Monoclonal antibody against streptomycin and use thereof

Abstract

The application discloses a kind of monoclonal antibody of streptomycin resistance and application thereof, it is related to streptomycin detection technical field.The application filters out the hybridoma cell strain that can stably secrete anti-streptomycin monoclonal antibody, and its preservation number is CGMCC NO.46126.Specific, sensitive streptomycin monoclonal antibody is obtained by screening.The experiment proves that the streptomycin monoclonal antibody provided in the application has high specificity to streptomycin, and has high detection sensitivity to streptomycin, and wide linear detection range.Therefore, the anti-streptomycin monoclonal antibody provided in the application can be used for developing streptomycin detection kit, test strip and other products, and the application provides detection tools and means for rapid detection of streptomycin and other antibiotic residues in livestock products, aquatic products and agriculture.

Description

Technical Field

[0001] This invention relates to the field of streptomycin detection technology, and more specifically, to an anti-streptomycin monoclonal antibody and its application. Background Technology

[0002] Streptomycin (Str) is an aminoglycoside antibiotic, the second antibiotic produced and used clinically after penicillin. Due to its effective killing of Gram-negative bacteria and Mycobacterium tuberculosis, and its low cost and good efficacy, streptomycin is widely used in animal husbandry, aquaculture, and agriculture as a feed additive and therapeutic agent, leading to its misuse. Long-term consumption of food containing streptomycin residues poses a significant risk to human health. GB 31650-2019 specifies the maximum residue limit for streptomycin as 200 μg / kg in cow / sheep milk, 600 μg / kg in livestock and poultry muscle, fat, and liver, and 1000 μg / kg in kidneys.

[0003] Currently, the main methods for detecting streptomycin residues include three types: microbiological, instrumental analysis, and immunoassay. Among them, microbiological methods suffer from low detection sensitivity and long detection times, and their application is limited by the requirement to be performed in a laboratory. Instrumental analysis methods, mainly high-performance liquid chromatography (HPLC), gas chromatography (GC), and liquid chromatography-mass spectrometry (LC-MS), have problems such as expensive equipment, cumbersome operation, time and labor costs, high expenses, and inability to conduct large-scale detection. They also require technicians to have certain operational and analytical skills, thus hindering their widespread adoption.

[0004] Enzyme-linked immunosorbent assay (ELISA) and immunoassay strips have the advantages of being simple, fast, sensitive, inexpensive, and capable of large-scale and on-site testing. They are especially suitable for detecting antibiotic residues such as streptomycin in livestock products during production and distribution. However, the establishment of these two immunological detection methods requires the preparation of specific and sensitive antibodies against streptomycin.

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

[0006] The purpose of this invention is to provide an anti-streptomycin monoclonal antibody and its application to solve the above-mentioned technical problems.

[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.46126.

[0009] Secondly, the present invention provides an anti-streptomycin monoclonal antibody or its antigen-binding fragment thereof, comprising: a heavy chain complementarity-determining region and a light chain complementarity-determining region, wherein the heavy chain complementarity-determining region comprises: CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 1-3 in sequence, and the light chain complementarity-determining region comprises: CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is DVS.

[0010] Thirdly, the present invention provides an antibody conjugate formed by conjugating the above-mentioned anti-streptomycin monoclonal antibody 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, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0011] Fourthly, the present invention provides the use of an anti-streptomycin monoclonal antibody or its antigen-binding fragment, or the antibody-conjugate thereof, in any of the following:

[0012] (1) Detection of streptomycin or dihydrostreptomycin;

[0013] (2) Prepare streptomycin or dihydrostreptomycin detection products;

[0014] The testing products include reagents, kits, test strips, antibody chips, antibody probes, or testing instruments.

[0015] Fifthly, the present invention provides a streptomycin or dihydrostreptomycin 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 anti-streptomycin monoclonal antibody or its antigen-binding fragment, or the above-mentioned antibody secreted by hybridoma cells.

[0016] Sixthly, the present invention provides a method for detecting streptomycin or dihydrostreptomycin, comprising any one of the following methods:

[0017] (1) Add diluent and the above-mentioned anti-streptomycin monoclonal antibody or its antigen-binding fragment to the control well of the microplate coated with streptomycin antigen, and incubate; add streptomycin standard or test sample and the above-mentioned anti-streptomycin monoclonal antibody or its antigen-binding fragment to the inhibition well of the microplate coated with streptomycin antigen, and incubate; by adding enzyme-labeled secondary antibody to the control well and inhibition well respectively and incubating, the absorbance of the control well and inhibition well is detected by colorimetric reaction, and the concentration of streptomycin in the test sample is obtained by plotting the standard curve of absorbance versus streptomycin concentration;

[0018] (2) Load the sample to be tested onto the sample pad of the test strip, and determine whether the sample contains streptomycin based on the chromatographic results of the test strip.

[0019] In a seventh aspect, the present invention provides a nucleic acid molecule that encodes the above-mentioned anti-streptomycin monoclonal antibody or its antigen-binding fragment.

[0020] The present invention has the following beneficial effects:

[0021] This invention involves immunizing mice with streptomycin antigen, followed by hybridoma fusion, and screening for hybridoma cell lines that stably secrete anti-streptomycin monoclonal antibodies. Specific and sensitive streptomycin monoclonal antibodies are then obtained through screening. Immunoassay experiments demonstrate that the streptomycin monoclonal antibody provided by this invention has high specificity for streptomycin, high detection sensitivity for streptomycin, and a wide linear detection range. Therefore, the anti-streptomycin monoclonal antibody provided by this invention can be used to develop streptomycin detection kits, test strips, and other products. This invention provides a detection tool and method for the rapid detection of streptomycin and other antibiotic residues in livestock products, aquatic products, and agriculture. Attached Figure Description

[0022] 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.

[0023] Figure 1 A standard curve for the detection of streptomycin;

[0024] Figure 2 The graph shows the detection results of 5 ng / mL and 10 ng / mL streptomycin and dihydrostreptomycin in milk samples using colloidal gold test strips. Detailed Implementation

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Definition of noun

[0029] 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."

[0030] 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, as long as they exhibit the desired biological activity, such as specific binding to streptomycin antigens or fragments thereof.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.46126. This hybridoma cell line stably secretes monoclonal antibodies against streptomycin. The submitted biological material is LMS-1D11H8, scientifically described as a mouse hybridoma cell line, deposited on October 23, 2024, and identified as viable.

[0037] Secondly, the present invention provides an anti-streptomycin monoclonal antibody or its antigen-binding fragment thereof, comprising: a heavy chain complementarity-determining region and a light chain complementarity-determining region, wherein the heavy chain complementarity-determining region comprises: CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 1-3 in sequence, and the light chain complementarity-determining region comprises: CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is DVS.

[0038] Immunoassay experiments demonstrated that the anti-streptomycin monoclonal antibody provided by this invention exhibits high specificity for streptomycin, high detection sensitivity, and a wide linear detection range. Therefore, the anti-streptomycin monoclonal antibody provided by this invention can be used to develop streptomycin detection kits, test strips, and other products. This invention provides a detection tool and method for the rapid detection of streptomycin and other antibiotic residues in livestock products.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

[0044] 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.

[0045] 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.

[0046] Thirdly, the present invention provides an antibody conjugate formed by conjugating the above-mentioned anti-streptomycin monoclonal antibody 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, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0047] 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.

[0048] 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.).

[0049] 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.

[0050] 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 and 18 F.

[0051] 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.

[0052] 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.

[0053] In a preferred embodiment of the present invention, the colloid is selected from colloidal gold, colloidal silver, or colloidal selenium.

[0054] Fourthly, the present invention provides the use of an anti-streptomycin monoclonal antibody or its antigen-binding fragment, or the antibody-conjugate thereof, in any of the following:

[0055] (1) Detection of streptomycin or dihydrostreptomycin;

[0056] (2) Prepare streptomycin or dihydrostreptomycin detection products;

[0057] The testing products include reagents, kits, test strips, antibody chips, antibody probes, or testing instruments.

[0058] 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.

[0059] Antibody chips are chips formed by immobilizing the aforementioned anti-streptomycin antibodies or their antigen-binding fragments on a carrier.

[0060] 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; for example, by chemical coupling, the antibody or its antigen-binding fragment is linked to the solid phase.

[0061] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates, and membranes;

[0062] 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.

[0063] Antibodies against streptomycin or their antigen-binding fragments have the property of specifically binding to streptomycin. These antibodies can be used for the rapid detection of antibiotic residues such as streptomycin in livestock products, aquatic products, and agriculture.

[0064] Fifthly, the present invention provides a streptomycin or dihydrostreptomycin 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 anti-streptomycin monoclonal antibody or its antigen-binding fragment, or the above-mentioned antibody secreted by hybridoma cells.

[0065] In a preferred embodiment of the present invention, the detection product is an ELISA kit, which includes a microplate and an anti-streptomycin monoclonal antibody or its antigen-binding fragment, wherein the microplate is coated with streptomycin antigen; preferably, it is a competitive ELISA kit.

[0066] In a preferred embodiment of the present invention, the detection product is a test strip, which includes a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with an anti-streptomycin monoclonal antibody or its antigen-binding fragment. The strip is assembled in the order of sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper, and then cut into test strips of a certain width using a cutting machine.

[0067] In a preferred embodiment of the present invention, the binding pad is coated with a colloidal gold-labeled anti-streptomycin monoclonal antibody or its antigen-binding fragment.

[0068] In a preferred embodiment of the present invention, the nitrocellulose membrane has T lines and C lines, the T lines have streptomycin antigen, and the C lines have X anti-mouse antibody; X is sheep, rabbit, horse, monkey or chicken.

[0069] In a preferred embodiment of the present invention, the coating concentration of streptomycin 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. Good detection performance is achieved at these coating concentrations.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Furthermore, the kit may also include at least one of the following: buffer solution, detection reagent, diluent, washing solution, streptomycin antigen, and streptomycin standard, and is not limited thereto.

[0074] Sixthly, the present invention provides a method for detecting streptomycin or dihydrostreptomycin, comprising any one of the following methods:

[0075] (1) Add diluent and the above-mentioned anti-streptomycin monoclonal antibody or its antigen-binding fragment to the control well of the microplate coated with streptomycin antigen, and incubate; add streptomycin standard or test sample and the above-mentioned anti-streptomycin monoclonal antibody or its antigen-binding fragment to the inhibition well of the microplate coated with streptomycin antigen, and incubate; by adding enzyme-labeled secondary antibody to the control well and inhibition well respectively and incubating, the absorbance of the control well and inhibition well is detected by colorimetric reaction, and the concentration of streptomycin in the test sample is obtained by plotting the standard curve of absorbance versus streptomycin concentration;

[0076] (2) Load the sample to be tested onto the sample pad of the test strip, and determine whether the sample contains streptomycin based on the chromatographic results of the test strip.

[0077] 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.

[0078] When plotting a standard curve of absorbance versus streptomycin concentration, this includes, but is not limited to, plotting: a standard curve of the absorbance ratio of the inhibition well / control well versus the streptomycin standard concentration, or plotting a standard curve of the absorbance ratio of (control well - inhibition well) / control well versus the streptomycin standard concentration.

[0079] In a preferred embodiment of the present invention, the coating concentration of streptomycin antigen on the microplate is 1 × 10⁻⁶. -5 -2.5×10 -4 mg / mL; the dilution of the anti-streptomycin monoclonal antibody or its antigen-binding fragment is 1:1000-8000. Correspondingly, the added concentration of the anti-streptomycin monoclonal antibody or its antigen-binding fragment is 1×10⁻⁶ mg / mL. -4 -1×10 -3 mg / mL. At the above coating concentration and dilution, the inhibitory effect on streptomycin was the best. The higher the inhibition rate, the higher the binding activity of the antibody with streptomycin in the test sample, and the more accurate the detection result.

[0080] The coating concentration of streptomycin antigen on the microplate was 1×10⁻⁶. -5 mg / mL, 2×10 -5 mg / mL, 3×10 -5 mg / mL, 4×10 -5 mg / mL, 5×10 -5 mg / mL, 6×10 -5 mg / mL, 7×10 -5 mg / mL, 8×10 -5 mg / mL, 9×10 -5 mg / mL, 1×10 -4 mg / mL, 2×10 -4 mg / mL or 2.5×10 -4 mg / mL.

[0081] The concentration of the anti-streptomycin monoclonal antibody or its antigen-binding fragment added was 1 × 10⁻⁶. -4 mg / mL, 2×10 - 4 mg / mL, 3×10 -4 mg / mL, 4×10 -4mg / mL, 5×10 -4 mg / mL, 6×10 -4 mg / mL, 7×10 -4 mg / mL, 8×10 - 4 mg / mL, 9×10 -4 mg / mL or 1×10 -3 mg / mL.

[0082] In a seventh aspect, the present invention provides a nucleic acid molecule that encodes the above-mentioned anti-streptomycin monoclonal antibody or its antigen-binding fragment.

[0083] 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.

[0084] Eighthly, the present invention also provides a carrier containing the above-mentioned nucleic acid molecules.

[0085] 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.

[0086] 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.

[0087] In a ninth aspect, the present invention also provides a recombinant cell containing the aforementioned carrier.

[0088] 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.

[0089] In one alternative implementation, the recombinant cells are eukaryotic cells.

[0090] In one alternative implementation, the recombinant cells are mammalian cells.

[0091] In one alternative implementation, the recombinant cells are HEK293.

[0092] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0093] Example 1

[0094] This example demonstrates the preparation of monoclonal antibodies.

[0095] 1. Preparation of immunogens and coating agents

[0096] Following the method reported in the literature (Fan Guoying, Wang Shungang, Wang Ziliang, et al. Preparation and identification of artificial antigens of streptomycin [J]. Northwest Agricultural Journal, 2009, 18(2):45-50. DOI:10.3969 / j.issn.1004-1389.2009.02.011), streptomycin antigens were prepared. Using the aldehyde group on streptomycin, a carboxyl group was introduced by oxime treatment with O-carboxymethyl hydroxylamine to obtain streptomycin haptens. Then, two complete antigens, Str-BSA and Str-OVA, were synthesized separately by EDC method and aliquoted and stored at 4℃. Str-BSA was used as an immunogen for mouse immunization, and Str-OVA was used as a coating antigen for serum and antibody titer detection.

[0097] 2. Monoclonal antibody preparation:

[0098] (1) Three BALB / C female mice aged 6-8 weeks were selected as experimental animals.

[0099] (2) Basic immunization: The diluted complete antigen Str-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.

[0100] (3) Booster Immunization: Two weeks after the primary immunization, diluted complete antigen Str-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 the complete antigen Str-OVA.

[0101] (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.

[0102] (5) Cell fusion

[0103] 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 performed through limiting dilutions to obtain the streptomycin monoclonal antibody hybridoma cell line LMS-1D11H8. The antibodies secreted by this cell line showed good specificity for streptomycin, with a detection sensitivity of 1.1 μg / L. This cell line is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC NO. 46126.

[0104] (6) Cell cryopreservation and thawing

[0105] The obtained hybridoma cell line LMS-1D11H8 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.

[0106] (7) Preparation and purification of monoclonal antibodies.

[0107] 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 LMS-1D11H8, with approximately 10⁶ cells injected per mouse. Once 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 purified monoclonal antibodies secreted by the hybridoma cell line LMS-1D11H8.

[0108] Example 2

[0109] The monoclonal antibody obtained in Example 1 was tested for antibody efficacy.

[0110] The buffer solutions used in the following experiments are as follows:

[0111] Coating buffer (pH 9.6 0.05M carbonate buffer): Na2CO3 1.5g; NaHCO3 2.94g, add pure water to a final volume of 1000mL;

[0112] Phosphate-buffered saline (PBS) (0.01M pH 7.4): 0.2g KH2PO4; 8g NaCl; 2.92g NaH2PO4·12H2O, add pure water to 1000mL;

[0113] Washing buffer (PBST): Add 1 mL of Tween-20 to 1000 mL of prepared PBS solution;

[0114] 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;

[0115] Colorimetric solutions: TMB stock solution (375 mg TMB solid + 30 mL DMSO (prepared according to the ratio, stored at room temperature and protected from light)), BUFFER (0.1 g potassium sorbate + 46.04 g potassium dihydrogen citrate hydrate + 1 L pure water). The colorimetric solution is prepared fresh each time: 200 μ L TMB stock solution + 11 mL BUFFER + 3.34 μ L 30% hydrogen peroxide solution.

[0116] Termination solution (2M H2SO4): Add 445.6 mL of distilled water dropwise to 54.4 mL of concentrated sulfuric acid (98%) while stirring.

[0117] The following is a checkerboard experiment involving antigen and antibody:

[0118] 1) Wrapped in blankets:

[0119] 1 mg / mL of streptomycin complete antigen Str-OVA was serially diluted with coating buffer at concentrations of 1:2000, 1:4000, 1:8000, and 1:16000 to obtain coating antigen solutions of different concentrations of streptomycin complete antigen Str-OVA. 100 μL of the streptomycin complete antigen Str-OVA coating antigen solution prepared in step 1 was added to each well of a 96-well microplate and incubated overnight at 4°C. The plate was then washed three times with PBST.

[0120] 2) Competition:

[0121] Dilute the purchased 100 μg / mL streptomycin 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 streptomycin standard solution to each inhibition well.

[0122] Streptomycin antibody was serially diluted with PBSTG at 1:1000, 1:2000, 1:4000 and 1:8000 to obtain streptomycin antiserum dilution (50 uL / well). The solution was placed in a humidified chamber at 37°C for 30 min and washed 3 times.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] The results are shown in Table 1 below:

[0128]

[0129] Note: I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate.

[0130] The results in Table 1 show that the best inhibitory effect on streptomycin was achieved when the coating antigen dilution was 1:4000 and the antibody dilution was 1:8000, with an inhibition rate of 81.4%. The higher the inhibition rate, the higher the binding activity of the antibody with streptomycin in the test sample, and the more accurate the detection results. This indicates that the antibody produced by the hybridoma cells LMS-1D11H8 can detect streptomycin.

[0131] Example 3

[0132] This embodiment provides a standard curve for detecting streptomycin and tests the sensitivity of the detection method.

[0133] The streptomycin standard solution was diluted with sample diluent to the following concentrations: 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.31 ng / mL, and 0.15 ng / mL. A standard curve experiment was performed following the checkerboard assay procedure, with three replicates for each standard concentration. The coating antigen was diluted 1:4000, and the antibody was diluted 1:8000 for detection.

[0134] Plotting a standard curve: Using streptomycin 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 streptomycin 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 1 As shown.

[0135] The results show that its sensitivity (IC) 50 The concentration was 1.1 ng / mL, and the sensitivity (IC50) was 1.1 ng / mL. 50 () is the concentration value of streptomycin standard when the inhibition rate reaches 50%.

[0136] The detection range is 0.4 ng / mL to 5 ng / mL. This indicates that the streptomycin antibody prepared by the above method has high sensitivity and good detection performance.

[0137] Example 4

[0138] This embodiment performs specific detection on the antibody prepared in Example 1.

[0139] Following the preparation method of streptomycin standard in Example 3, standard samples of dihydrostreptomycin, spectinomycin, neomycin, gentamicin, and kanamycin were prepared.

[0140] The five drugs were diluted with sample diluent to the following concentrations: 5000 ng / mL, 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 200 ng / mL, and 100 ng / mL.

[0141] 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 streptomycin standard curve mentioned above.

[0142] Cross-reactivity rate (%) = (streptomycin IC50) 50 ) / (Similar IC 50 )×100%.

[0143] Drug Name <![CDATA[IC 50 (ng / mL)]]> Cross-reactivity rate (%) Streptomycin 1.1 100 dihydrostreptomycin 1.96 56.1 Spectinomycin >1000 <0.15 Kanamycin >1000 <0.15 Neomycin sulfate >1000 <0.15 Gentamicin sulfate >1000 <0.15 Chloramphenicol >1000 <0.15 sulfadiazine >1000 <0.15

[0144] The results showed that the antibody prepared in Example 1 had good specificity for streptomycin and cross-reactivity with dihydrostreptomycin.

[0145] Example 5

[0146] This embodiment provides a colloidal gold test strip.

[0147] 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 from 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).

[0148] 2) Scribing: Use a gold-spraying scribing instrument to scribble lines on the nitrocellulose membrane attached to the substrate. The concentration of T-line antigen (Str-BSA) is 1.0 mg / mL, and the concentration of C-line secondary antibody is 1.0 mg / mL.

[0149] 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.

[0150] 4) Detection: Prepare 5 ng / mL and 10 ng / mL streptomycin and dihydrostreptomycin standard solutions using fresh milk samples, respectively. Add 200 μL of each solution to a 96-well plate. Simultaneously, add 200 μL of fresh milk as a blank control to the same well. Then, insert six prepared test strips into the wells containing the test solutions and allow them to react for 10 min. The results are then compared with the control. Figure 2 As shown.

[0151] It is evident that the prepared test strip can be used to detect streptomycin and dihydrostreptomycin at 10 ng / mL in fresh milk. The detection sensitivity meets the detection requirements of GB 31650-2019, which specifies a maximum residue limit of 200 μg / kg in milk, 600 μg / kg in animal muscle, fat, and liver, and 1000 μg / kg in kidney.

[0152] The sequence information involved in this invention is as follows:

[0153]

[0154]

[0155] 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.46126.

2. A monoclonal antibody against streptomycin or its antigen-binding fragment, characterized in that, It includes: The heavy chain complementarity-determining region (CDR-H1), CDR-H2, and CDR-H3 have amino acid sequences as shown in SEQ ID NO: 1-3. The light chain complementarity-determining region (CDR-L1), CDR-L2, and CDR-L3 have amino acid sequences as shown in SEQ ID NO: 4-5. The amino acid sequence of CDR-L2 is DVS.

3. The anti-streptomycin monoclonal antibody 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, whose amino acid sequences are shown in SEQ ID NO:6-9; the light chain framework region includes LFR1, LFR2, LFR3 and LFR4, whose amino acid sequences are shown in SEQ ID NO:10-13.

4. The anti-streptomycin monoclonal antibody 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 the anti-streptomycin monoclonal antibody or its antigen-binding fragment as described in any one of claims 2-4 and a marker, wherein the marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

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 anti-streptomycin monoclonal antibody or its antigen-binding fragment as described in any one of claims 2-4, or the antibody conjugate as described in any one of claims 5-7, in any of the following: (1) Detection of streptomycin or dihydrostreptomycin; (2) Prepare streptomycin or dihydrostreptomycin detection products; The detection products include reagents, kits, test strips, antibody chips, antibody probes, or detection instruments.

9. A streptomycin or dihydrostreptomycin 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 anti-streptomycin monoclonal antibody or its antigen-binding fragment as described in any one of claims 2-4, or the antibody secreted by hybridoma cells as described in claim 1.

10. The streptomycin or dihydrostreptomycin detection product according to claim 9, characterized in that, The detection product is an ELISA kit, which includes a microplate and the anti-streptomycin monoclonal antibody or its antigen-binding fragment, wherein the microplate is coated with streptomycin antigen.

11. The streptomycin or dihydrostreptomycin detection product according to claim 9, characterized in that, The detection product is a test strip, which includes a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with the anti-streptomycin monoclonal antibody or its antigen-binding fragment.

12. The streptomycin or dihydrostreptomycin detection product according to claim 11, characterized in that, The nitrocellulose membrane has T lines and C lines, the T lines have streptomycin antigen, and the C lines have X anti-mouse antibody; X is sheep, rabbit, horse, monkey or chicken.

13. The streptomycin or dihydrostreptomycin detection product according to claim 12, characterized in that, The coating concentration of streptomycin 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.

14. A method for detecting streptomycin or dihydrostreptomycin, characterized in that, It includes any of the following methods: (1) Add diluent and the anti-streptomycin monoclonal antibody or its antigen-binding fragment as described in any one of claims 2-4 to the control wells of a microplate coated with streptomycin antigen, and incubate; add streptomycin standard or the sample to be tested and the anti-streptomycin monoclonal antibody or its antigen-binding fragment as described in any one of claims 2-4 to the inhibition wells of a microplate coated with streptomycin antigen, and incubate; by adding enzyme-labeled secondary antibody to the control wells and the inhibition wells respectively and incubating, the absorbance of the control wells and the inhibition wells is detected by colorimetric reaction; by plotting a standard curve of absorbance versus streptomycin concentration, the concentration of streptomycin in the sample to be tested is obtained according to the standard curve; (2) Load the sample to be tested onto the sample pad of the test strip, and determine whether the sample contains streptomycin based on the chromatographic results of the test strip.

15. The method for detecting streptomycin or dihydrostreptomycin according to claim 14, characterized in that, The coating concentration of streptomycin antigen on the microplate is 1×10⁻⁶. -5 -2.5×10 -4 mg / mL; the concentration of the anti-streptomycin monoclonal antibody or its antigen-binding fragment added is 1×10 mg / mL. -4 -1×10 -3 mg / mL.

16. A nucleic acid molecule, characterized in that, It encodes the anti-streptomycin monoclonal antibody or its antigen-binding fragment as described in any one of claims 2-4.

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