A method for detecting hybridoma cells and nitrofurantoin metabolites

Abstract

This invention discloses a method for detecting hybridoma cells and nitrofurantoin metabolites, relating to the field of nitrofurantoin metabolite detection technology. Immunoassay experiments demonstrate that the anti-nitrofurantoin metabolite monoclonal antibody provided by this invention has the technical advantage of high specificity against nitrofurantoin metabolite derivatives, and exhibits high detection sensitivity and a wide linear detection range. Therefore, the anti-nitrofurantoin metabolite monoclonal antibody provided by this invention can be used to develop nitrofurantoin metabolite detection kits, test strips, and other products. This invention provides a detection tool and method for the rapid detection of nitrofurantoin antibiotic residues in livestock and poultry products, aquatic products, and feed, which is of great significance for meeting practical detection needs, rapidly controlling risks, and ensuring the quality and safety of agricultural products.

Description

Technical Field

[0001] This invention relates to the field of nitrofurantoin metabolite detection technology, and more specifically, to a method for detecting hybridoma cells and nitrofurantoin metabolites. Background Technology

[0002] Nitrofuran is a class of nitrofuran drugs, a synthetic broad-spectrum antibacterial agent used to prevent and treat gastrointestinal infections caused by Escherichia coli and Salmonella. After entering the animal body, it is rapidly metabolized into its metabolite 1-amino-2-yldiurea (AHD), which remains stable in the animal body for a long period. Due to the potential carcinogenic and mutagenic effects of nitrofuran and its metabolites on humans, many countries prohibit its use in food production animals. my country's Ministry of Agriculture and Rural Affairs Announcement No. 250 explicitly prohibits the use of nitrofuran drugs in food animals. However, due to its low cost and significant effectiveness in preventing and treating bacterial infections, there remains a demand for nitrofuran residue detection. The National Food Safety Standard GB 31656.13—2021, "Determination of Multiple Residues of Nitrofuran Metabolites in Aquatic Products by Liquid Chromatography-Tandem Mass Spectrometry," specifies a method detection limit of 0.5 μg / kg and a quantitation limit of 1 μg / kg for AHD.

[0003] Early detection of nitrofurantoin primarily targeted the active ingredient, with unsatisfactory results. Currently, more advanced methods detect AHD levels in animal tissues to indirectly reflect residues of the active ingredient. The most widely used instrumental analytical methods include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). However, these instruments are expensive, complex to operate, and time-consuming, requiring specialized personnel and failing to meet the timeliness requirements of agricultural product market access and origin-based export regulations.

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

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

[0006] The purpose of this invention is to provide a method for detecting hybridoma cells and nitrofurantoin metabolites, thereby developing highly sensitive nitrofurantoin metabolite antibody raw materials and detection products. This is of great significance for meeting actual detection needs, quickly controlling risks, and ensuring the quality and safety of agricultural products.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a hybridoma cell, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.46123.

[0009] Secondly, the present invention provides an antibody or antigen-binding fragment thereof against a nitrofurantoin metabolite, wherein the nitrofurantoin metabolite is 1-amino-2-lactamase, and the antibody or antigen-binding fragment thereof includes a heavy chain complementarity-determining region and a light chain complementarity-determining region. The heavy chain complementarity-determining region includes CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 1-3 in sequence. The light chain complementarity-determining region includes CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is VTR.

[0010] Thirdly, the present invention provides an antibody conjugate formed by conjugating a monoclonal antibody against the above-mentioned anti-nitrofurantoin metabolite or its antigen-binding fragment with a label, wherein the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0011] Fourthly, the present invention provides the use of monoclonal antibodies against nitrofurantoin metabolites or their antigen-binding fragments or the antibody conjugates thereof in any of the following:

[0012] (1) Detection of nitrofurantoin residues;

[0013] (2) Detection of nitrofurantoin metabolites;

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

[0015] (4) Prepare a nitrofurantoin residue detection product, a nitrofurantoin metabolite detection product, or a detection product for derivatives of nitrofurantoin metabolites; wherein the derivatives of nitrofurantoin metabolites are selected from 2-NP-AHD;

[0016] The testing products include reagents, kits, test strips, antibody chips, antibody probes, or testing instruments.

[0017] Fifthly, the present invention provides a product for detecting nitrofurantoin metabolites, wherein the detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector; the detection product includes the above-mentioned monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment, or the above-mentioned antibody secreted by hybridoma cells;

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

[0019] Alternatively, the test product is a test strip, which includes a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with a monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment, and the nitrocellulose membrane has T lines and C lines, with a derivative antigen of nitrofurantoin metabolites on the T lines and an X anti-mouse antibody on the C lines; X is sheep, rabbit, horse, monkey or chicken.

[0020] Sixthly, the present invention provides a method for detecting nitrofurantoin metabolites, comprising any one of the following methods:

[0021] (1) Add diluent and the above-mentioned monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment to the control wells of a microplate coated with antigens of nitrofurantoin metabolites, and incubate; add the above-mentioned monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment, as well as nitrofurantoin metabolite derivative standards or test samples to the inhibition wells of a microplate coated with antigens of nitrofurantoin metabolites, and incubate; by adding enzyme-labeled secondary antibody to the control wells and inhibition wells respectively and incubating, the absorbance of the control wells and inhibition wells is detected by colorimetric reaction; by plotting a standard curve of absorbance versus concentration of nitrofurantoin metabolite derivatives, the concentration of nitrofurantoin metabolite derivatives in the test sample is obtained according to the standard curve; the test sample is derivatized before loading.

[0022] (2) The derivatized sample is loaded onto the sample pad of the test strip, and the chromatographic results of the test strip are used to determine whether the sample contains nitrofurantoin metabolites.

[0023] In a seventh aspect, the present invention provides a nucleic acid molecule that encodes a monoclonal antibody or an antigen-binding fragment thereof of the aforementioned anti-nitrofurantoin metabolite.

[0024] Eighthly, the present invention provides a recombinant vector comprising the above-described nucleic acid molecules.

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

[0026] This invention involves immunizing mice with an antigen of 1-amino-2-lactamase, a metabolite of nitrofurantoin, followed by hybridoma fusion. Hybridoma cell lines that stably secrete monoclonal antibodies against nitrofurantoin metabolites are then screened, resulting in specific and sensitive monoclonal antibodies against nitrofurantoin metabolites. Immunoassay experiments demonstrate that the monoclonal antibodies against nitrofurantoin metabolites provided by this invention have a high specificity against nitrofurantoin metabolite derivatives, exhibiting high detection sensitivity and a wide linear detection range.

[0027] Therefore, the monoclonal antibody against nitrofurantoin metabolites provided by this invention can be used to develop products such as detection kits and test strips for nitrofurantoin metabolites. The invention provides a detection tool and means for the rapid detection of nitrofurantoin antibiotic residues in livestock and poultry products, aquatic products, and feed, which is of great significance for meeting actual detection needs, quickly controlling risks, and ensuring the quality and safety of agricultural products. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used 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 a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The preparation route for the 4-CPAHD-BSA complete antigen;

[0030] Figure 2 Standard curve for detecting derivatives of nitrofurantoin metabolites;

[0031] Figure 3 The image shows the detection results of nitrofurantoin metabolite AHD in three samples of shrimp, sea bass, and grass carp using test strips.

[0032] Figure 4 This is a graph showing the results of the intra-batch stability test of the test strips. Detailed Implementation

[0033] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0034] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0036] Definition of noun

[0037] The term "antigen-binding fragment" broadly refers to all proteins / protein fragments containing a CDR region, particularly antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-binding fragments of the aforementioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. Antibody types can include IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0038] The term “antibody” as used in this article is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, provided they exhibit the desired biological activity, such as antigens or fragments thereof that specifically bind to derivatives of nitrofurantoin metabolites.

[0039] In this invention, the terms "complementarity-determining region" or "CDR" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically 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 specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0040] In this invention, the heavy chain complementarity-determining region (CDR) is represented by HCDR, which includes HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region (LCDR) is represented by LCDR, which includes 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 led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of this invention.

[0041] Typically, the variable region (VH) of the antibody heavy chain is obtained by linking the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0042] The variable region (VL) of the antibody light chain can be obtained by linking the following numbered CDRs with FRs in the following combination: 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 interglycosylation (backbone) bonds. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of this invention can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences and may contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases may also be present. Examples of these modified bases include nitrogenous and denitrogenated adenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine.

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

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

[0046] In a first aspect, this invention provides a hybridoma cell line deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO.46123. This hybridoma cell line stably secretes antibodies against nitrofurantoin metabolites. The biological material submitted for deposit is AHD-1E9G3, named "Mouse Hybridoma Cell Line," with a deposit date of October 23, 2024, and is identified as viable.

[0047] Secondly, the present invention provides an antibody or antigen-binding fragment thereof against a nitrofurantoin metabolite, wherein the nitrofurantoin metabolite is 1-amino-2-lactamase, and the antibody or antigen-binding fragment thereof includes a heavy chain complementarity-determining region and a light chain complementarity-determining region. The heavy chain complementarity-determining region includes CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 1-3 in sequence. The light chain complementarity-determining region includes CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is VTR.

[0048] Immunoassay experiments have demonstrated that the anti-nitrofurantoin metabolite monoclonal antibody provided by this invention possesses the technical advantage of high specificity against nitrofurantoin metabolite derivatives, along with high detection sensitivity and a wide linear detection range. Therefore, the anti-nitrofurantoin metabolite monoclonal antibody provided by this invention can be used to develop detection kits, test strips, and other products for nitrofurantoin metabolites. This invention provides a detection tool and method for the rapid detection of nitrofurantoin antibiotic residues in livestock and poultry products, aquatic products, and feed, which is of great significance for meeting practical detection needs, rapidly controlling risks, and ensuring the quality and safety of agricultural products.

[0049] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a heavy chain framework region and a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 having at least 80% homology with the amino acid sequences shown in SEQ ID NO:6-9; for example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NO:6-9.

[0050] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:10-13; 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 in SEQ ID NO:10-13.

[0051] In one embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:14, and the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:15.

[0052] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region, which 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 region.

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

[0054] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0055] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0056] Thirdly, the present invention provides an antibody conjugate formed by conjugating a monoclonal antibody against the above-mentioned anti-nitrofurantoin metabolite or its antigen-binding fragment with a label, wherein 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 aforementioned markers refer to substances possessing properties that can be directly observed with the naked eye or detected by instruments, such as luminescence, color development, and radioactivity. These properties enable qualitative or quantitative detection of the corresponding target analytes. In practical applications, those skilled in the art can select appropriate markers based on detection conditions or actual needs. Regardless of the marker used, it falls within the scope of protection of this 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), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5...). .5, Cy3, etc. or similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

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

[0060] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 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 optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0062] In a preferred embodiment of the present invention, the nanoparticle marker is selected from nanoparticles or colloids; in a preferred embodiment of the present invention, the nanoparticle marker 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 present invention, the colloid is selected from colloidal gold, colloidal silver, or colloidal selenium.

[0064] Fourthly, the present invention provides the use of monoclonal antibodies against nitrofurantoin metabolites or their antigen-binding fragments or the antibody conjugates thereof in any of the following:

[0065] (1) Detection of nitrofurantoin residues;

[0066] (2) Detection of nitrofurantoin metabolites;

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

[0068] (4) Prepare a nitrofurantoin residue detection product, a nitrofurantoin metabolite detection product, or a detection product for derivatives of nitrofurantoin metabolites; wherein the derivatives of nitrofurantoin metabolites are selected from 2-NP-AHD;

[0069] The testing products include reagents, kits, test strips, antibody chips, antibody probes, or testing instruments.

[0070] To improve reagent stability and extend shelf life, those skilled in the art can add functional components such as stabilizers and protectants as needed. Protein stabilizers are selected from sucrose, trehalose, BSA, glycerol, mannitol, Triton X-100, and Tween-20. Protectants are selected from cryoprotectants, such as polyols and sugars. Polyols may be selected from sorbitol, mannitol, or mixtures thereof. The reagent may be in the form of, but is not limited to, solid, liquid, or semi-solid.

[0071] Antibody chips are chips formed by immobilizing antibodies or antigen-binding fragments of the aforementioned anti-nitrofurantoin metabolites on a carrier.

[0072] In a preferred embodiment of the present invention, the kit includes a solid phase on which an antibody or its antigen-binding fragment is coated; or, an antigen of a nitrofurantoin metabolite is coated onto the solid phase. For example, an antibody against a nitrofurantoin metabolite or an antigen of a nitrofurantoin metabolite may be chemically coupled to the solid phase.

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

[0074] In a preferred embodiment of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

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

[0076] In a preferred embodiment 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] Eggs and egg-containing products include, but are not limited to: chicken eggs, duck eggs, goose eggs, quail eggs, ostrich eggs, and egg yolk powder, egg liquid, and dried chicken eggs of any of the above.

[0078] Aquatic products include, but are not limited to: live fish, shrimp, crab, and shellfish.

[0079] Fish such as ribbonfish, squid, yellow croaker, cod, sardines, shark, whale, salmon, tuna, sea bass, saury, ray, conger eel, mackerel, and yellow croaker, etc.

[0080] Livestock and poultry meat products include, but are not limited to, meat products from cattle, sheep, horses, donkeys, camels, pigs, chickens, ducks, geese, etc. Meat products include, but are not limited to, fresh meat, frozen meat, dried meat, minced meat, etc.

[0081] Feeds can be selected from mixed feeds or silage.

[0082] Fifthly, the present invention provides a nitrofurantoin metabolite detection product, wherein the detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector; the detection product includes the above-mentioned monoclonal antibody against nitrofurantoin metabolite or its antigen-binding fragment, or the above-mentioned antibody secreted by hybridoma cells.

[0083] The aforementioned chip can also be called a suspension array or 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.

[0084] The aforementioned carrier can be made of various materials and in various forms, such as preferably a container with a flat bottom. A more typical preferred example is multi-well plates, microplates, microfluidic-based devices (e.g., microfluidic chips), petri dish-like containers, etc., which are widely used in biochemical assays, and are not limited thereto.

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

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

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

[0088] Alternatively, the test product is a test strip, which includes a conjugate pad and a nitrocellulose membrane. The conjugate pad is coated with a monoclonal antibody against a nitrofurantoin metabolite or its antigen-binding fragment. The nitrocellulose membrane has T lines and C lines. The T lines contain a derivative antigen of the nitrofurantoin metabolite, and the C lines contain an X anti-mouse antibody; X can be sheep, rabbit, horse, monkey, or chicken. The strips are assembled in the order of sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper, and then cut into strips of a certain width using a cutting machine.

[0089] In a preferred embodiment of the present invention, the coating concentration of the nitrofurantoin metabolite derivative antigen on the T line is 0.1-2 mg / mL, and the coating concentration of the X anti-mouse antibody on the C line is 0.2-1 mg / mL; the nitrofurantoin metabolite derivative antigen is a conjugate of the nitrofurantoin metabolite derivative and the carrier protein; at this coating concentration, it has good detection effect.

[0090] For example, the coating concentration of nitrofurantoin metabolite derivative antigens on the T line is 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, while the coating concentration of X anti-mouse antibody on the C line is 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 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 present invention, the conjugate pad is coated with a colloidally labeled monoclonal antibody against a nitrofurantoin metabolite or its antigen-binding fragment; each mL of the colloid contains 5-20 μg of the monoclonal antibody against a 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] Sixthly, the present invention provides a method for detecting nitrofurantoin metabolites, comprising any one of the following methods:

[0094] (1) Add diluent and the above-mentioned monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment to the control wells of a microplate coated with antigens of nitrofurantoin metabolites, and incubate; add the above-mentioned monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment, as well as nitrofurantoin metabolite derivative standards or test samples to the inhibition wells of a microplate coated with antigens of nitrofurantoin metabolites, and incubate; by adding enzyme-labeled secondary antibody to the control wells and inhibition wells respectively and incubating, the absorbance of the control wells and inhibition wells is detected by colorimetric reaction; by plotting a standard curve of absorbance versus concentration of nitrofurantoin metabolite derivatives, the concentration of nitrofurantoin metabolite derivatives in the test sample is obtained according to the standard curve; before loading the test sample, derivatization treatment is performed;

[0095] (2) The derivatized sample is loaded onto the sample pad of the test strip, and the chromatographic results of the test strip are used to determine whether the sample contains nitrofurantoin metabolites.

[0096] Derivatization involves mixing the sample with a derivatization reagent, such as reacting the sample with o-nitrobenzaldehyde solution, benzaldehyde solution, 2-nitrobenzaldehyde, or o-phthalaldehyde dimethyl sulfoxide solution in a water bath at 80-90°C for 10-20 minutes. The nitrofurantoin metabolite 1-amino-2-lactamase (AHD) in the sample, after derivatization, such as with 2-nitrobenzaldehyde, forms 2-NP-AHD, which can be detected by antibody binding. Since 2-NP-AHD and 4-CPAHD have similar hapten structures, and hybridoma cell screening is based on 2-NP-AHD, the antibodies obtained during screening can specifically bind to 2-NP-AHD.

[0097] Enzyme-labeled secondary antibodies include, but are not limited to, secondary antibodies labeled with horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

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

[0099] In a preferred embodiment of the present invention, the coating concentration of the nitrofurantoin metabolite derivative antigen on the microplate is 2.5 × 10⁻⁶. -5 -1×10 -3 mg / mL; the concentration of monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment added is 1×10 mg / mL. -5- 1×10 -3 The concentration of the anti-nitrofurantoin metabolite monoclonal antibody at a concentration of mg / mL, or a dilution of its antigen-binding fragment at 1:2000-8000, was optimal. At these coating concentrations, the anti-nitrofurantoin metabolite monoclonal antibody exhibited the best inhibitory effect against nitrofurantoin metabolite derivatives. A higher inhibition rate indicated higher binding activity of the antibody to the nitrofurantoin metabolite derivatives in the test sample, leading to more accurate detection results. Specifically, a dilution of 1:4000 for the anti-nitrofurantoin metabolite monoclonal antibody or its antigen-binding fragment resulted in a superior inhibition rate against nitrofurantoin metabolite derivatives under these conditions.

[0100] In a seventh aspect, the present invention provides a nucleic acid molecule that encodes a monoclonal antibody or an antigen-binding fragment thereof of the aforementioned anti-nitrofurantoin metabolite.

[0101] In one embodiment, the coding sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:16, and the coding sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:17.

[0102] Eighthly, the present invention provides a recombinant vector comprising the above-described nucleic acid molecules.

[0103] The term "vector" is used herein in its most common sense and includes any intermediate medium for nucleic acids that enables the nucleic acids to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. Vectors of this type preferably replicate and / or are expressed in cells. The term "vector" refers to bacterial plasmids, bacteriophages, 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, typically a circular double-stranded DNA that can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0104] In one alternative embodiment, the vector is an expression vector, and an important feature of an expression vector is that it typically 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 containing the aforementioned carrier.

[0106] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. According to the invention, the term "recombinant cell" includes prokaryotic (e.g., *Escherichia coli*) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells, and insect cells). Mammalian cells, such as those derived from humans, mice, hamsters, pigs, goats, or primates, are particularly preferred. Cells can originate from multiple tissue types and comprise primary cells and cell lines. Nucleic acids may be present in the host cell in single-copy or two or more copies, and in one embodiment, are expressed in the recombinant cell.

[0107] In one alternative implementation, the recombinant cells are eukaryotic cells.

[0108] In one alternative implementation, the recombinant cells are mammalian cells.

[0109] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0110] Example 1

[0111] This example demonstrates the preparation of monoclonal antibodies.

[0112] 1. Preparation of haptens and complete antigens

[0113] Following the method reported in the literature (Preparation and identification of monoclonal antibodies against nitrofurantoin metabolite AHD, Chinese Journal of Biological Products), the hapten and complete antigen of AHD were prepared. 150 mg of AHD was weighed into a beaker, and 5 ml of purified water was added and stirred to dissolve, yielding solution A. 150 mg of 4-CBA was added to a beaker, and DMF was slowly added dropwise until the precipitate was completely dissolved, yielding solution B. Solution B was added to a beaker equipped with a magnetic stirrer, followed by dropwise addition of solution A. The reaction was carried out at 37°C in the dark for 2 hours. The resulting mixture was centrifuged at 22000 g for 10 minutes, the precipitate was collected, washed three times with distilled water, filtered dry, transferred to an evaporating dish, and dried in a vacuum drying oven at 60°C to obtain a white powder, which was the hapten 4-CBAHD.

[0114] Complete antigen was prepared using the active ester method (synthetic route as described above). Figure 1 (As shown). 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 hours. Centrifuge the mixture at 10,000 rpm for 10 minutes. The supernatant obtained is the active ester intermediate, known as 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 while maintaining the temperature at 4°C. After all solution A has been added, react at 4°C for 24 hours. Transfer the mixture to a dialysis bag and dialyze with 0.01 M PBS (pH 7.4) buffer at 4°C in the dark for 3 days, changing the dialysis buffer 3 times a day. After dialysis, centrifuge the reaction solution to obtain the supernatant, which is the complete antigen 4-CPAHD-BSA, and store at 4°C. Prepare the coated antigen 4-CPAHD-OVA using the same method. 4-CPAHD-BSA was used as an immunogen for mouse immunization, and 4-CPAHD-OVA was used as a coating agent for the detection of serum and antibody titers.

[0115] 2. Monoclonal antibody preparation:

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

[0117] (2) Basic immunization: The diluted complete antigen 4-CPAHD-BSA solution (concentration of 1 mg / mL) was emulsified with an equal volume of Freund's complete adjuvant using a magnetic stirrer and then injected subcutaneously at multiple points on the back of the mice to immunize them. The immunization dose was 0.1 mg antigen / mouse.

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

[0119] (4) Sprint immunization: Select mice with high titer and good inhibition and perform sprint immunization on the 10th day after the 5th immunization. 1 mg / mL 100uL is injected intraperitoneally without adjuvant.

[0120] (5) Cell fusion

[0121] On day 3 post-immunization, hybridoma fusion was performed by fusing mouse spleen cells with mouse myeloma cells sp2 / 0 using PEG 1450. The cells were then selectively cultured in 2% HAT medium. Indirect competitive ELISA was used to screen wells with high titers and good inhibition. Subcloning was then performed using limiting dilution to obtain the AHD monoclonal antibody hybridoma cell line AHD-1E9G3. The antibodies secreted by this cell line showed good specificity for AHD, with a detection sensitivity of 0.5 μg / L. This cell line is deposited at the China General Microbiological Culture Collection Center, accession number: 46123.

[0122] (6) Cell cryopreservation and thawing

[0123] The obtained hybridoma cell line AHD-1E9G3 was expanded and cultured, then resuspended in DMEM and centrifuged. Cells were then frozen in cell cryopreservation medium at a concentration of 1×10⁻⁶. 9 Cryopreservation was performed using liquid nitrogen at a rate of 1 / mL for long-term storage. Upon thawing, the cryopreservation tubes were removed from the liquid nitrogen tank and immediately placed in a 37°C water bath for thawing. They were then transferred to preheated 10mL DMEM via pipette, centrifuged to remove the cryopreservation solution, and finally transferred to culture plates for incubation.

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

[0125] Eight 10-12 week old female BALB / c mice were intraperitoneally injected with sterile paraffin oil, 0.3 mL per mouse. One week later, each mouse was intraperitoneally injected with the monoclonal cell line AHD-1E9G3, with approximately 10 cells injected per mouse. 6Once the mice's abdomens swelled, ascites fluid was collected and purified using the saturated ammonium sulfate method before being stored at -20°C. This yielded a purified monoclonal antibody (i.e., 2-NP-AHD antibody) secreted by the hybridoma cell line AHD-1E9G3.

[0126] Example 2

[0127] The monoclonal antibody obtained in Example 1 was tested for antibody efficacy.

[0128] The buffer solutions used in the following experiments are as follows:

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

[0130] 2) Phosphate-buffered saline (PBS) (0.01M pH 7.4): 0.2g KH2PO4; 8g NaCl; 2.92g NaH2PO4·12H2O, add pure water to 1000mL;

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

[0132] 4) Sample dilution buffer (PBSTG): Add 1 mL Tween-20 and 1 g gelatin (melted by microwave heating) to the prepared PBS, and bring the volume to 1 L;

[0133] 5) Colorimetric solution: TMB stock solution (375mg TMB solid + 30mL DMSO (prepared according to the ratio, stored at room temperature and protected from light)), BUFFER (0.1g potassium sorbate + 46.04g potassium dihydrogen citrate hydrate + 1L pure water). The colorimetric solution should be prepared fresh each time: 200μL TMB stock solution + 11mL BUFFER + 3.34μL 30% hydrogen peroxide solution;

[0134] 6) Termination solution (2M H2SO4): Add 445.6mL of distilled water dropwise to 54.4mL of concentrated sulfuric acid (98%) while stirring.

[0135] The following is a checkerboard experiment involving antigen and antibody:

[0136] 1) Wrapped in blankets:

[0137] 1 mg / mL of the complete antigen 4-CPAHD-OVA was serially diluted with coating buffer at 1:1000, 1:2000, 1:4000, and 1:8000 to obtain coated antigen solutions of different concentrations of the complete antigen 4-CPAHD-OVA. 100 μL of each of the prepared 4-CPAHD-OVA coated antigen solutions was added to each well of a 96-well microplate, incubated overnight at 4°C, and washed three times with PBST.

[0138] 2) Competition:

[0139] Dilute the purchased 100 μg / mL 2-NP-AHD standard (from Tanmo Quality Inspection) to 5 ng / mL using PBSTG. Add 50 μL of sample diluent to each zero well and 50 μL of the diluted 5 ng / mL 2-NP-AHD standard solution to each inhibition well. Zero wells, also known as control wells, contain neither 2-NP-AHD standard nor the test sample, while inhibition wells contain either 2-NP-AHD standard or the test sample. Since the 2-NP-AHD standard or the test sample can competitively bind to the antibody on the pre-coated complete antigen 4-CPAHD-OVA on the ELISA plate, thereby inhibiting the absorbance of the plate, these wells are called inhibition wells.

[0140] The AHD-1E9G3 monoclonal antibody (1 mg / mL) in Example 1 was serially diluted with PBST at 1:1000, 1:2000, 1:4000 and 1:8000 to obtain a monoclonal antibody dilution buffer (50 μL / well). The buffer was placed in a humidified chamber at 37°C for 30 min and washed 3 times.

[0141] 3) Add enzyme-labeled secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 10000 times with PBSTG, add 100uL to each well, place in a humidified chamber at 37℃ for 30 min, and wash the plate 3 times.

[0142] 4) Color development: The color development solution should be prepared fresh for use. Mix the prepared TMB solution with hydrogen peroxide in the specified ratio, add 100 μL to each well, and develop the color at room temperature in the dark for 10 min.

[0143] 5) Termination: Add 50 μL of 2M H2SO4 to each well and measure the OD value of each well at 450 nm using a microplate reader.

[0144] The formula for calculating the inhibition rate is: Inhibition rate = (B0-B) / B0×100%), where B0 is the OD value of the control well and B is the OD value of the inhibition well.

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

[0146]

[0147] Note: I represents the inhibition well in the ELISA plate, and CK represents the control well in the ELISA plate.

[0148] Table 1 shows that when the coating antigen dilution is 1:1000 and the antibody dilution is 1:4000 (2.5 × 10⁻⁶), the antibody concentration is significantly higher than that of the target antibody. -4 At a concentration of 5 ng / mL, 2-NP-AHD showed the best inhibitory effect on the antibody, with an inhibition rate of 93.6%, indicating that the antibody produced by the hybridoma cells AHD-1E9G3 can detect 2-NP-AHD. A higher inhibition rate indicates higher antibody binding activity to derivatives of nitrofurantoin metabolites, resulting in more accurate detection results, further demonstrating that the antibody produced by the hybridoma cells can detect derivatives of nitrofurantoin metabolites.

[0149] Example 3

[0150] This embodiment provides a standard curve for detecting derivatives of nitrofurantoin metabolites and tests the sensitivity of the detection method.

[0151] The 2-NP-AHD standard solution was diluted with sample diluent to the following concentrations: 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.31 ng / mL, 0.15 ng / mL, and 0.075 ng / mL. A standard curve experiment was performed following the checkerboard assay procedure, with three replicates for each standard concentration. The antigen dilution was set at 1:4000, and the antibody dilution at 1:8000 for detection.

[0152] Plotting a standard curve: Using 2-NP-AHD standard solutions of 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 wells) as the Y-axis, plot the standard curve. The experiment was repeated three times, and the average of the three results was taken. The resulting standard curve is shown below. Figure 2 As shown.

[0153] The results show that its sensitivity (IC) 50 The concentration was 0.22 ng / mL, and the detection range was 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 performance.

[0154] Example 4

[0155] This embodiment performs specific detection on the antibody prepared in Example 1.

[0156] Following the preparation method for 2-NP-AHD standards, diluents were prepared for standard samples of 2-NP-AHD, 2-NP-AOZ, 2-NP-AMOZ, 2-NP-SEM, AHD, AOZ, AMOZ, SEM, furazolidone, furazolidone, nitrofurazone, and sulfadiazine. The five drugs were then diluted with the sample diluents to the following concentrations: 200 ng / mL, 100 ng / mL, 50 ng / mL, 250 ng / mL, 12.5 ng / mL, and 6.25 ng / mL, respectively.

[0157] Establish a standard curve and determine the inhibitory concentration (IC50) at the intermediate concentration. 50 (Standard concentration value with an inhibition rate of 50%), the method for establishing the standard curve is the same as the method for establishing the 2-NP-AHD standard curve mentioned above.

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

[0159]

[0160]

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

[0162] Example 5

[0163] This embodiment describes the preparation of a colloidal gold test strip.

[0164] 1) Preparation of gold-labeled antibody pads: Take 1 mL of colloidal gold solution (30 nm) into a 2 mL centrifuge tube (rinse with pure water), add 2 μL of 0.2 M K2CO3 solution, then add 5 μg of the antibody prepared in Example 1, mix by inverting, and let stand for 20 min. Then add 50 μL of 3% BSA for blocking, mix by inverting, and let stand for 20 min. Then centrifuge the mixture at 10000 r / min for 10 min (4℃) using a high-speed refrigerated centrifuge. 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 onto a 1 cm × 15 cm sample pad, and dry in a 37℃ oven (1.5 h).

[0165] 2) Scribing: The nitrocellulose membrane attached to the substrate was scribed using a gold-spraying scribing instrument. The concentration of T-line antigen (4-CPAHD-BSA) was 1.0 mg / mL, and the concentration of C-line secondary antibody was 1.0 mg / mL.

[0166] 3) Assembly: Assemble the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent paper in that order, and then cut them into 4mm wide test strips using a chopper for subsequent testing.

[0167] 4) Testing:

[0168] Animal tissue samples were chopped, homogenized for 5 minutes at 4000 rpm using a homogenizer, and then subjected to derivatization and extraction.

[0169] Derivatization: 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, and place in an 80 °C water bath for 10 min.

[0170] Extraction process: After derivatization, the sample was removed and brought to room temperature. 5 mL of PBS (0.2 M) and 1.6 mL of NaOH (1 mol / L) solution were added to adjust the pH to neutral. Then, 7 mL of ethyl acetate was added, vortexed for 30 s, and centrifuged at 5000 r / min for 10 min. The upper layer, i.e., the ethyl acetate layer, was collected in a 10 mL centrifuge tube and dried under nitrogen at 50 °C. After redissolving in 2 mL of n-hexane, 1 mL of PBS buffer solution was added, vortexed, and centrifuged at 5000 r / min for 10 min. The upper n-hexane layer was discarded, and the lower layer solution was used for analysis.

[0171] Upon inspection, Figure 3 The results showed that the tissues of shrimp, sea bass, and grass carp did not contain the nitrofurantoin metabolite AHD. AHD standards (obtained by derivatization of the AHD standards to obtain 2-NP-AHD, thus making them detectable) were added to the samples at concentrations of 0 μg / kg and 0.5 μg / kg, respectively. Using the above treatment method, the test strips showed a significant decrease in the intensity of the T line in the spiked samples, indicating that the prepared test strips can be used for the detection of nitrofurantoin metabolites in animal tissues.

[0172] Example 6

[0173] The intra-batch stability of the test strips provided in Example 5 was evaluated.

[0174] A 0.5 ppb 2-NP-AHD standard (from the same source as Example 5) was prepared using 0.02 M PB. Ten test strips prepared in Example 5 were randomly selected for testing against the standard solution. The color values ​​of the T line and C line on each test strip were measured using a test strip reader, and the ratio of the T line to the C line was obtained and marked on the test strips. Figure 4As shown. The intra-batch stability is calculated as follows: (STDEV.S (10 test data) / AVERAGE (10 test data)) * 100 (i.e., the ratio of the standard deviation to the mean of all test data). The test results show that the intra-batch stability is 5.91%, indicating that the colloidal gold test strip has good stability.

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

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

[0177]

[0178]

[0179] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hybridoma cell, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.46123.

2. An antibody against a nitrofurantoin metabolite or an antigen-binding fragment thereof, characterized in that, The nitrofurantoin metabolite is 1-amino-2-lactamase, and the antibody or its antigen-binding fragment includes a heavy chain complementarity-determining region (CDR) and a light chain complementarity-determining region (LCD). The heavy chain LCD includes CDR-H1, CDR-H2, and CDR-H3, whose amino acid sequences are shown in SEQ ID NO: 1-3. The light chain LCD includes CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5, and the amino acid sequence of CDR-L2 is VTR.

3. The monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain framework region and a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 in sequence as shown in SEQ ID NO:6-9; the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 in sequence as shown in SEQ ID NO:10-13.

4. The monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment according to claim 3, characterized in that, The antibody or its antigen-binding fragment further includes a constant region, which includes a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the κ-type or λ-type light chain constant region. The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

5. An antibody conjugate, characterized in that, It is formed by conjugation of a monoclonal antibody against a nitrofurantoin metabolite as described in any one of claims 2-4 or its antigen-binding fragment and a label, wherein the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

6. The antibody conjugate according to claim 5, characterized in that, The nanoparticle markers are selected from nanoparticles or colloids.

7. The antibody conjugate according to claim 6, characterized in that, The colloid is selected from colloidal gold, colloidal silver, or colloidal selenium.

8. The use of the monoclonal antibody against the anti-nitrofurantoin metabolite as described in any one of claims 2-4, or the antigen-binding fragment thereof, or the antibody conjugate as described in any one of claims 5-7, in any of the following: (1) Detection of nitrofurantoin residues; (2) Detection of nitrofurantoin metabolites; (3) Detection of derivatives of nitrofurantoin metabolites; (4) Prepare a nitrofurantoin residue detection product, a nitrofurantoin metabolite detection product, or a detection product for derivatives of nitrofurantoin metabolites; wherein the derivatives of nitrofurantoin metabolites are selected from 2-NP-AHD; The detection products include reagents, kits, test strips, antibody chips, antibody probes, or detection instruments.

9. The application according to claim 8, characterized in that, The samples to be tested are animal-derived foods, feed, or feed additives.

10. The application according to claim 9, characterized in that, The animal-derived foods are selected from livestock and poultry meat products, eggs and egg-containing foods or aquatic products.

11. A product for detecting nitrofurantoin metabolites, characterized in that, The detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector; the detection product includes a monoclonal antibody against nitrofurantoin metabolites as described in any one of claims 2-4 or its antigen-binding fragment, or an antibody secreted by hybridoma cells as described in claim 1.

12. The nitrofurantoin metabolite detection product according to claim 11, characterized in that, The detection product is an ELISA kit, which includes a microplate and a monoclonal antibody or antigen-binding fragment of the anti-nitrofurantoin metabolite. The microplate is coated with a derivative antigen of the nitrofurantoin metabolite. Alternatively, the detection product is a test strip, which includes a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with a monoclonal antibody or its antigen-binding fragment against the anti-nitrofurantoin metabolite; the nitrocellulose membrane has a T line and a C line; the T line has a derivative antigen of the nitrofurantoin metabolite; and the C line has an X anti-mouse antibody; where X is sheep, rabbit, horse, monkey, or chicken.

13. The nitrofurantoin metabolite detection product according to claim 12, characterized in that, The coating concentration of the nitrofurantoin metabolite derivative antigen on the T line is 0.1-2 mg / mL, and the coating concentration of the X anti-mouse antibody on the C line is 0.2-1 mg / mL; the nitrofurantoin metabolite derivative antigen is a conjugate of nitrofurantoin metabolite derivative and carrier protein.

14. The nitrofurantoin metabolite detection product according to claim 13, characterized in that, The carrier protein is selected from any one of bovine serum albumin, ovalbumin, human serum albumin, or hemocyanin.

15. The nitrofurantoin metabolite detection product according to claim 12, characterized in that, The coating concentration of the monoclonal antibody or its antigen-binding fragment against the anti-nitrofurantoin metabolite on the conjugation pad is 5-20 μg / mL.

16. A method for detecting nitrofurantoin metabolites, characterized in that, It includes any of the following methods: (1) Add diluent and the monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment as described in any one of claims 2-4 to the control wells of a microplate coated with nitrofurantoin metabolite antigen, and incubate; add the monoclonal antibody against nitrofurantoin metabolites or its antigen-binding fragment as described in any one of claims 2-4, and nitrofurantoin metabolite derivative standards or test samples to the inhibition wells of a microplate coated with nitrofurantoin metabolite antigen, and incubate; by adding enzyme-labeled secondary antibody to the control wells and inhibition wells respectively and incubating, the absorbance of the control wells and inhibition wells is detected by colorimetric reaction, and the concentration of nitrofurantoin metabolite derivatives in the test sample is obtained by plotting a standard curve of absorbance versus concentration of nitrofurantoin metabolite derivatives; the test sample is derivatized before loading. (2) The sample to be tested after derivatization is loaded onto the sample pad of the test strip as described in claim 12, and the presence of nitrofurantoin metabolites in the sample is determined based on the chromatographic results of the test strip.

17. The method for detecting nitrofurantoin metabolites according to claim 16, characterized in that, The coating concentration of the nitrofurantoin metabolite derivative antigen on the microplate was 2.5 × 10⁻⁶. -5 -1×10 -3 mg / mL; the concentration of the monoclonal antibody against the anti-nitrofurantoin metabolite or its antigen-binding fragment added is 1×10 mg / mL. -5- 1×10 -3 mg / mL.

18. A nucleic acid molecule, characterized in that, It encodes a monoclonal antibody or antigen-binding fragment thereof against the anti-nitrofurantoin metabolite as described in any one of claims 2-4.

19. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 18.

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