Anti-streptomycin monoclonal antibody and application thereof

Abstract

The invention discloses an anti-streptomycin monoclonal antibody and application thereof, and relates to the technical field of streptomycin detection. According to the invention, a hybridoma cell strain capable of stably secreting an anti-streptomycin monoclonal antibody is screened, and the preservation number of the hybridoma cell strain is CGMCC (China General Microbiological Culture Collection Center And screening to obtain the specific and sensitive streptomycin monoclonal antibody. Immunodetection experiments prove that the streptomycin monoclonal antibody provided by the invention has high specificity to streptomycin, high detection sensitivity to streptomycin and wide linear detection range. Therefore, the anti-streptomycin monoclonal antibody provided by the invention can be used for developing streptomycin detection kits, test strips and other products, and a detection tool and means are provided for rapid detection of streptomycin and other antibiotic residues in animal products, aquatic products and agriculture.

Description

Technical Field

[0001] The present invention relates to the technical field of streptomycin detection, and in particular, to a monoclonal antibody against streptomycin and its application. Background Art

[0002] Streptomycin (Str) is an aminoglycoside antibiotic and is the second antibiotic produced and used clinically after penicillin. Since streptomycin has good bactericidal effects on Gram-negative bacteria and Mycobacterium tuberculosis, and has low cost and good curative effect, it is often used as a feed additive and therapeutic agent in aquaculture, fisheries and agriculture, which has led to the problem of abuse of streptomycin. Long-term intake of foods with streptomycin residues is greatly harmful to human health. GB 31650-2019 stipulates that the maximum residue limit of streptomycin in bovine / goat milk is 200 μg / kg, in livestock and poultry muscle, fat and liver is 600 μg / kg, and in kidneys is 1000 μg / kg.

[0003] At present, the main detection methods for streptomycin residues are microbiology, instrumental analysis and immunoassay. Among them, the microbiological method has the defects of low detection sensitivity and long time, and its application is limited because it must be carried out in the laboratory; the instrumental analysis methods mainly include high performance liquid chromatography (HPLC), gas chromatography (GC) and liquid chromatography-mass spectrometry (LC-MS). They have problems such as expensive instruments, cumbersome operation, time-consuming and laborious, high cost, and inability to detect on a large scale, and also require technicians to have certain operation and analysis skills, so they cannot be well promoted.

[0004] The enzyme-linked immunosorbent assay (ELISA) and immunochromatographic strip both have the advantages of simplicity, rapidity, sensitivity, low cost, and the ability to detect on a large scale and on-site. They are particularly suitable for detecting the residues of antibiotics such as streptomycin in livestock products during production and circulation. To establish these two immunological detection methods, antibodies specific and sensitive to streptomycin need to be prepared.

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

[0006] The purpose of the present invention is to provide a monoclonal antibody against streptomycin and its application to solve the above technical problems.

[0007] The present invention is implemented as follows:

[0008] In the first aspect, the present invention provides a hybridoma cell, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC NO.46126.

[0009] In a second aspect, the present invention provides a streptomycin-resistant monoclonal antibody or an antigen-binding fragment thereof, which comprises: heavy-chain complementarity-determining regions and light-chain complementarity-determining regions. The heavy-chain complementarity-determining regions include: CDR-H1, CDR-H2, and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light-chain complementarity-determining regions include: CDR-L1, CDR-L2, and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is DVS.

[0010] In a third aspect, the present invention provides an antibody conjugate, which is formed by conjugating the above streptomycin-resistant monoclonal antibody or an antigen-binding fragment thereof with a labeling agent, and the labeling agent is selected from at least one of fluorescent dyes, enzymes that catalyze substrate chromogenic reactions, radioisotopes, chemiluminescent reagents, and nanoparticle-based labeling agents.

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

[0012] (1) Detecting streptomycin or dihydrostreptomycin;

[0013] and (2) preparing a streptomycin or dihydrostreptomycin detection product;

[0014] The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe, or a detector.

[0015] In a fifth aspect, the present invention provides a streptomycin or dihydrostreptomycin detection product. The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe, or a detector; the detection product comprises the above streptomycin-resistant monoclonal antibody or an antigen-binding fragment thereof, or an antibody secreted by the above hybridoma cells.

[0016] In a sixth aspect, the present invention provides a method for detecting streptomycin or dihydrostreptomycin, which comprises any one of the following methods:

[0017] (1) Adding a diluent and the above streptomycin-resistant monoclonal antibody or an antigen-binding fragment thereof to a control well of a microplate coated with a streptomycin antigen, and incubating; adding a streptomycin standard or a test sample and the above streptomycin-resistant monoclonal antibody or an antigen-binding fragment thereof to an inhibition well of a microplate coated with a streptomycin antigen, and incubating; adding an enzyme-labeled secondary antibody to the control well and the inhibition well respectively for incubation, and after a chromogenic reaction, detecting the absorbance of the control well and the inhibition well, and obtaining the concentration of streptomycin in the measured sample by plotting a standard curve of absorbance versus streptomycin concentration and according to the standard curve;

[0018] (2) Load the sample to be tested onto the sample pad of the test strip, and determine whether streptomycin is contained in the sample according to the chromatographic result of the test strip.

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

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

[0021] In the present invention, mice are immunized with the antigen of streptomycin, and after hybridoma fusion, hybridoma cell lines that can stably secrete streptomycin-resistant monoclonal antibodies are screened out, and specific and sensitive streptomycin monoclonal antibodies are obtained through screening. The streptomycin monoclonal antibody provided by the present invention is proved to have high specificity for streptomycin, high detection sensitivity for streptomycin, and a wide linear detection range through immunodetection experiments. Therefore, the streptomycin-resistant monoclonal antibody provided by the present invention can be used to develop products such as detection kits and test strips for streptomycin. The present invention provides detection tools and means for the rapid detection of antibiotic residues such as streptomycin in livestock products, aquatic products, and agriculture. Description of the Drawings

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

[0023] Figure 1 It is a standard curve graph for detecting streptomycin;

[0024] Figure 2 It is a detection result graph for detecting 5 ng / mL and 10 ng / mL of streptomycin and dihydrostreptomycin milk standards with a colloidal gold test strip. Detailed Embodiments

[0025] References to embodiments of the present invention will now be provided in detail, with one or more examples described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0026] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Coligan et al., eds., 1991), each of which is hereby incorporated by reference in its entirety.

[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. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer are conventional products that can be obtained commercially.

[0028] Definition of Terms

[0029] The term "antigen-binding fragment" generally refers to any protein / protein fragment containing CDR regions, especially antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-compound binding fragments of these antibodies, including Fab, F(ab’)2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the minimum antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0030] The term "antibody" 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 required biological activity, such as specifically binding to the streptomycin antigen or its fragments.

[0031] In the present invention, the term "complementary determining region or complementarity-determining region", "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to regions containing one or more or even all of the major amino acid residues that play a role in the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0032] In the present invention, the heavy-chain complementarity-determining regions are denoted as HCDR and include HCDR1, HCDR2, and HCDR3; the light-chain complementarity-determining regions are denoted as LCDR and include LCDR1, LCDR2, and LCDR3. Commonly used CDR labeling methods in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label the CDR regions, but CDR regions labeled by other methods also fall within the protection scope of the present invention.

[0033] Under normal circumstances, the variable region VH of the heavy chain of an antibody can be obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0034] The variable region VL of the light chain of an antibody can be obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: 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 sugar-sugar (backbone) linkages. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of the present invention can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and can contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases can also be included. Examples of these modified bases include nitrogen-containing and de-nitrogenated adenine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine.

[0036] In a first aspect, the present invention provides a hybridoma cell, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO. 46126. This hybridoma cell can stably secrete a monoclonal antibody against streptomycin. The submitted biological material is LMS-1D11H8, scientific description: mouse hybridoma cell line, the deposit date is October 23, 2024, and the identification result is viable.

[0037] In a second aspect, the present invention provides a monoclonal antibody against streptomycin or an antigen-binding fragment thereof, which includes: heavy chain complementarity-determining regions and light chain complementarity-determining regions. The heavy chain complementarity-determining regions include: CDR-H1, CDR-H2, and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light chain complementarity-determining regions include: CDR-L1, CDR-L2, and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is DVS.

[0038] The monoclonal antibody against streptomycin provided by the present invention is proven by immunoassay experiments to have high specificity for streptomycin, high detection sensitivity for streptomycin, and a wide linear detection range. Therefore, the monoclonal antibody against streptomycin provided by the present invention can be used to develop products such as detection kits and test strips for streptomycin. The present invention provides detection tools and means for the rapid detection of antibiotic residues such as streptomycin in livestock products.

[0039] In a preferred embodiment of the application of the present invention, the antibody or its antigen-binding fragment further comprises a heavy-chain framework region and a light-chain framework region; the heavy-chain framework region comprises HFR1, HFR2, HFR3 and HFR4 which have at least 80% homology with the amino acid sequences shown in SEQ ID NOs: 6-9 in sequence; for example, the heavy-chain framework region comprises HFR1, HFR2, HFR3 and HFR4 which have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NOs: 6-9 in sequence.

[0040] The light-chain framework region comprises LFR1, LFR2, LFR3 and LFR4 which have at least 80% homology with the amino acid sequences shown in SEQ ID NOs: 10-13 in sequence; for example, the light-chain framework region comprises LFR1, LFR2, LFR3 and LFR4 which have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NOs: 10-13 in sequence.

[0041] In one embodiment, the heavy-chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 14, and the light-chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 15.

[0042] In a preferred embodiment of the application of the present invention, the antibody or its antigen-binding fragment further comprises a constant region, and the constant region comprises a heavy-chain constant region, and / or, a light-chain constant region. The heavy-chain constant region is selected from the heavy-chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light-chain constant region is selected from the κ-type or λ-type light-chain constant regions;

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

[0044] The antigen-binding fragments of the above-mentioned antibodies generally have the same binding specificity as the antibodies from which they are derived. It is readily understood by those skilled in the art from the content described in the present invention that the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds.

[0045] The antigen-binding fragments of the above-mentioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0046] In a third aspect, 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, and the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate chromogenesis, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0047] The above-mentioned label refers to a class of substances having characteristics such as luminescence, chromogenesis, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through these characteristics. In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs, and no matter which label is used, it falls within the protection scope of the present invention.

[0048] Fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (such as, for example, but not limited to fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, for example, but not limited to red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, for example, but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, for example, but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, for example, but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0049] In an alternative embodiment, the enzymes that catalyze substrate chromogenesis include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0050] In alternative embodiments, the radioisotopes include, but are not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0051] In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, rhodamine and its derivatives, and peroxyoxalate and its derivatives.

[0052] In a preferred embodiment of the application of the present invention, the nanoparticle markers are 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 application 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 a monoclonal antibody against streptomycin or its antigen-binding fragment or the above-mentioned antibody conjugate in any one of the following:

[0055] (1) Detecting streptomycin or dihydrostreptomycin;

[0056] And (2) preparing a streptomycin or dihydrostreptomycin detection product;

[0057] The test product is a reagent, kit, test strip, antibody chip, antibody probe or detector.

[0058] In order to improve the stability of the reagent and extend its shelf life, those skilled in the art can add functional components such as stabilizers and protectants to the reagent as needed. The protein stabilizer is selected from: sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100 and Tween-20. The protectant is selected from cryoprotectants, such as polyols and sugars. The polyol is selected from sorbitol, mannitol or a mixture thereof. The form of the reagent includes, but is not limited to, solid, liquid, and semi-solid.

[0059] The antibody chip refers to a chip formed by immobilizing the above-mentioned anti-streptomycin antibody or its antigen-binding fragment on a carrier.

[0060] In a preferred embodiment of the application of the present invention, the kit includes a solid phase, and the antibody or its antigen-binding fragment is coated on the solid phase; for example, by chemical coupling, the antibody or its antigen-binding fragment is connected to the solid phase.

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

[0062] In a preferred embodiment of the application of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic particles, latex microspheres, microtiter plates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0063] Based on the property that the anti-streptomycin antibody or its antigen-binding fragment specifically binds to streptomycin, the antibody can be used for the rapid detection of antibiotic residues such as streptomycin in livestock products, aquatic products and agriculture.

[0064] In a fifth aspect, the present invention provides a streptomycin or dihydrostreptomycin detection product, and 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 antibody secreted by the above-mentioned hybridoma cells.

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

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

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

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

[0069] In a preferred embodiment of the application of the present invention, the coating concentration of the streptomycin antigen on the T line is 0.1-2 mg / mL, and the coating concentration of the anti-mouse antibody of X on the C line is 0.2-1 mg / mL. Good detection effects are achieved at such coating concentrations.

[0070] The above-mentioned chip can also be called a suspension array or a liquid array. It includes a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound to the surface of the carrier.

[0071] The aforementioned carrier can be of various materials and forms. For example, it can preferably be selected from containers with a flat bottom. A more typical preferred example is the multi-well plates, microplates, microfluidics-based devices (such as microfluidic chips), petri-dish-like containers, etc. widely used in biochemical detection, and is not limited thereto.

[0072] The microfluidic chip is selected from PDMS chips or metal droplet generators of T-type chips, flow-focusing type chips or coaxial flow type chips, or PMMA microfluidic chips.

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

[0074] In a sixth aspect, the present invention provides a method for detecting streptomycin or dihydrostreptomycin, which includes any one of the following methods:

[0075] (1) Add a diluent and the above-mentioned monoclonal antibody against streptomycin or an antigen-binding fragment thereof to the control wells of a microplate coated with a streptomycin antigen, and incubate; add a streptomycin standard product or a sample to be tested and the above-mentioned monoclonal antibody against streptomycin or an antigen-binding fragment thereof to the inhibition wells of a microplate coated with a streptomycin antigen, and incubate; add an enzyme-labeled secondary antibody to the control wells and the inhibition wells respectively for incubation, and after a color reaction, detect the absorbance of the control wells and the inhibition wells, and obtain the concentration of streptomycin in the sample to be measured according to the standard curve 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 streptomycin is contained in the sample according to the chromatography result of the test strip.

[0077] The enzyme-labeled secondary antibody includes, but is not limited to, secondary antibodies labeled with horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

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

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

[0080] The coating concentration of streptomycin antigen on the microplate is 1×10 -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 added concentration of the monoclonal antibody against streptomycin or its antigen-binding fragment is 1×10 -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 encoding the above-mentioned streptomycin-resistant monoclonal antibody or an antigen-binding fragment thereof.

[0083] In one embodiment, the coding sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 16, and the coding sequence of the light chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 17.

[0084] In an eighth aspect, the present invention further provides a vector containing the above-mentioned nucleic acid molecule.

[0085] Here, the term "vector" is used in its most common sense and includes any intermediate agent for nucleic acids, which can enable the nucleic acids to be introduced into prokaryotic and / or eukaryotic cells, for example, and integrated into the genome where appropriate. This type of vector preferably replicates and / or expresses in cells. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, which can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and be stable in the host.

[0086] In an alternative embodiment, the above-mentioned vector is an expression vector, and an important feature of an expression vector is usually to contain an origin of replication, a promoter, a marker gene and translation control elements.

[0087] In a ninth aspect, the present invention further provides a recombinant cell containing the above-mentioned vector.

[0088] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid. The term "recombinant cell" according to the present invention includes prokaryotes (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells, especially human cells, yeast cells, and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats, or primates. The cells can be derived from multiple tissue types and include primary cells and cell lines. The nucleic acid can be present in the host cell in single copy or in two or more copies, and in one embodiment, is expressed in the recombinant cell.

[0089] In an alternative embodiment, the recombinant cell is a eukaryotic cell.

[0090] In an alternative embodiment, the recombinant cell is a mammalian cell.

[0091] In an alternative embodiment, the recombinant cell is HEK293.

[0092] The features and properties of the present invention are further described in detail below in conjunction with examples.

[0093] Example 1

[0094] This example is for the preparation of monoclonal antibodies.

[0095] 1. Preparation of immunogen and coating antigen

[0096] Referring to the method reported in the literature (Fan Guoying, Wang Shungang, Wang Ziliang, et al. Preparation and identification of streptomycin artificial antigen [J]. Acta Agriculturae Boreali-occidentalis Sinica, 2009, 18(2): 45-50. DOI: 10.3969 / j.issn.1004-1389.2009.02.011), the antigen preparation of streptomycin was carried out. Using the aldehyde group on streptomycin, oximation treatment with O-carboxymethylhydroxylamine was carried out to introduce a carboxyl group to obtain the streptomycin hapten. Then, two complete antigens, Str-BSA and Str-OVA, were synthesized by the EDC method respectively and stored separately at 4°C. Str-BSA was used as the immunogen for immunizing mice, and Str-OVA was used as the coating antigen for detecting the serum and antibody titers.

[0097] 2. Preparation of monoclonal antibodies:

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

[0099] (2) Primary immunization: The diluted complete antigen Str-BSA solution (concentration 1 mg / mL) and an equal volume of Freund's complete adjuvant were fully emulsified with a magnetic stirrer and then injected subcutaneously at multiple points on the back of the mice for immunization. The immunization dose was 0.1 mg antigen / mouse.

[0100] (3) Booster immunization: Two weeks after the primary immunization, the diluted complete antigen Str-BSA solution (concentration: 1 mg / mL) and an equal volume of Freund's incomplete adjuvant were fully emulsified with a magnetic stirrer and then used to immunize mice by multiple subcutaneous injections in the back. The immunization dose was 0.1 mg antigen per mouse. Booster immunization was performed every two weeks. Starting from the third booster immunization, orbital blood sampling was carried out on the 7th day after each immunization, and the antibody titer and inhibition effect were detected by the indirect competitive ELISA method. The coated antigen used in the method was the complete antigen Str-OVA.

[0101] (4) Boost immunization: Select mice with high titer and good inhibition, and perform boost immunization on the 10th day after the 5th immunization by intraperitoneal injection of 100 μL of 1 mg / mL without adjuvant.

[0102] (5) Cell fusion

[0103] On the 3rd day after the boost immunization, hybridoma fusion was carried out. The spleen cells of mice were fused with mouse myeloma cells sp2 / 0 by PEG 1450 and cultured selectively in 2% HAT medium. The cell wells with high titer and good inhibition were screened by the indirect competitive ELISA method and subcloned by limited dilution. Finally, the monoclonal antibody hybridoma cell line LMS-1D11H8 against streptomycin was obtained. The antibodies secreted by this cell line all had good specificity for streptomycin, and the detection sensitivity could reach 1.1 μg / L. This cell line was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO.46126.

[0104] (6) Cryopreservation and resuscitation of cells

[0105] After the obtained hybridoma cell line LMS-1D11H8 was expanded in culture, it was resuspended by pipetting with DMEM and centrifuged. It was cryopreserved in liquid nitrogen at a density of 1×10 9 cells / mL for long-term storage. For resuscitation, the cryopreservation tube was taken out from the liquid nitrogen tank and immediately placed in a 37°C water bath for thawing. Then it was transferred to pre-warmed 10 mL DMEM by pipette and centrifuged to remove the cryopreservation solution, and then transferred to a culture plate for culture.

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

[0107] Eight 10 - 12 week - old female BALB / C mice were taken and injected intraperitoneally with sterile paraffin oil, 0.3 mL per mouse. One week later, each mouse was injected intraperitoneally with the monoclonal cell line LMS - 1D11H8, and the number of injected cells per mouse was approximately 10^6. When the abdomen of the mouse became swollen, ascites of the mouse was collected and purified by the saturated ammonium sulfate method and then stored at - 20 °C. Thus, the purified monoclonal antibody secreted by the hybridoma cell line LMS - 1D11H8 was obtained.

[0108] Example 2

[0109] The antibody effect of the monoclonal antibody obtained in Example 1 was detected.

[0110] The various buffers used in the following experiments are as follows:

[0111] Coating buffer (pH 9.6 0.05M carbonate buffer): 1.5 g of Na2CO3; 2.94 g of NaHCO3, made up to 1000 mL with pure water;

[0112] Phosphate - buffered saline PBS (0.01M pH7.4): 0.2 g of KH2PO4; 8 g of NaCl; 2.92 g of NaH2PO4·12H2O, made up to 1000 mL with pure water;

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

[0114] Sample diluent (PBSTG): Add 1 mL of Tween - 20 and 1 g of gelatin (melted by microwave heating) to the prepared PBS, and make up to 1 L;

[0115] Chromogenic solution: TMB stock solution (375 mg of TMB solid + 30 mL of DMSO (prepared according to the ratio, stored in the dark at room temperature)), BUFFER (0.1 g of potassium sorbate + 46.04 g of potassium dihydrogen citrate hydrate + 1 L of pure water), the chromogenic solution is prepared immediately before use: 200 μL of TMB stock solution + 11 mL of BUFFER + 3.34 μL of 30% hydrogen peroxide solution;

[0116] Stop solution (2M H2SO4): 445.6 mL of distilled water, add 54.4 mL of concentrated sulfuric acid (98%) drop by drop and stir.

[0117] The following checkerboard experiment of antigen and antibody was carried out:

[0118] 1) Coating:

[0119] The complete antigen of streptomycin Str-OVA at 1 mg / mL was serially diluted at ratios of 1:2000, 1:4000, 1:8000, and 1:16000 with coating buffer to obtain coating antigen solutions of the complete antigen of streptomycin Str-OVA at different concentrations. 100 μL of the coating antigen solution of the complete antigen of streptomycin Str-OVA prepared in step 1 was added to each well of a 96-well ELISA plate and incubated overnight at 4°C, followed by washing 3 times with PBST;

[0120] 2) Competition:

[0121] The purchased 100 μg / mL streptomycin standard from Tanmo Quality Inspection was diluted to 5 ng / mL with PBSTG. 50 μL of sample diluent was added to each zero well, and 50 μL of the diluted 5 ng / mL streptomycin standard solution was added to each inhibition well.

[0122] The streptomycin antibody was serially diluted at ratios of 1:1000, 1:2000, 1:4000, and 1:8000 with PBSTG to obtain antiserum dilutions of streptomycin (50 μL / well), which were placed in a humid box and incubated at 37°C for 30 min, followed by washing the plate 3 times.

[0123] 3) Addition of enzyme-labeled secondary antibody: The goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) was diluted 10,000-fold with PBSTG, and 100 μL was added to each well. The plate was placed in a humid box and incubated at 37°C for 30 min, followed by washing the plate 3 times.

[0124] 4) Color development: The color developer was prepared immediately before use. After mixing the prepared TMB solution and hydrogen peroxide in proportion, 100 μL was added to each well, and color development was carried out at room temperature in the dark for 10 min.

[0125] 5) Termination: 50 μL of 2 M H2SO4 was added to each well, and the OD value of each well was measured at 450 nm using an ELISA reader.

[0126] Calculation formula for inhibition rate: Inhibition rate = (B0 - B) / B0 × 100%, where B0 is the OD value of the control well; B is the OD value of the inhibition well.

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

[0128]

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

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

[0131] Example 3

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

[0133] The streptomycin standard solution was diluted with the sample diluent into the following different concentrations: 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.31 ng / mL, 0.15 ng / mL. Referring to the checkerboard experiment procedure, a standard curve experiment was carried out. Three parallels were made for each standard concentration, and the coating antigen dilution factor of 1:4000 and the antibody dilution of 1:8000 were selected for detection.

[0134] Draw a standard curve: Using the streptomycin standard solution with different concentrations (ng / mL) as the X-axis and the ratio of absorbance values (B / B0, where B is the average absorbance value of the streptomycin standard solution and B0 is the average absorbance value of the control well) as the Y-axis, draw a standard curve graph. The experiment was set to be repeated 3 times, and the average value of the three experimental results was taken. The obtained standard curve graph is as Figure 1 shown.

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

[0136] The detection range is 0.4 ng / mL - 5 ng / mL. It shows that the streptomycin antibody prepared by the above method has high sensitivity and good detection effect.

[0137] Example 4

[0138] This example conducts a specificity test on the antibody prepared in Example 1.

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

[0140] Dilute the above 5 drugs with a sample diluent to the following concentrations: 5000 ng / mL, 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL.

[0141] Establish a standard curve and determine the median inhibitory concentration IC 50 (The standard sample concentration value at which the inhibition rate reaches 50%). The method for establishing the standard curve is the same as that for establishing the streptomycin standard curve described above.

[0142] Cross - reaction rate (%) = (Streptomycin IC 50 ) / (Analog IC 50 ) × 100%.

[0143] Drug Name <![CDATA[IC 50 (ng / mL)]]> Cross-reaction 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 show that the antibody prepared in Example 1 has good specificity for streptomycin and cross - reacts with dihydrostreptomycin.

[0145] Example 5

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

[0147] 1) Preparation of the gold - labeled antibody pad: Take 1 mL of colloidal gold solution (30 nm) in a 2 - mL centrifuge tube (rinsed with pure water), add 2 μL of 0.2 M K2CO3 solution to it, then add 5 μg of the antibody from Example 1. Mix well by inverting up and down and let it stand for 20 min. Then add 50 μL of 3% BSA for blocking, mix well by inverting up and down and let it stand for 20 min. Then use a high - speed refrigerated centrifuge to centrifuge the mixture at 10000 r / min for 10 min (4°C). Discard the supernatant, add 0.5 mL of reconstitution solution (0.01 M PBS containing 3% sucrose) to the precipitate for re - dissolution, spread the solution onto a 1 cm × 15 cm sample pad, and dry it in an oven at 37°C (1.5 h).

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

[0149] 3) Assembly: Assemble in the order of sample pad, colloidal gold pad, nitrocellulose membrane, and absorbent paper in sequence, and then use a cutting machine to cut it into test strips 4 mm wide for subsequent testing.

[0150] 4) Detection: Prepare streptomycin and dihydrostreptomycin standard solutions of 5 ng / mL and 10 ng / mL respectively with fresh milk samples. Take 200 μL of each and add them to a 96-well plate. At the same time, take 200 μL of fresh milk as a blank control and also add it to the 96-well plate. Then, take 6 prepared test strips and insert them into the wells with the detection solution respectively. React for 10 min, and refer to the results Figure 2 as shown.

[0151] It can be clearly seen that the prepared test strips can be used to detect 10 ng / mL of streptomycin and dihydrostreptomycin in fresh milk, and the detection sensitivity can meet the detection requirements of the maximum residue limits specified in GB 31650-2019, which are 200 μg / kg for milk, 600 μg / kg for livestock and poultry muscle, fat, and liver, and 1000 μg / kg for kidneys.

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

[0153]

[0154]

[0155] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hybridoma cell, characterized in that, It is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC NO. 46126.

2. A monoclonal antibody against streptomycin or an antigen-binding fragment thereof, characterized in that, It includes: Heavy chain complementarity-determining regions and light chain complementarity-determining regions. The heavy chain complementarity-determining regions include CDR-H1, CDR-H2, and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light chain complementarity-determining regions include CDR-L1, CDR-L2, and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is DVS.

3. The monoclonal antibody against streptomycin or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment also includes heavy chain framework regions and light chain framework regions; the heavy chain framework regions include HFR1, HFR2, HFR3, and HFR4 that have at least 80% homology with the amino acid sequences shown as SEQ ID NO: 6-9 in sequence; the light chain framework regions include LFR1, LFR2, LFR3, and LFR4 that have at least 80% homology with the amino acid sequences shown as SEQ ID NO: 10-13 in sequence; Preferably, the antibody or its antigen-binding fragment also includes a constant region, and the constant region includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant regions; The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, and scFv of the antibody.

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

5. Use of the monoclonal antibody against streptomycin or its antigen-binding fragment described in any one of claims 2-3 or the antibody conjugate described in claim 4 in any one of the following: (1) Detecting streptomycin or dihydrostreptomycin; and (2) Preparing a streptomycin or dihydrostreptomycin detection product; The detection product is a reagent, kit, test strip, antibody chip, antibody probe, or detector.

6. 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 monoclonal antibody against streptomycin or its antigen-binding fragment described in any one of claims 2-3, or the antibody secreted by the hybridoma cell described in claim 1; Preferably, the detection product is an ELISA kit, and the ELISA kit includes a microplate and the monoclonal antibody against streptomycin or its antigen-binding fragment, and the microplate is coated with a streptomycin antigen. Preferably, the detection product is a test strip, and the test strip includes a conjugate pad and a nitrocellulose membrane; the conjugate pad is coated with the monoclonal antibody against streptomycin or its antigen-binding fragment.

7. The streptomycin or dihydrostreptomycin detection product according to claim 6, characterized in that, The nitrocellulose membrane has a T line and a C line, the T line has streptomycin antigen, and the C line has an anti-mouse antibody of X; X is sheep, rabbit, horse, monkey or chicken; Preferably, the coating concentration of streptomycin antigen on the T line is 0.1-2 mg / mL, and the coating concentration of the anti-mouse antibody of X on the C line is 0.2-1 mg / mL.

8. A method for detecting streptomycin or dihydrostreptomycin, characterized in that, It includes any one of the following methods: (1) Add diluent and the monoclonal antibody against streptomycin or its antigen-binding fragment according to any one of claims 2-3 to the control well of the microplate coated with streptomycin antigen, and incubate; add streptomycin standard or the sample to be tested and the monoclonal antibody against streptomycin or its antigen-binding fragment according to any one of claims 2-3 to the inhibition well of the microplate coated with streptomycin antigen, and incubate; add enzyme-labeled secondary antibody to the control well and the inhibition well respectively for incubation, and after color reaction, detect the absorbance of the control well and the inhibition well, and obtain the concentration of streptomycin in the sample to be tested by drawing a standard curve of absorbance vs. streptomycin concentration and according to the standard curve; (2) Load the sample to be tested onto the sample pad of the test strip, and judge whether the sample contains streptomycin according to the chromatographic result of the test strip.

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

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

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