeks. Starting from the third booster immunization, orbital blood collection is performed on the 7th day after each immunization, and the antibody titer and inhibition effect are detected by the indirect competitive ELISA method. The coated antigen used is the complete antigen FF-HS-OVA prepared in step 1.

[0112] (4) Boost immunization: Select mice with high titer and good inhibition. Perform boost immunization on the 10th day after the 5th immunization. Inject 100 μL of 1 mg / mL intraperitoneally without adjuvant.

[0113] (5) Cell fusion

[0114] On the 3rd day after boost immunization, perform hybridoma fusion. Fuse spleen cells of mice with mouse myeloma cells sp2 / 0 using PEG 1450. Selectively culture through 2% HAT medium. Screen cell wells with high titer and good inhibition using the indirect competitive ELISA method. Perform subcloning by limited dilution. Finally, obtain the monoclonal antibody hybridoma cell line JFMS-3B12H11 against thiamphenicol. The antibody secreted by this cell line has good specificity for both thiamphenicol and florfenicol, and the detection sensitivity can reach 1 μg / L. This cell line is preserved in the China General Microbiological Culture Collection Center, preservation number: 46125.

[0115] (6) Cryopreservation and resuscitation of cells

[0116] After expanding the obtained hybridoma cell line JFMS-3B12H11, resuspend it by pipetting with DMEM and centrifuge. Freeze it in liquid nitrogen at 1×10 9 cells / mL for long-term preservation. When resuscitating, take out the cryopreservation tube from the liquid nitrogen tank and immediately place it in a 37°C water bath for thawing. Then transfer it to pre-warmed 10 mL DMEM using a pipette, centrifuge to remove the cryopreservation solution, and then transfer it to a culture plate for cultivation.

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

[0118] Take 8 female BALB / C mice at 10 - 12 weeks old, inject 0.3 mL of sterile paraffin oil intraperitoneally per mouse. One week later, inject each mouse intraperitoneally with the monoclonal cell line JFMS-3B12H11, with approximately 10 6 cells per injection. When the abdomen of the mice becomes swollen, start collecting mouse ascites and purify the ascites by the saturated ammonium sulfate method and store it at -20°C. Thus, obtain the purified monoclonal antibody secreted by the hybridoma cell line JFMS-3B12H11.

[0119] Example 2

[0120] Detect the antibody effect of the monoclonal antibody obtained in Example 1.

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

[0122] 1) Coating buffer (0.05M carbonate buffer, pH 9.6): 1.5 g of Na2CO3; 2.94 g of NaHCO3, make up to 1000 mL with pure water;

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

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

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

[0126] 5) Chromogenic solution: TMB stock solution (375 mg of TMB solid + 30 mL of DMSO (prepared according to proportion, stored in the dark at room temperature)), BUFFER (0.1 g of potassium sorbate + 46.04 g of potassium dihydrogen citrate hydrate + 1 L of pure water), the chromogenic solution is prepared immediately before use: 200 μL of TMB stock solution + 11 mL of BUFFER + 3.34 μL of 30% hydrogen peroxide solution;

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

[0128] The checkerboard experiment of antigen and antibody is carried out as follows:

[0129] 1) Coating:

[0130] Gradiently dilute the complete antigen FF-HS-OVA at 1 mg / mL with coating buffer at 1:2000, 1:4000 and 1:8000 to obtain coating solutions of different concentrations of the complete antigen FF-HS-OVA. Add 100 μL of the above-prepared coating solution to each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate, incubate overnight at 4°C, and wash 3 times with PBST;

[0131] 2) Competition:

[0132] Dilute the purchased 1 μg / mL thiamphenicol reference standard from Tanmo Quality Inspection with PBSTG to 10 ng / mL. Add 50 μL of sample diluent to each zero well, and add 50 μL of the diluted 10 ng / mL thiamphenicol reference standard solution to each inhibition well.

[0133] The antibody against thiamphenicol prepared in Example 1 was serially diluted with PBSTG at ratios of 1:2000, 1:4000, 1:8000, and 1:16000 to obtain antibody dilutions of thiamphenicol (50 μL / well), placed in a humid box at 37 °C for 30 min, and the plates were washed 3 times.

[0134] 3) Add enzyme-labeled secondary antibody: Dilute the goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 10,000-fold with PBSTG, add 100 μL to each well, place in a humid box at 37 °C for 30 min, and wash the plates 3 times.

[0135] 4) Color development: Prepare the color development solution freshly. After mixing the prepared TMB solution and hydrogen peroxide in proportion, add 100 μL to each well and develop color at room temperature in the dark for 10 min.

[0136] 5) Termination: Add 50 μL of 2 M H2SO4 to each well and measure the OD value of each well at 450 nm using an enzyme-linked immunosorbent assay reader.

[0137] Calculation formula for inhibition rate: Inhibition rate = (B0 - B) / B0 × 100%, where 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 wells in the enzyme-linked immunosorbent assay plate, and C represents the control wells in the enzyme-linked immunosorbent assay plate.

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

[0142] Example 3

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

[0144] The thiamphenicol and florfenicol standard solutions were respectively diluted into the following different concentrations with the sample diluent: 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. Referring to the checkerboard experiment procedure, a standard curve experiment was carried out. Three parallels were made for each standard concentration. The dilution factor of the coated antigen was selected as 1:4000, and the antibody dilution was 1:4000 for detection.

[0145] Drawing the standard curve: Using the thiamphenicol and florfenicol standard solutions with different concentrations (ng / mL) as the X-axis, and the ratio of absorbance values (B / B0, where B is the average absorbance value of the thiamphenicol / florfenicol standard solution, and B0 is the average absorbance value of the control well) as the Y-axis, draw the standard curve graph. The experiment was set to be repeated 3 times, and the average value of the three experimental results was taken. The obtained standard curve graph was referred to Figure 2 as shown.

[0146] The results showed that the sensitivity of thiamphenicol (IC 50 ) was 1.14 ng / mL, and the linear range was 0.4 ng / mL - 5 ng / mL. The sensitivity of florfenicol (IC 50 ) was 0.66 ng / mL, and the linear range was 0.4 ng / mL - 5 ng / mL. It indicated that the antibody prepared by the above method had high sensitivity to thiamphenicol and florfenicol, and the detection effect was good.

[0147] Example 4

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

[0149] Referring to the preparation method of the thiamphenicol standard in Example 3, standard samples of chloramphenicol and florfenicol amine were prepared.

[0150] The above two analogs were respectively diluted into the following concentrations with the sample diluent: 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] Establish a standard curve and determine the median inhibitory concentration IC 50 (the standard sample concentration value when the inhibition rate reaches 50%). The method for establishing the standard curve was the same as the method for establishing the thiamphenicol standard curve above.

[0152] Cross-reactivity rate (%) = (thiamphenicol IC 50 ) / (analog IC 50 ) × 100%.

[0153]

[0154]

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

[0156] Example 5

[0157] This example 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 (rinsed with pure water), add 2 μL of 0.2 M K2CO3 solution to it, then add 5 μL of 1 mg / mL antibody, mix well by inverting up and down and let stand for 20 min, then add 50 μL of 3% BSA blocking solution, mix well by inverting up and down and 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, add 0.4 mL of reconstitution solution (0.01 M PBS containing 3% sucrose) to the precipitate for reconstitution, spread the solution on a 1 cm × 15 cm sample pad, and dry it in an oven at 37 °C (1.5 h).

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

[0160] 3) Egg spiking detection: Break a fresh egg 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 residue limit requirements in GB 31650.1—2022, add 100 μL of 10 ng / mL thiamphenicol and florfenicol standard products respectively, vortex for 1 min to mix well, then add 4 mL of sample diluent, and vortex for 2 min with a vortex mixer to obtain the sample solution to be detected, and it should be detected immediately. Take 100 μL of the sample solution to be detected and drop it onto the test strip for detection. The detection results are as shown in Figure 3 shown, CK is repeated 2 times, MRL refers to the maximum residue limit in GB 31650.1—2022. It can be clearly seen that when the test strip developed with the antibody prepared by the present invention is used to detect egg samples, it can be used for the rapid detection of thiamphenicol and florfenicol in fresh egg samples.

[0161] 4) Pork spiking detection: Take the defatted 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 requirements 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 to mix evenly, then add 2 mL of sample diluent and vortex for 2 min to mix evenly. Centrifuge at 3000 r / min or more for 5 min at room temperature (20 - 25 °C). The upper clear part is the sample solution. After dilution with the sample diluent, take 100 μL and drop it onto the test strip for detection. The specific dilution method is as shown in the following table. The test results are referred to Figure 4 As shown, MRL refers to the maximum residue limit in GB 31650—2019, 1 / 2MRL is 1 / 2 of the maximum residue limit, and CK is the treatment group without added standard (repeated 3 times). The national standard limits of thiamphenicol and florfenicol in pork are 50 μg / kg and 200 μg / kg respectively. It can be clearly seen that the test strip developed with the antibody prepared by the present invention can be used for the rapid detection of thiamphenicol and florfenicol in meat samples. It can be used to develop detection kits, test strips and other products for thiamphenicol or florfenicol. The present invention provides detection tools and means for the 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 0.02M PB. Randomly take 10 test strips prepared in Example 5 to detect the standard solution. Detect the chromaticity values of the T line and C line on each test strip through a test strip reader, and obtain the ratio of the T line to the C line, and mark them on the test strip respectively, referring to Figure 5 As shown. The calculation method of within - batch stability is as follows (STDEV.S(10 detection data) / AVERAGE(10 detection data))*100 (that is, the ratio of the standard deviation to the average value of all detection data). The test results show that the within - batch stability is 2.9%, indicating that the colloidal gold test strip has good stability.

[0166] In summary, through immunological detection experiments, it is proved that the thiamphenicol or florfenicol monoclonal antibody provided by the present invention has high specificity for thiamphenicol or florfenicol, high detection sensitivity for thiamphenicol or florfenicol, and a wide linear detection range.

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

[0168]

[0169]

[0170] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hybridoma cell, characterized in that, It is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC NO. 46125.

2. A monoclonal antibody against thiamphenicol and / or florfenicol or an antigen-binding fragment thereof, characterized in that, It includes: Heavy chain complementarity determining regions and light chain complementarity determining regions. The heavy chain complementarity determining regions include CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light chain complementarity determining regions include CDR-L1, CDR-L2 and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, 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 its antigen-binding fragment further includes heavy chain framework regions and light chain framework regions; the heavy chain framework regions include 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; the light chain framework regions include 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; Preferably, the antibody or its antigen-binding fragment further includes a constant region, and the constant region includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant regions; The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

4. An antibody conjugate, characterized in that, It is formed by conjugating the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment described in any one of claims 2-3 with a label, and the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate chromogenesis, radioisotopes, chemiluminescent reagents and nanoparticle-based labels; Preferably, the nanoparticle-based label is selected from nanoparticles or colloids; Preferably, the colloid is selected from colloidal gold, colloidal silver or colloidal selenium.

5. Use of the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment described in any one of claims 2-3 or the antibody conjugate described in claim 4 in any one of the following: (1) Detecting thiamphenicol and / or florfenicol; and (2) Preparing a detection product for thiamphenicol and / or florfenicol; The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; Preferably, the sample to be tested is an animal-derived food; Preferably, the animal-derived food is selected from livestock and poultry meat products, eggs, dairy products, egg-containing foods or aquatic products.

6. A thiamphenicol and / or florfenicol detection product, characterized in that, The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; the detection product includes the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment described in any one of claims 2-3, or the antibody secreted by the hybridoma cell described in claim 1; Preferably, the detection product is an ELISA kit, which includes a microplate and the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment, and florfenicol antigen is coated on the microplate. Alternatively, the detection product is a test strip, which includes a sample pad, a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment.

7. The thiamphenicol and / or florfenicol detection product according to claim 6, characterized in that The nitrocellulose membrane has a T line and a C line, florfenicol antigen is on the T line, and the antibody against mouse of X is on the C line; X is sheep, rabbit, horse, monkey or chicken. Preferably, the coating concentration of florfenicol antigen on the T line is 0.1-2 mg / mL, and the coating concentration of the antibody against mouse of X on the C line is 0.2-1.0 mg / mL. Preferably, the florfenicol antigen is a conjugate of florfenicol succinic anhydride hapten and a carrier protein. Preferably, the carrier protein is selected from BSA or OVA. Preferably, the conjugate pad is coated with the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment labeled with colloid; the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment in each milliliter of colloid is 5-20 μg.

8. A method for detecting thiamphenicol and / or florfenicol, characterized in that, It includes any one of the following methods: (1) Add diluent and the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment according to any one of claims 2-3 to the control wells of the microplate coated with florfenicol antigen, and incubate; add the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment according to any one of claims 2-3, as well as thiamphenicol standard, florfenicol standard or the sample to be tested to the inhibition wells of the microplate coated with florfenicol antigen, and incubate; add enzyme-labeled secondary antibody to the control wells and the inhibition wells respectively for incubation, through color reaction, detect the absorbance of the control wells and the inhibition wells, and obtain the concentration of thiamphenicol and / or florfenicol in the tested sample by drawing a standard curve of absorbance vs. the concentration of thiamphenicol or florfenicol. (2) Make the sample to be tested contact with the sample pad of the test strip according to claim 6, and judge whether the sample contains thiamphenicol and / or florfenicol according to the chromatography result of the test strip, or draw a standard curve of the ratio of the chromaticity values of the T line and the C line on the test strip vs. the concentration of thiamphenicol and / or florfenicol, and obtain the concentration of thiamphenicol and / or florfenicol in the tested sample according to the standard curve.

9. The method for detecting thiamphenicol and / or florfenicol according to claim 8, wherein On the microplate, the coating concentration of florfenicol antigen is 2.5×10 -5 -1×10 -4 mg / mL; the addition concentration of the monoclonal antibody against thiamphenicol or its antigen-binding fragment is 2.5×10 -5 -1×10 -3 mg / mL.

10. A nucleic acid molecule, characterized in that, It encodes the monoclonal antibody against thiamphenicol and / or florfenicol or its antigen-binding fragment according to any one of claims 2-3.

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