Hybridoma cell and nitrofurantoin metabolite detection method

Abstract

The invention discloses a hybridoma cell and a nitrofurantoin metabolite detection method, and relates to the technical field of nitrofurantoin metabolite detection. Immunodetection experiments prove that the monoclonal antibody for resisting the nitrofurantoin metabolite has the technical advantage of high specificity on derivatives of the nitrofurantoin metabolite, the detection sensitivity on the derivatives of the nitrofurantoin metabolite is high, and the linear detection range is wide. Therefore, the monoclonal antibody for resisting the macrodantin metabolite provided by the invention can be used for developing products such as a detection kit, a test strip and the like of the macrodantin metabolite, a detection tool and means are provided for rapid detection of macrodantin antibiotic residues in livestock and poultry products, aquatic products and feeds, and the monoclonal antibody has important significance in meeting actual detection requirements. Risks are quickly controlled, and the method is of great significance in guaranteeing agricultural product quality safety.

Description

Technical Field

[0001] The present invention relates to the technical field of nitrofurantoin metabolite detection, and more specifically, to a hybridoma cell and a detection method for nitrofurantoin metabolites. Background Art

[0002] Nitrofurantoin belongs to the nitrofurans class of drugs, which are a type of synthetic broad-spectrum antibacterial agents used for the prevention and treatment of gastrointestinal infections caused by Escherichia coli and Salmonella. After entering the animal body, it is quickly metabolized into the nitrofurantoin metabolite 1-amino-2-hydantoin (AHD), which can stably exist in the animal body for a long time. Since nitrofurantoin and its metabolites have potential carcinogenic and mutagenic effects on the human body, many countries have prohibited their use in food-producing animals. The 250th Announcement of the Ministry of Agriculture and Rural Affairs of China clearly stipulates that nitrofurans are prohibited from being used in food animals. However, due to their low cost and obvious effects in preventing and treating bacterial infections, there is still a need for the detection of nitrofurans residues. The national food safety standard GB 31656.13—2021 "Determination of Multi-residues of Nitrofurans Metabolites in Aquatic Products - Liquid Chromatography-Tandem Mass Spectrometry" stipulates that the method detection limit of AHD is 0.5 μg / kg, and the quantification limit is 1 μg / kg.

[0003] Most of the early detections of nitrofurantoin were for the original drug, and the results were not satisfactory. Now, the content of AHD in animal tissues is mostly detected to indirectly reflect the residue situation of the original drug use. Currently, the most widely used instrumental analysis methods mainly include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), etc. However, the instrument cost is high, the operation is complex, the detection time is long, and it requires professional personnel to operate, which cannot meet the demand for the timeliness of detection methods after the implementation of the market access and origin exit systems for agricultural products.

[0004] Immunoassay, as a specific and sensitive detection method, has been widely used in the detection of drug residues in food.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a hybridoma cell and a detection method for nitrofurantoin metabolites, so as to develop highly sensitive nitrofurantoin metabolite antibody raw materials and detection products, which is of great significance for meeting the actual detection needs, quickly controlling risks, and ensuring the quality and safety of agricultural products.

[0007] The present invention is implemented as follows:

[0008] 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. 46123.

[0009] In a second aspect, the present invention provides an antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof, wherein the nitrofurantoin metabolite is l-amino-2-hydantoin, and the antibody or the antigen-binding fragment thereof comprises: heavy-chain complementarity-determining regions and light-chain complementarity-determining regions. The heavy-chain complementarity-determining regions comprise: 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 comprise: 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 VTR.

[0010] In a third aspect, the present invention provides an antibody conjugate which is formed by conjugating the monoclonal antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof described above with a labeling agent, and the labeling agent is selected from at least one of fluorescent dyes, enzymes for catalytic substrate chromogenesis, radioisotopes, chemiluminescent reagents and nanoparticle-based labeling agents.

[0011] In a fourth aspect, the present invention provides the use of the monoclonal antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof or the antibody conjugate described above in any one of the following:

[0012] (1) Detecting nitrofurantoin residues;

[0013] (2) Detecting metabolites of nitrofurantoin;

[0014] (3) Detecting derivatives of metabolites of nitrofurantoin;

[0015] and (4) Preparing a product for detecting nitrofurantoin residues, a product for detecting metabolites of nitrofurantoin or a product for detecting derivatives of metabolites of nitrofurantoin; the derivatives of metabolites of nitrofurantoin are selected from 2-NP-AHD;

[0016] The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

[0017] In a fifth aspect, the present invention provides a product for detecting nitrofurantoin metabolites, and 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 monoclonal antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof described above, or an antibody secreted by the hybridoma cell described above;

[0018] In a preferred embodiment of the application of the present invention, the detection product is an ELISA kit, which includes a microplate and a monoclonal antibody against furantoin metabolite or its antigen-binding fragment, and a derivative antigen of furantoin metabolite is coated on the microplate.

[0019] Alternatively, the detection product is a test strip, which includes a conjugate pad and a nitrocellulose membrane; a monoclonal antibody against furantoin metabolite or its antigen-binding fragment is coated on the conjugate pad, and a T line and a C line are provided on the nitrocellulose membrane. A derivative antigen of furantoin metabolite is provided on the T line, and an anti-mouse antibody of X is provided on the C line; X is sheep, rabbit, horse, monkey or chicken.

[0020] In a sixth aspect, the present invention provides a method for detecting furantoin metabolite, which includes any one of the following methods:

[0021] (1) Add a diluent and the above-mentioned monoclonal antibody against furantoin metabolite or its antigen-binding fragment to the control well of the microplate coated with the derivative antigen of furantoin metabolite, and incubate; add the above-mentioned monoclonal antibody against furantoin metabolite or its antigen-binding fragment, and a derivative standard or a test sample of furantoin metabolite to the inhibition well of the microplate coated with the derivative antigen of furantoin metabolite, and incubate; add an enzyme-labeled secondary antibody to the control well and the inhibition well respectively for incubation, and after a color reaction, detect the absorbance of the control well and the inhibition well. By plotting a standard curve of absorbance versus the concentration of the derivative of furantoin metabolite, the concentration of the derivative of furantoin metabolite in the measured sample is obtained according to the standard curve; before the test sample is loaded, derivatization treatment is performed.

[0022] (2) Load the derivatized test sample onto the sample pad of the test strip, and judge whether the sample contains furantoin metabolite according to the chromatography result of the test strip.

[0023] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody against furantoin metabolite or its antigen-binding fragment.

[0024] In an eighth aspect, the present invention provides a recombinant vector including the above-mentioned nucleic acid molecule.

[0025] The present invention has the following beneficial effects:

[0026] In the present invention, mice are immunized with the antigen of furantoin metabolite 1-amino-2-hydantoin. Through hybridoma fusion, hybridoma cell lines that can stably secrete monoclonal antibodies against furantoin metabolites are screened out, and specific and sensitive monoclonal antibodies against furantoin metabolites are obtained through screening. The immunodetection experiment proves that the monoclonal antibody against furantoin metabolite provided by the present invention has the technical advantages of high specificity for derivatives of furantoin metabolite, high detection sensitivity for derivatives of furantoin metabolite, and a wide linear detection range.

[0027] Therefore, the monoclonal antibody against furantoin metabolite provided by the present invention can be used to develop products such as detection kits and test strips for furantoin metabolites. The proposal of the present invention provides detection tools and means for the rapid detection of furantoin antibiotic residues in livestock and poultry products, aquatic products, and feeds, which is of great significance for meeting the actual detection needs, quickly controlling risks, and ensuring the quality and safety of agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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 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.

[0029] Figure 1 It is a preparation route diagram of 4-CPAHD-BSA complete antigen;

[0030] Figure 2 It is a standard curve diagram for detecting derivatives of furantoin metabolites;

[0031] Figure 3 It is a detection result diagram for detecting AHD of furantoin metabolite in 3 sample tissues of shrimp, perch, and grass carp respectively using a test strip;

[0032] Figure 4 It is a test result diagram for the within-batch stability of the test strip. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] Reference will now be provided in detail to embodiments of the present invention, one or more examples of which are 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.

[0034] 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 well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); and "Current Protocols in Immunology" (ed. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0035] 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 clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0036] Definition of Terms

[0037] The term "antigen-binding fragment" generally refers to any protein / protein fragment that contains CDR regions, particularly 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 minimal recognition units of antibodies, 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".

[0038] 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, provided that they exhibit the desired biological activity, such as specifically binding to an antigen or a fragment of a derivative of a nitrofurantoin metabolite.

[0039] 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 an immunoglobulin, and refers to the region that contains one or more or even all of the main 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.

[0040] 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 the variable regions of immunoglobulins. 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 scope of protection of the present invention.

[0041] 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.

[0042] 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.

[0043] As used herein, the term "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers composed of natural bases, sugars, and inter-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.

[0044] In the present invention, derivatives of nitrofurantoin metabolites are also equivalent to nitrofurantoin metabolite structural analogs.

[0045] 2-NP-AHD, with the molecular formula C 10 H8N4O4, and the CAS number: 623145-57-3, is a derivative of nitrofurantoin metabolite.

[0046] 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.46123. This hybridoma cell can stably secrete an antibody against nitrofurantoin metabolite. The biological material submitted for deposit is: AHD-1E9G3, named: mouse hybridoma cell line, the deposit date is October 23, 2024, and the identification result is viable.

[0047] Second aspect, the present invention provides an antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof, the nitrofurantoin metabolite being 1-amino-2-hydantoin, and the antibody or the antigen-binding fragment thereof comprising: 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 VTR.

[0048] The immunodetection experiment proves that the monoclonal antibody against the nitrofurantoin metabolite provided by the present invention has the technical advantages of high specificity for derivatives of the nitrofurantoin metabolite, high detection sensitivity for derivatives of the nitrofurantoin metabolite, and a wide linear detection range. Therefore, the monoclonal antibody against the nitrofurantoin metabolite provided by the present invention can be used to develop products such as detection kits and test strips for the nitrofurantoin metabolite. The proposal of the present invention provides detection tools and means for the rapid detection of nitrofurantoin antibiotic residues in livestock and poultry products, aquatic products, and feeds, which is of great significance for meeting the actual detection needs, quickly controlling risks, and ensuring the quality and safety of agricultural products.

[0049] In a preferred embodiment of the application of the present invention, 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 includes 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; for example, the heavy-chain framework region includes 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 as SEQ ID NO: 6-9 in sequence.

[0050] The light-chain framework region includes 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; for example, the light-chain framework region includes 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 as SEQ ID NO: 10-13 in sequence.

[0051] 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.

[0052] 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 kappa-type or lambda-type light chain constant regions.

[0053] The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

[0054] The antigen-binding fragment of the above antibody generally has the same binding specificity as its source antibody. It is easy for those skilled in the art to understand from the content recorded in the present invention that the functional fragment of the above antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemically reducing and cleaving disulfide bonds.

[0055] The antigen-binding fragment of the above antibody 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 the automated peptide synthesizers sold by Applied BioSystems and the like.

[0056] In a third aspect, the present invention provides an antibody conjugate formed by conjugating the above-mentioned monoclonal antibody against a nitrofurantoin metabolite or its antigen-binding fragment with a label, and the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

[0057] The above-mentioned label refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by instruments, and qualitative or quantitative detection of the corresponding target can be achieved through these characteristics. In the actual use process, 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.

[0058] Fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (e.g., including but not limited to rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (e.g., including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (e.g., including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0059] In alternative embodiments, the enzymes that catalyze the substrate to develop color include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0060] 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 and18 F.

[0061] In an alternative embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rosalic acid and its derivatives, and peroxyoxalate and its derivatives.

[0062] 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.

[0063] In a preferred embodiment of the application of the present invention, the colloid is selected from colloidal gold, colloidal silver, or colloidal selenium.

[0064] In a fourth aspect, the present invention provides the use of a monoclonal antibody against a nitrofurantoin metabolite, or an antigen-binding fragment thereof, or the above-mentioned antibody conjugate in any one of the following:

[0065] (1) Detecting nitrofurantoin residues;

[0066] (2) Detecting metabolites of nitrofurantoin;

[0067] (3) Detecting derivatives of metabolites of nitrofurantoin;

[0068] and (4) Preparing a detection product for nitrofurantoin residues, a detection product for nitrofurantoin metabolites, or a detection product for derivatives of metabolites of nitrofurantoin; the derivatives of metabolites of nitrofurantoin are selected from 2-NP-AHD;

[0069] The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe, or a detector.

[0070] In order to improve the stability of the reagent and extend the shelf life, 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, Triton X-100, and Tween-20. The protectant is selected from cryoprotectants such as polyols and sugars. The polyol is selected from sorbitol, mannitol, or a mixture thereof. The form of the reagent includes, but is not limited to, solid, liquid, and semi-solid.

[0071] An antibody chip refers to a chip formed by immobilizing the above-mentioned antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof on a carrier.

[0072] In a preferred embodiment of the application of the present invention, the kit comprises a solid phase, to which an antibody or its antigen-binding fragment is coated; or, an antigen of a metabolite of nitrofurantoin is coated on the solid phase. For example, by means of chemical coupling, an antibody against a metabolite of nitrofurantoin or an antigen of a metabolite of nitrofurantoin is connected to the solid phase.

[0073] In a preferred embodiment of the application of the present invention, the solid phase is selected from microspheres, plates and membranes;

[0074] 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.

[0075] In a preferred embodiment of the application of the present invention, the sample to be tested is animal-derived food, feed or feed additive;

[0076] In a preferred embodiment of the application of the present invention, the animal-derived food is selected from livestock and poultry meat products, eggs and egg-containing foods or aquatic products.

[0077] The eggs and egg-containing foods include, but are not limited to: eggs, duck eggs, goose eggs, quail eggs, ostrich eggs and egg yolk powder, egg liquid, dried eggs of any of the above eggs.

[0078] The aquatic products include, but are not limited to: fresh fish, shrimps, crabs, shellfish.

[0079] 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.

[0080] The 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.

[0081] The feed is, for example, selected from compound feed, silage feed.

[0082] In a fifth aspect, the present invention provides a nitrofurantoin metabolite 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 comprises the above-mentioned monoclonal antibody against a metabolite of nitrofurantoin or its antigen-binding fragment, or the antibody secreted by the above-mentioned hybridoma cells.

[0083] The above-mentioned chip can also be called 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 to the surface of the carrier.

[0084] The foregoing carriers can be of various materials and forms. For example, they can preferably be 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.

[0085] The microfluidic chip is selected from a PDMS chip or a metal droplet generator of a T-type chip, a flow-focusing type chip or a coaxial flow type chip, or a PMMA microfluidic chip.

[0086] Furthermore, the kit may further include at least one of a buffer, a detection reagent, a diluent, a washing solution, a nitrofurantoin metabolite antigen, a standard of a derivative of a nitrofurantoin metabolite, and is not limited thereto.

[0087] In a preferred embodiment of the application of the present invention, the detection product is an ELISA kit. The ELISA kit includes a microplate and a monoclonal antibody against a nitrofurantoin metabolite or its antigen-binding fragment, and a derivative antigen of a nitrofurantoin metabolite is coated on the microplate; preferably a competitive ELISA kit.

[0088] Alternatively, the detection product is a test strip. The test strip includes a conjugate pad and a nitrocellulose membrane; a monoclonal antibody against a nitrofurantoin metabolite or its antigen-binding fragment is coated on the conjugate pad, and a T line and a C line are provided on the nitrocellulose membrane. A derivative antigen of a nitrofurantoin metabolite is provided on the T line, and an anti-mouse antibody of X is provided on the C line; X is sheep, rabbit, horse, monkey or chicken. It is assembled in the order of a sample pad, a conjugate pad, a nitrocellulose membrane and a blotting paper, and then cut into test strips of a certain width by a cutting machine.

[0089] In a preferred embodiment of the application of the present invention, the coating concentration of the derivative antigen of a nitrofurantoin metabolite 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 mg / mL; the derivative antigen of a nitrofurantoin metabolite is a conjugate of a derivative of a nitrofurantoin metabolite and a carrier protein; a good detection effect is achieved at this coating concentration.

[0090] For example, the coating concentrations of the derivative antigen of the nitrofurantoin metabolite on the T line are 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL or 2 mg / mL, and the coating concentrations of the antibody against mouse X on the C line are 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1 mg / mL.

[0091] In a preferred embodiment of the application of the present invention, the carrier protein is selected from any one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin;

[0092] In a preferred embodiment of the application of the present invention, the conjugate pad is coated with a colloidal-labeled monoclonal antibody against the nitrofurantoin metabolite or its antigen-binding fragment; each mL of the colloid contains 5-20 μg of the monoclonal antibody against the nitrofurantoin metabolite or its antigen-binding fragment. The coating concentration is, for example, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL or 20 μg / mL.

[0093] In a sixth aspect, the present invention provides a method for detecting a nitrofurantoin metabolite, which includes any one of the following methods:

[0094] (1) Add a diluent and the above-mentioned monoclonal antibody against the nitrofurantoin metabolite or its antigen-binding fragment to the control wells of a microplate coated with the derivative antigen of the nitrofurantoin metabolite, and incubate; add the above-mentioned monoclonal antibody against the nitrofurantoin metabolite or its antigen-binding fragment, and the derivative standard of the nitrofurantoin metabolite or the test sample to the inhibition wells of the microplate coated with the derivative antigen of the nitrofurantoin metabolite, 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. By plotting a standard curve of the absorbance against the concentration of the derivative of the nitrofurantoin metabolite, obtain the concentration of the derivative of the nitrofurantoin metabolite in the measured sample according to the standard curve; before loading the test sample, perform derivatization treatment;

[0095] (2) Load the derivatized test sample onto the sample pad of the test strip, and judge whether the sample contains the nitrofurantoin metabolite according to the chromatographic result of the test strip.

[0096] Derivatization treatment is specifically carried out by mixing a derivatizing reagent with a sample. For example, the sample to be tested is mixed with o-nitrobenzaldehyde solution, benzaldehyde solution, 2-nitrobenzaldehyde or o-phthalaldehyde dimethyl sulfoxide solution and reacted in a water bath at 80 - 90 °C for 10 - 20 min. After derivatization of 1-amino-hydantoin (AHD), a metabolite of nitrofurantoin in the sample, such as formation of 2-NP-AHD after derivatization with 2-nitrobenzaldehyde, it can be detected by antibody binding. Since the hapten structures of 2-NP-AHD and 4-CPAHD are similar, and hybridoma cells are screened based on 2-NP-AHD. Therefore, the obtained antibody can specifically bind to 2-NP-AHD.

[0097] 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-phosphoglucose dehydrogenase.

[0098] When drawing a standard curve of absorbance vs. the concentration of the derivative of the nitrofurantoin metabolite, it includes but is not limited to drawing: a standard curve of the absorbance ratio of the inhibition well / control well vs. the concentration of the derivative standard of the nitrofurantoin metabolite, or a standard curve of the ratio of (control well - inhibition well) / control well absorbance vs. the concentration of the derivative standard of the nitrofurantoin metabolite.

[0099] In a preferred embodiment of the application of the present invention, on the microplate, the coating concentration of the derivative antigen of the nitrofurantoin metabolite is 2.5×10 -5 -1×10 -3 mg / mL; the addition concentration of the monoclonal antibody against the nitrofurantoin metabolite or its antigen-binding fragment is 1×10 -5- 1×10 -3 mg / mL, or the dilution of the monoclonal antibody against the nitrofurantoin metabolite or its antigen-binding fragment is 1:2000 - 8000. At the above coating concentration, the monoclonal antibody against the nitrofurantoin metabolite has the best inhibitory effect on the derivative of the nitrofurantoin metabolite. The higher the inhibition rate, the higher the binding activity of the antibody to the derivative of the nitrofurantoin metabolite in the test sample, and the more accurate the detection result. In particular, the dilution of the monoclonal antibody against the nitrofurantoin metabolite or its antigen-binding fragment is 1:4000. Under this condition, the monoclonal antibody against the nitrofurantoin metabolite has a better inhibition rate for the derivative of the nitrofurantoin metabolite.

[0100] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody against the nitrofurantoin metabolite or its antigen-binding fragment.

[0101] 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.

[0102] In an eighth aspect, the present invention provides a recombinant vector, which comprises the above nucleic acid molecule.

[0103] The term "vector" is used herein in its most general sense and includes any intermediate agent for nucleic acids, which can enable the nucleic acids to be introduced into prokaryotic and / or eukaryotic cells, for example, and integrated into the genome where appropriate. 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.

[0104] In an alternative embodiment, the above 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.

[0105] In a ninth aspect, the present invention also provides a recombinant cell, which contains the above vector.

[0106] 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.

[0107] In an alternative embodiment, the recombinant cell is a eukaryotic cell.

[0108] In an alternative embodiment, the recombinant cell is a mammalian cell.

[0109] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0110] Example 1

[0111] In this example, monoclonal antibody preparation was carried out.

[0112] 1. Preparation of hapten and complete antigen

[0113] Refer to the method reported in the literature (Preparation and identification of monoclonal antibody against furantoin metabolite AHD, Chinese Journal of Biologicals), and prepare the hapten and complete antigen of AHD. Weigh 150 mg of AHD in a beaker, add 5 ml of pure water and stir to dissolve to obtain solution A. Weigh 150 mg of 4-carboxybenzaldehyde (4-CBA) in a beaker, slowly add DMF dropwise until the precipitate is completely dissolved to obtain solution B. Add solution B to a beaker equipped with a magnetic stirrer, and add solution A dropwise. Control the temperature at 37 °C and react in the dark for 2 h. Centrifuge the reacted mixture at 22,000 g for 10 min, take the precipitate, wash it 3 times with distilled water, filter it dry, transfer it to an evaporating dish, and place it in a vacuum drying oven to dry at 60 °C to obtain a white powder, which is the hapten 4-CPAHD.

[0114] The active ester method is used for the preparation of the complete antigen (the synthetic route is shown in Figure 1 ). Dissolve 30 mg of 4-CPAHD in 1 ml of DMF with magnetic stirring, add 60 mg of DCC and 25 mg of NHS, stir at room temperature in the dark for 12 h, centrifuge the reacted mixture at 10,000 rpm for 10 min, and the obtained supernatant is the active ester intermediate, which is solution A. Dissolve 50 mg of BSA in 5 ml of 0.1 M PBS (pH 8.0) to obtain solution B. Slowly add solution A dropwise to solution B, maintain the temperature at 4 °C. After all of solution A is added, react at 4 °C for 24 h; transfer the mixture to a dialysis bag and dialyze it against 0.01 M PBS (pH 7.4) buffer at 4 °C in the dark for 3 d, changing the dialysis solution 3 times a day. After dialysis, centrifuge the reaction solution to obtain the supernatant, which is the complete antigen 4-CPAHD-BSA, and store it at 4 °C. Prepare the coating antigen 4-CPAHD-OVA in the same way. 4-CPAHD-BSA is used as an immunogen for immunizing mice, and 4-CPAHD-OVA is used as a coating antigen for detecting the titer of serum and antibody.

[0115] 2. Preparation of monoclonal antibody:

[0116] (1) Select 3 female BALB / C mice aged 6 - 8 weeks as experimental animals.

[0117] (2) Primary immunization: Thoroughly emulsify the diluted complete antigen 4-CPAHD-BSA solution (concentration 1 mg / mL) with an equal volume of Freund's complete adjuvant using a magnetic stirrer, and then perform multiple subcutaneous injections on the back of the mice for immunization. The immunization dose is 0.1 mg of antigen per mouse.

[0118] (3) Booster immunization: Two weeks after the primary immunization, the diluted complete antigen 4-CPAHD-BSA solution (concentration: 1 mg / mL) was emulsified with an equal volume of Freund's incomplete adjuvant using a magnetic stirrer and then injected subcutaneously at multiple points on the back of the mice. The immunization dose was 0.1 mg antigen per mouse. Booster immunization was performed every two weeks. Starting from the third booster immunization, blood was collected from the orbital sinus on the 7th day after each immunization, and the antibody titer and inhibition effect were detected by the indirect competitive ELISA method. The coating antigen used in the method was the complete antigen 4-CPAHD-OVA.

[0119] (4) Boost immunization: Mice with high titer and good inhibition were selected for boost immunization on the 10th day after the 5th immunization. 100 μL of 1 mg / mL was injected intraperitoneally without adjuvant.

[0120] (5) Cell fusion

[0121] On the 3rd day after the boost immunization, hybridoma fusion was performed. The spleen cells of the mice were fused with the mouse myeloma cell sp2 / 0 using PEG 1450 and then cultured in a 2% HAT medium for selection. The wells with high titer and good inhibition were screened by the indirect competitive ELISA method, and then subcloned by the limiting dilution method. Finally, the monoclonal antibody hybridoma cell line AHD-1E9G3 against AHD was obtained. The antibody secreted by this cell line had good specificity for AHD, and the detection sensitivity could reach 0.5 μg / L. This cell line was deposited in the China General Microbiological Culture Collection Center with the deposit number: 46123.

[0122] (6) Cryopreservation and resuscitation of cells

[0123] After the obtained hybridoma cell line AHD-1E9G3 was expanded in culture, it was resuspended by pipetting with DMEM and centrifuged. It was cryopreserved in liquid nitrogen at a density of 1×10 9 cells / mL for long-term storage. During resuscitation, the cryopreservation tube was taken out from the liquid nitrogen tank and immediately thawed in a 37 °C water bath, then transferred to pre-warmed 10 mL DMEM using a pipette for centrifugation to remove the cryopreservation solution, and then transferred to a culture plate for cultivation.

[0124] (7) Preparation and purification of monoclonal antibody.

[0125] Eight 10- to 12-week-old female BALB / C mice were intraperitoneally injected with sterile paraffin oil at a dose of 0.3 mL per mouse. One week later, each mouse was intraperitoneally injected with the monoclonal cell line AHD-1E9G3, and the number of injected cells per mouse was approximately 10 6Individuals. When the abdomen of the mice became swollen, ascites of the mice was collected and purified by the saturated ammonium sulfate method and then stored at -20°C. Thus, the purified monoclonal antibody secreted by the hybridoma cell line AHD-1E9G3 (i.e., 2-NP-AHD antibody) was obtained.

[0126] Example 2

[0127] The antibody effect of the monoclonal antibody obtained in Example 1 was detected.

[0128] The various buffers used in the following experiments are as follows:

[0129] 1) Coating buffer (0.05M carbonate buffer, pH 9.6): 1.5 g of Na2CO3; 2.94 g of NaHCO3, made up to 1000 mL with pure water;

[0130] 2) Phosphate buffer PBS (0.01M, pH 7.4): 0.2 g of KH2PO4; 8 g of NaCl; 2.92 g of NaH2PO4·12H2O, made up to 1000 mL with pure water;

[0131] 3) Washing buffer (PBST): Add 1 mL of Tween-20 to 1000 mL of the prepared PBS solution;

[0132] 4) Sample diluent (PBSTG): Add 1 mL of Tween-20 and 1 g of gelatin (melted by microwave heating) to the prepared PBS and make up to 1 L;

[0133] 5) Chromogenic solution: TMB stock solution (375 mg of TMB solid + 30 mL of DMSO (prepared according to the ratio, 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;

[0134] 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.

[0135] The following checkerboard experiment of antigen and antibody was carried out:

[0136] 1) Coating:

[0137] The complete antigen 4-CPAHD-OVA at 1 mg / mL was serially diluted at ratios of 1:1000, 1:2000, 1:4000, and 1:8000 with coating buffer to obtain coating antigen solutions of the complete antigen 4-CPAHD-OVA at different concentrations. 100 μL of the prepared 4-CPAHD-OVA coating antigen solutions at different dilution multiples was added to each well of a 96-well ELISA plate and incubated overnight at 4 °C, followed by washing 3 times with PBST;

[0138] 2) Competition:

[0139] The purchased 100 μg / mL 2-NP-AHD standard from Tanmo Quality Inspection was diluted to 5 ng / mL with PBSTG. 50 μL of sample diluent was added to the zero well, and 50 μL of the diluted 5 ng / mL 2-NP-AHD standard solution was added to each inhibition well. The zero well, i.e., the control well, did not add the 2-NP-AHD standard or the sample to be tested, while the inhibition wells added the 2-NP-AHD standard or the sample to be tested. Since the 2-NP-AHD standard or the sample to be tested can competitively bind to the antibody on the ELISA plate pre-coated with the complete antigen 4-CPAHD-OVA, thereby inhibiting the absorbance of the ELISA plate, it is called the inhibition well.

[0140] The AHD-1E9G3 monoclonal antibody (1 mg / mL) of Example 1 was serially diluted at ratios of 1:1000, 1:2000, 1:4000, and 1:8000 with PBST to obtain dilutions of the monoclonal antibody (50 μL / well), placed in a humid box and incubated at 37 °C for 30 min, followed by washing the plate 3 times.

[0141] 3) Addition of enzyme-labeled secondary antibody: The goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) was diluted 10,000-fold with PBSTG, 100 μL was added to each well, placed in a humid box and incubated at 37 °C for 30 min, followed by washing the plate 3 times.

[0142] 4) Color development: The color development solution was prepared immediately before use. After mixing the prepared TMB solution and hydrogen peroxide in proportion, 100 μL was added to each well and developed at room temperature in the dark for 10 min.

[0143] 5) Termination: 50 μL of 2 M H2SO4 was added to each well, and the OD value of each well was measured at 450 nm using an ELISA reader.

[0144] 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.

[0145] The results are shown in the following table:

[0146]

[0147] Note: I represents the inhibition wells in the enzyme-linked immunosorbent assay (ELISA) plate, and CK represents the control wells in the ELISA plate.

[0148] Table 1 results show that when the coating antigen dilution is 1:1000 and the antibody dilution is 1:4000 (2.5×10 -4 mg / mL), 5 ng / mL of 2-NP-AHD has the best inhibitory effect on this antibody, with an inhibition rate of 93.6%. This indicates that the antibody produced by the above hybridoma cell AHD-1E9G3 can detect 2-NP-AHD. The higher the inhibition rate, the higher the binding activity of the antibody to the derivative of the metabolite of nitrofurantoin, and the more accurate the detection result. This shows that the antibody produced by the above hybridoma cells can detect the derivative of the metabolite of nitrofurantoin.

[0149] Example 3

[0150] This example provides a standard curve for detecting the derivative of the metabolite of nitrofurantoin and tests the sensitivity of the detection method.

[0151] The 2-NP-AHD standard solution was diluted with the sample diluent to the following different concentrations: 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.31 ng / mL, 0.15 ng / mL, 0.075 ng / mL. Referring to the checkerboard experiment procedure, a standard curve experiment was carried out. Three parallels were made for each standard concentration, and the coating antigen dilution factor of 1:4000 and the antibody dilution of 1:8000 were selected for detection.

[0152] Draw a standard curve: Using the 2-NP-AHD standard solution 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 AHD standard solution and B0 is the average absorbance value of the control well) as the Y-axis, draw a 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 is as Figure 2 shown.

[0153] The results show that its sensitivity (IC 50 ) is 0.22 ng / mL, and the detection range is 0.08 ng / mL - 0.68 ng / mL. This indicates that the 2-NP-AHD antibody prepared by the above method has high sensitivity and good detection effect.

[0154] Example 4

[0155] This example conducts a specificity test on the antibody prepared in Example 1.

[0156] Referring to the preparation method of the 2-NP-AHD standard sample, diluents of standard samples of 2-NP-AHD, 2-NP-AOZ, 2-NP-AMOZ, 2-NP-SEM, AHD, AOZ, AMOZ, SEM, furaltadone, furazolidone, nitrofurazone and sulfadiazine drugs were prepared respectively. The above five drugs were diluted into the following concentrations with the sample diluent: 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL.

[0157] Establish a standard curve and determine the median inhibitory concentration IC 50 (The standard sample concentration value when the inhibition rate reaches 50%), and the method for establishing the standard curve is the same as that for establishing the 2-NP-AHD standard curve above.

[0158] Cross-reactivity rate (%) = (2-NP-AHD IC 50 ) / (analogue IC 50 ) × 100%.

[0159]

[0160]

[0161] The results show that the antibody provided by the present invention can specifically bind to 2-NP-AHD and has no cross-reaction with other analogues.

[0162] Example 5

[0163] In this example, a colloidal gold test strip was prepared.

[0164] 1) Preparation of the 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 thereto, and then add 5 μg of the antibody prepared in Example 1. Mix well by inverting up and down and let stand for 20 min. Then add 50 μL of 3% BSA for blocking, 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 0.5 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).

[0165] 2) Membrane scribing: Use a gold-spraying membrane scribing instrument to scribe the nitrocellulose membrane attached to the bottom plate. The concentration of the T-line antigen (4-CPAHD-BSA) is 1.0 mg / mL, and the concentration of the C-line secondary antibody is 1.0 mg / mL.

[0166] 3) Assembly: Assemble in the order of sample pad, colloidal gold pad, nitrocellulose membrane and absorbent paper, and then cut it into test strips with a width of 4 mm using a cutting machine for subsequent testing.

[0167] 4) Detection:

[0168] Chop the sample of animal tissue, homogenize it with a homogenizer at 4000 r / min for 5 min, and then perform derivatization treatment and extraction treatment.

[0169] Derivatization treatment: Weigh 5.0 g of the sample into a 50 mL centrifuge tube, add 10 mL of deionized water and 2 mL of 1 mol / L HCl solution, mix well, then add 300 μL of 2-nitrobenzaldehyde solution (50 mmol / L), mix well, place it in a water bath at 80 °C, and react for 10 min.

[0170] Extraction treatment: Take out the derivatized sample and let it return to room temperature, add 5 mL of PBS (0.2 M) and 1.6 mL of NaOH (1 mol / L) solution, adjust the pH to neutral; then add 7 mL of ethyl acetate, vortex mix for 30 s, and centrifuge at 5000 r / min for 10 min. Take the upper layer, i.e., the ethyl acetate layer, into a 10 mL centrifuge tube, and dry it with nitrogen at 50 °C; redissolve it with 2 mL of n-hexane, add 1 mL of PBS buffer solution, shake well, and centrifuge at 5000 r / min for 10 min. Discard the upper n-hexane layer, and use the lower layer solution for detection.

[0171] After inspection, Figure 3 The results show that the tissues of 3 kinds of samples, namely shrimp, perch, and grass carp, do not contain furantoin metabolite AHD. Add AHD standard products (obtain 2-NP-AHD through the above derivatization treatment so that it can be detected) to the samples respectively, with concentrations of 0 μg / kg and 0.5 μg / kg. Process using the above treatment method and detect with test strips. It can be clearly seen that the intensity of the T line of the spiked samples is significantly weakened, indicating that the prepared test strips can be used for the detection of furantoin metabolites in animal tissues.

[0172] Example 6

[0173] Evaluate the within-batch stability of the test strips provided in Example 5.

[0174] Prepare a 0.5 ppb 2-NP-AHD standard product (the source is the same as that in Example 5) with 0.02 M 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, obtain the ratio of the T line to the C line, and mark them on the test strips respectively for reference Figure 4As shown below. The within-batch stability calculation method is as follows: (STDEV.S(10 test data) / AVERAGE(10 test data))*100 (i.e., the ratio of the standard deviation to the average value of all test data). The test results show that the within-batch stability is 5.91%, indicating that the colloidal gold test strip has good stability.

[0175] In summary, the monoclonal antibody against furantoin metabolite provided by the present invention can be used to develop products such as test kits and test strips for furantoin metabolite detection. The present invention provides detection tools and means for the rapid detection of furantoin antibiotic residues in livestock and poultry products, aquatic products, and feeds, which is of great significance for meeting the actual detection needs, quickly controlling risks, and ensuring the quality and safety of agricultural products.

[0176] The sequence information involved in the present invention is as follows:

[0177]

[0178]

[0179] 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 in the protection scope of the present invention.

Claims

1. A hybridoma cell, characterized in that, It is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO. 46123.

2. An antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof, characterized in that, The nitrofurantoin metabolite is l-amino-2-hydantoin. The antibody or its antigen-binding fragment 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 VTR.

3. The monoclonal antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof 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 that 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 that 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. 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 nitrofurantoin metabolite or its antigen-binding fragment described in any one of claims 2-3 with a label. The label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, 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 nitrofurantoin metabolite 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 nitrofurantoin residues; (2) Detecting metabolites of nitrofurantoin; (3) Detecting derivatives of metabolites of nitrofurantoin; And (4) Preparing a detection product for nitrofurantoin residues, a detection product for metabolites of nitrofurantoin, or a detection product for derivatives of metabolites of nitrofurantoin. The derivative of the metabolite of nitrofurantoin is selected from 2-NP-AHD. 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, feed, or feed additive. Preferably, the animal-derived food is selected from livestock and poultry meat products, eggs and egg-containing foods, or aquatic products.

6. A nitrofurantoin metabolite detection product, characterized in that, The detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector; the detection product includes the monoclonal antibody against furantoin metabolite or its antigen-binding fragment according to any one of claims 2-3, or the antibody secreted by the hybridoma cell according to claim 1; Preferably, the detection product is an ELISA kit, and the ELISA kit includes a microplate and the monoclonal antibody against furantoin metabolite or its antigen-binding fragment, and a derivative antigen of furantoin metabolite is coated on the microplate; 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 the monoclonal antibody against furantoin metabolite or its antigen-binding fragment, the nitrocellulose membrane has a T line and a C line, the T line has a derivative antigen of furantoin metabolite, and the C line has an anti-mouse antibody of X; X is sheep, rabbit, horse, monkey or chicken.

7. The nitrofurantoin metabolite detection product according to claim 6, wherein The coating concentration of the derivative antigen of furantoin metabolite 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 mg / mL; the derivative antigen of furantoin metabolite is a conjugate of a derivative of furantoin metabolite and a carrier protein; Preferably, the carrier protein is selected from any one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin; Preferably, the coating concentration of the monoclonal antibody against furantoin metabolite or its antigen-binding fragment on the conjugate pad is 5-20 μg / mL.

8. A method for detecting nitrofurantoin metabolites, characterized in that, It includes any one of the following methods: (1) Add a diluent and the monoclonal antibody against furantoin metabolite or its antigen-binding fragment according to any one of claims 2-3 to the control wells of the microplate coated with the derivative antigen of furantoin metabolite, and incubate; add the monoclonal antibody against furantoin metabolite or its antigen-binding fragment according to any one of claims 2-3, and a derivative standard or a test sample of furantoin metabolite to the inhibition wells of the microplate coated with the antigen of furantoin metabolite, 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 obtain the concentration of the derivative of furantoin metabolite in the measured sample by plotting a standard curve of absorbance vs. the concentration of the derivative of furantoin metabolite; before the test sample is loaded, it is subjected to derivatization treatment; (2) Load the derivatized test sample onto the sample pad of the test strip, and judge whether the sample contains furantoin metabolite according to the chromatography result of the test strip; Preferably, on the microplate, the coating concentration of the derivative antigen of nitrofurantoin metabolite is 2.5×10 -5 -1×10 - 3 mg / mL; the addition concentration of the monoclonal antibody against nitrofurantoin metabolite or its antigen-binding fragment is 1×10 -5- 1×10 -3 mg / mL.

9. A nucleic acid molecule, characterized in that, It encodes the monoclonal antibody against furantoin metabolite or its antigen-binding fragment according to any one of claims 2-3.

10. A recombinant vector, characterized in that, It includes the nucleic acid molecule according to claim 9.

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