Anti-gm antibodies and uses thereof

Abstract

The application discloses an anti-GM antibody and application thereof, and relates to the technical field of antibiotic residue immunoassay. The anti-GM antibody, i.e. an anti-gentamicin antibody, targets a gentamicin (GM) antigen epitope; the anti-GM antibody comprises HCDR1, HCDR2, HCDR3 with an amino acid sequence as shown in SEQ ID No. 1, 2, 3 and / or LCDR1, LCDR2, LCDR3 with an amino acid sequence as shown in SEQ ID No. 4, 5, 6, and specifically binds to the gentamicin antigen epitope. The application further discloses polynucleotides encoding the anti-GM antibody and a kit / non-diagnostic method and application related to the anti-GM antibody. Compared with the prior art, the anti-GM antibody can highly specifically bind to the gentamicin antigen epitope without cross-reaction; and the polynucleotides, the kit / non-diagnostic method and the application are also beneficial to rapid and accurate screening of gentamicin residues in samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibiotic residue immunoassay, and more particularly, to an anti-GM antibody and application thereof. BACKGROUND

[0002] Gentamicin (GM) is a broad-spectrum antibiotic of aminoglycoside, which is also referred to as GM in the industry. GM is a standard abbreviation of gentamicin in the fields of medicine, detection and scientific research. In specific practices such as antibiotic residue analysis, GM is directly used as a simple name and an identifier of gentamicin. The so-called anti-GM antibody is an anti-gentamicin antibody.

[0003] The core mechanism of gentamicin for killing and inhibiting bacteria is to inhibit bacterial protein synthesis and destroy the integrity of bacterial cell membranes. Therefore, it has good killing and inhibiting effect on most gram-negative bacteria and part of gram-positive bacteria, such as common Escherichia coli, Klebsiella, Salmonella, Shigella, and part of subtypes of Staphylococcus aureus (such as methicillin-sensitive Staphylococcus aureus MSSA). In addition, gentamicin is a heat-stable antibiotic. Compared with non-heat-stable antibiotics, its pharmaceutical process is relatively simple, the production cost is lower, and it can break through the limitation of storage conditions and does not depend on cold chain storage, thereby maximizing the accessibility of antibiotic drugs. Due to the above advantages, since its advent, gentamicin has been widely used in the fields of medical treatment and agricultural breeding.

[0004] However, with the confirmation of the ototoxicity, nephrotoxicity and other toxic side effects of gentamicin, and the problem of antibiotic drug resistance caused by antibiotic drug residues, gentamicin has been regarded as a restricted antibiotic in the fields of medical treatment, agricultural breeding, etc. Specifically, in the fields of food safety supervision, environmental pollution monitoring, etc., gentamicin residues are listed as an index of antibiotic residues to monitor the residual pollution of related substances. However, the traditional liquid chromatography-tandem mass spectrometry (LC-MS / MS) is time-consuming and complicated to operate, which is not helpful for rapid screening of gentamicin residues in samples. Although the immunoassay is based on the specific binding of antigen and antibody to qualitatively / quantitatively detect target substances, the theory and related practice also support that this method can realize rapid and accurate detection of target substances. However, the core tool is a high-specificity antibody targeting the target substance. In this regard, directly expressing a full-length protein such as an antibody targeting the gentamicin antigen epitope with complex functions is a core technical challenge in current biopharmaceutical and protein expression basic research, which is beyond the current level of technology. Moreover, the anti-gentamicin monoclonal antibody (i.e., anti-GM mAb) obtained by traditional hybridoma cell technology often has different performances due to the uncertainty in the preparation process. For example, the monoclonal antibody produced by the CCTCC C201135 hybridoma cell strain disclosed in CN102776151A shows affinity to multiple aminoglycoside antibiotics such as streptomycin, neomycin, gentamicin and kanamycin, which is obviously not suitable for specific detection targeting only gentamicin residues. SUMMARY

[0005] In view of the above problems in the prior art, the present application provides an anti-GM antibody, i.e., an anti-gentamicin antibody, targeting the gentamicin antigen epitope. The anti-GM antibody comprises at least one complementarity determining region (CDR) and specifically binds to the gentamicin antigen epitope. Based on this, a series of applications of the anti-GM antibody in the field of immunoassay technology are also proposed, such as polynucleotides encoding the anti-GM antibody, kits / non-diagnostic methods for detecting gentamicin residues, and applications of the anti-GM antibody and the polynucleotides, kits / non-diagnostic methods in food safety supervision / environmental pollution monitoring, etc.

[0006] The first aspect of the present application provides an anti-GM antibody.

[0007] The anti-GM antibody provided in the embodiments of the present aspect comprises:

[0008] HCDR1, HCDR2, HCDR3; the HCDR1, HCDR2, HCDR3 refer to a group of complementarity determining regions (CDRs) of a heavy chain variable region (VH) of the anti-GM antibody; wherein the amino acid sequence of the HCDR1 is shown as SEQ ID No. 1; the amino acid sequence of the HCDR2 is shown as SEQ ID No. 2; the amino acid sequence of the HCDR3 is shown as SEQ ID No. 3;

[0009] and / or,

[0010] LCDR1, LCDR2, LCDR3; the LCDR1, LCDR2, LCDR3 refer to a group of complementarity determining regions (CDRs) of a light chain variable region (VL) of the anti-GM antibody; wherein the amino acid sequence of the LCDR1 is shown as SEQ ID No. 4; the amino acid sequence of the LCDR2 is shown as SEQ ID No. 5; the amino acid sequence of the LCDR3 is shown as SEQ ID No. 6.

[0011] In a further aspect, in addition to the complementarity determining regions (CDRs), the heavy chain variable region (VH) / light chain variable region (VL) of the antibody further comprises a framework region (FR). Therefore, the anti-GM antibody provided in the embodiments of the present aspect can be: the heavy chain variable region thereof comprises a heavy chain variable region framework region (HFR); the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 7;

[0012] and / or,

[0013] the light chain variable region thereof comprises a light chain variable region framework region (LFR); the amino acid sequence of the light chain variable region is shown as SEQ ID No. 8.

[0014] In a further aspect, in addition to the heavy chain variable region / light chain variable region, the antibody can further comprise a constant region (C region). Therefore, the anti-GM antibody provided in the embodiments of the present aspect can further comprise: a heavy chain constant region (CH) and / or a light chain constant region (CL).

[0015] In a further aspect, the antibody can be obtained by stimulating an immune model animal to produce an immune response, and the typical immune model animal is a mouse or the like. Therefore, the anti-GM antibody provided in the embodiments of the present aspect can be a mouse-derived antibody.

[0016] The second aspect of the present application provides a polynucleotide encoding the anti-GM antibody described in the first aspect.

[0017] In a further aspect, the anti-GM antibody of the first aspect, whose heavy chain variable region has an amino acid sequence as set forth in SEQ ID No. 7 and / or whose light chain variable region has an amino acid sequence as set forth in SEQ ID No. 8, the polynucleotide provided in the embodiments of the present aspect comprises: a nucleotide fragment encoding the heavy chain variable region of the anti-GM antibody, the sequence of which is as set forth in SEQ ID No. 9;

[0018] and / or,

[0019] a nucleotide fragment encoding the light chain variable region of the anti-GM antibody, the sequence of which is as set forth in SEQ ID No. 10.

[0020] The third aspect of the present application provides a kit for detecting gentamicin residues.

[0021] The kit for detecting gentamicin residues provided in the embodiments of the present aspect comprises: the anti-GM antibody of the first aspect, which is intended to capture gentamicin residues by specifically binding with free gentamicin in the sample to be tested; and a reagent for detecting the specific binding of free gentamicin with the anti-GM antibody, which is intended to convert the specific binding event of free gentamicin with the anti-GM antibody into a detectable signal (such as an optical, electrochemical or colorimetric signal, etc.), so as to realize qualitative or quantitative analysis of gentamicin residues.

[0022] In a further aspect, the Enzyme-Linked Immunosorbent Assay (ELISA) is the mainstream technology in the field of immunoassay technology; its core is to realize qualitative / quantitative detection of target substances through solid-phase antibody / antigen and enzyme labels; wherein, the enzyme labels refer to complexes formed by specific enzyme molecules (such as horseradish peroxidase) and antibodies / antigens, such as enzyme-labeled secondary antibodies, enzyme-labeled primary antibodies and enzyme-labeled antigens, etc. Therefore, the kit provided in the embodiments of the present aspect can be an ELISA kit; the ELISA kit comprises: a solid-phase carrier pre-coated with the anti-GM antibody or the anti-GM antibody labeled with an enzyme or the anti-GM antibody and an enzyme-labeled secondary antibody adapted thereto.

[0023] In a further aspect, the immunochromatographic assay (ICA) is also an important branch of the development of immunoassay technology; it is also based on the core logic of the specific binding of antigen-antibody in the framework of the immunoassay technology, and specifically realizes the separation and capture of target substances through a chromatographic carrier (such as a nitrocellulose membrane), and then realizes the qualitative / quantitative detection of target substances; among them, the immunochromatographic test strip is the core functional carrier of the immunochromatographic kit, and the immunochromatographic kit mainly carries the label through the conjugate pad / conjugate zone of the immunochromatographic test strip or a carrier independent of the immunochromatographic test strip. Therefore, the kit provided in the embodiments of the current aspect can be an immunochromatographic kit; the immunochromatographic kit comprises: an immunochromatographic test strip; wherein the immunochromatographic kit coats a label through the conjugate pad / conjugate zone of the immunochromatographic test strip or independently loads a label; the label is an anti-GM antibody labeled by a chromogenic or fluorescent nanoparticle; wherein the chromogenic nanoparticle is colloidal gold, colloidal carbon or latex microspheres; the fluorescent nanoparticle is a quantum dot or a fluorescent microsphere.

[0024] The fourth aspect of the present application provides a non-diagnostic method for detecting gentamicin residues.

[0025] The non-diagnostic method for detecting gentamicin residues provided in the embodiments of the current aspect comprises:

[0026] incubating the anti-GM antibody of the first aspect or the anti-GM antibody in the kit for detecting gentamicin residues of the first aspect with a sample to be tested;

[0027] and further detecting the binding of free gentamicin in the sample to be tested after incubation and the anti-GM antibody, so as to determine the gentamicin residue condition in the sample to be tested.

[0028] The fifth aspect of the present application provides an application of the anti-GM antibody of the first aspect or the polynucleotide of the second aspect or the kit for detecting gentamicin residues of the third aspect or the non-diagnostic method for detecting gentamicin residues of the fourth aspect in food safety supervision / environmental pollution monitoring.

[0029] The present application provides an anti-GM antibody (i.e. an anti-Gentamicin antibody) which specifically targets a Gentamicin (GM) antigen epitope, avoids cross-reactions as much as possible, and thus realizes competitive recognition and capture of the Gentamicin antigen. On this basis, the present application also applies the anti-GM antibody to the field of immunoassay technology, specifically by providing polynucleotides encoding the anti-GM antibody, a kit / non-diagnostic method for detecting Gentamicin residues, and applications in food safety supervision / environmental pollution monitoring, etc., to promote rapid and high-precision detection of Gentamicin residues in samples. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 SDS-PAGE electrophoretogram of the anti-GM antibody 3A3-G3 provided by the present application;

[0031] Figure 2 Specificity test result schematic diagram of the Gentamicin (GM) rapid detection kit provided by the present application. DETAILED DESCRIPTION

[0032] Unless otherwise defined, the practice of the present application will employ, unless otherwise defined, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987 and periodic updates thereto); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0033] The term "antibody" (Ab) herein includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments (especially antigen-binding fragments; an antigen-binding fragment is a functional antibody fragment contained in an antibody that retains the ability to bind an antigen). The terms "immunoglobulin" (Ig) and antibody can be used interchangeably herein.

[0034] The term "antigen" (Ag) includes small-molecule antigens and complete antigens. Small-molecule antigens, also known as haptens or incomplete antigens, mainly refer to a class of compounds with small molecular weight (usually 100-1000 Da), which lack immunogenicity (Immunogenicity) when present alone, but have high reactogenicity (Reactogenicity), such as drugs, hormones, pesticides, toxins, etc.; "complete antigen" refers to a substance with immunogenicity and immunoreactivity. Complete antigens are abundant and widely distributed in nature, such as most proteins, bacteria, viruses, bacterial exotoxins, animal sera, etc. Among them, the term "immunogenicity" refers to the ability to stimulate the immune system to produce a specific immune response; the term "reactogenicity" refers to the ability to specifically bind to immune response products (such as antibodies).

[0035] The gentamicin involved in the present application is a small-molecule antigen. Small-molecule antigens can acquire immunogenicity by combining with a large-molecule protein carrier to transform into a complete antigen with both immunoreactivity and immunogenicity. Among them, the term "protein carrier" refers to any immunologically acceptable protein used to form a complete antigen, including but not limited to: bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, human serum albumin (HSA), and artificially synthesized polylysine (PLL), etc., preferably bovine serum albumin (BSA) or ovalbumin (OVA).

[0036] In immunoassay technology, complete antigens can be divided into immunogens and coating antigens according to their different functional emphases. Among them, immunogens refer to complete antigens that can induce immune responses; coating antigens refer to complete antigens that are pre-fixed on the surface of a solid-phase carrier during immune detection.

[0037] The "variable region" or "variable domain" (V region) of an antibody refers to the amino-terminal (N-terminal) domains of the heavy or light chain of an antibody. The variable regions of the heavy chain and light chain can be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of an antibody and contain the antigen binding sites.

[0038] The term "variable" refers to the broad specificity of certain segments in the variable region, which are usually referred to as complementarity determining regions (CDRs), are the key fragments of the antibody variable region that directly bind to the antigen, and determine the specificity and affinity of the antibody. Generally, a complete antibody molecule contains 6 complementarity determining region segments, namely HCDR1, HCDR2 and HCDR3 of the heavy chain variable region (VH) and LCDR1, LCDR2 and LCDR3 of the light chain variable region (VL); the aforementioned 6 complementarity determining region segments cooperatively form an antigen binding pocket (paratope) through spatial conformation to recognize the antigen epitope; however, there are exceptions, for example, there is a single domain antibody (VHH), which only contains a heavy chain variable region (lacks a light chain, etc.), and only through the 3 complementarity determining region segments (HCDR1, HCDR2, HCDR3) of the heavy chain variable region to achieve antigen binding. In addition to the complementarity determining region, the relatively conserved part of the variable region is called the framework region (FR); the framework region is intended to provide structural support for the complementarity determining region to maintain the stability of the spatial conformation of the antibody variable region; the variable regions of the native heavy chain and light chain each contain four framework region segments (FR1, FR2, FR3, FR4); the structure of the heavy chain variable region is usually HFR1―HCDR1―HFR2―HCDR2―HFR3―HCDR3―HFR4; the structure of the light chain variable region is usually LFR1―LCDR1―LFR2―LCDR2―LFR3―LCDR3―LFR4.

[0039] Antibody CDRs can be determined by various encoding systems, such as CCG, Kabat, AbM, Chothia, IMGT, etc. These encoding systems are known in the art. For example, the amino acid sequence numbering of the antigen binding protein referred to herein can follow the IMGT numbering scheme. V(D)J recombination generates a coding sequence for a variable region (V region), which codes for the variable region (V region) of an antibody molecule, rather than the entire amino acid sequence of the antibody heavy chain or light chain. Among them, V(D)J recombination is a gene rearrangement process that occurs during the development of vertebrate B cells and T cells, and is the core mechanism for generating diverse antigen binding proteins (antibodies and T cell receptors). This process combines V (variable region), D (diversity region), J (joining region) gene fragments to form a unique antigen binding domain, enabling specific recognition of a large number of pathogens.

[0040] The "constant region" or "constant domain" (C region) of an antibody refers to the carboxy-terminal structural domains of an antibody heavy or light chain. The constant regions / domains of the heavy and light chains can be referred to as "CH" and "CL", respectively. The amino acid sequences of these domains are relatively conserved among antibodies, and primarily serve to mediate the antibody's effector functions. The "Fc" (Fragment Crystallizable), or "Fc fragment" or "Fc region" or "Fc domain" refers to the C-terminal region of an antibody heavy chain.

[0041] The antibodies of the present application can be prepared using methods conventional in the art, such as hybridoma technology, phage display technology, cell line expression, all of which are well known in the art. The present application also provides fusion proteins comprising the antibodies or antigen-binding fragments thereof of the present application as an expression target, active molecule or targeting molecule. For example, fusion proteins formed by adding a tag (e.g., His6 tag) at both ends of the antibody to facilitate protein expression or purification, etc. The tag does not affect the function of the target protein, and can be easily removed.

[0042] The present application also provides polynucleotides encoding the antibodies or antigen-binding fragments thereof described herein. Polynucleotides encoding the variable region of the heavy chain, the variable region of the light chain, the heavy chain, the light chain, and each complementary determining region segment are provided herein. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The present application also includes degenerate variants of the nucleotide sequences encoding the antibodies or antigen-binding fragments thereof, fusion proteins or antibody conjugates, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

[0043] According to the amino acid sequence and codon, the skilled person in the art can easily obtain the coding sequence of the antibody or its fragment. The skilled person in the art can also change the expression of polypeptides in different species by codon optimization, and the codon bias of different species belongs to the conventional techniques in the art.

[0044] In some embodiments, in the ELISA kit provided, in addition to the solid phase carrier pre-coated with the anti-GM antibody, the anti-GM antibody labeled with an enzyme, and the anti-GM antibody and the enzyme-labeled secondary antibody matched therewith, correspondingly, the following are also included: enzyme-labeled gentamicin, solid phase carrier pre-coated with gentamicin complete antigen, and solid phase carrier pre-coated with gentamicin complete antigen. In addition, in some embodiments, in the ELISA kit provided, further, the following can also be included: lysing medium for dissolving the sample, general reagents and buffers required for detection, such as substrate, blocking solution, washing solution, sample diluent, etc. Of course, the above experimental materials can also be prepared by oneself.

[0045] The present application will be described in greater detail by way of specific examples. It is to be understood that these examples are merely illustrative, and are not intended to limit the scope of the present application. The methods and materials used in the examples are those that are conventional in the art, unless otherwise specified.

[0046] Example

[0047] I. Example 1: Preparation of Anti-GM Antibody

[0048] 1. Preparation of gentamicin complete antigen

[0049] The current example will use gentamicin (GM) to couple with bovine serum albumin (BSA), ovalbumin (OVA) to synthesize the immunogen and coating antigen of gentamicin, respectively.

[0050] 1.1 Preparation of immunogen

[0051] Sulfated gentamicin and bovine serum albumin (BSA) were prepared in a molar ratio of 50:1 to 500:1 to prepare the immunogen of gentamicin.

[0052] The current example uses the following ratio to prepare the gentamicin immunogen, and the specific process is as follows:

[0053] Take 1000 mg of sulfated gentamicin and 200 mg of BSA, dissolve in 5 ml of phosphate buffer (pH=7.4); 9 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is dissolved in 5 ml of pure water, and it is added dropwise to the gentamicin system, and after 1.5 h of reaction at room temperature, it is reacted at 0~5℃ for 3 d. Then the reaction solution is transferred into a dialysis bag, and dialysis is performed at 4℃ using 0.01 mol / L phosphate buffer for 2 d, and the dialysis solution is changed every 6 h. After dialysis, centrifugation is performed at 4℃ using a high-speed centrifuge at 4500 rpm for 15 min, and then it is aliquoted and stored at -80℃ for standby.

[0054] 1.2 Preparation of coating antigen

[0055] According to the method and steps of preparing the immunogen in the current example 1.1, replace BSA with ovalbumin (OVA), and other conditions remain unchanged, to prepare the coating antigen of gentamicin.

[0056] 2. Animal immunization

[0057] Four 6-8 week old female BALB / C mice were selected and immunized by subcutaneous multi-point injection of the immunogen prepared in 1.1, once every two weeks, for a total of three immunizations. The immunogen was emulsified with an equal volume of Freund's complete adjuvant for the first immunization, and emulsified with an equal volume of Freund's incomplete adjuvant for the second and third immunizations. The immunization dose (50-100 μg per mouse) and method were kept constant. One week after the third immunization, the mice were subjected to tail vein blood collection, and their serum was obtained and subjected to indirect ELISA to determine the titer. The serum titer of the immunized mice is shown in Table 1.

[0058] Table 1 Serum titer and inhibition rate of immunized mice

[0059]

[0060] The mouse with the best inhibition effect was selected from the mice with qualified serum titer (usually, an individual with an inhibition rate of more than 90% at 10 ppb of the standard product was taken as the standard for qualified serum titer) for boost immunization. In the present example, the mouse with the best inhibition effect (i.e., mouse 3) was selected for boost immunization according to the serum titer and inhibition rate data in Table 5: the immunogen was diluted to 200 uL with 1xPBS (i.e., 0.01 mol / L phosphate buffer), and the selected mouse was subjected to boost immunization by intraperitoneal injection.

[0061] The mouse after boost immunization was subjected to orbital blood collection, and the collected blood was placed in an EP tube and subjected to centrifugation at 4000 rpm for 10 min after standing at 37°C for 2 h. The collected serum was used as a positive control for subsequent screening of monoclonal antibodies.

[0062] Three days after boost immunization, the mouse was subjected to cell fusion to prepare hybridoma cells.

[0063] 3. Hybridoma cell preparation

[0064] 3.1 Culture of SP2 / 0 myeloma cells

[0065] The SP2 / 0 myeloma cell cryopreservation tube was quickly thawed in a 37°C constant temperature water bath, and 10 mL of complete culture medium was added to a 50 mL sterile centrifuge tube under sterile conditions. The tube was centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. The cells were resuspended in complete culture medium. 5 mL of culture medium was added to a cell culture bottle containing 5 mL of culture medium, and the bottle was placed in a CO2 cell culture incubator and cultured at 37°C. The cell growth and state were observed under a microscope, and when the cell density was about 80%, the SP2 / 0 myeloma cells were subcultured.

[0066] 3.2 Obtain B lymphocytes

[0067] After asphyxiation with carbon dioxide, the cervical dislocation of selected mice was determined, and their spleens were removed. The mouse spleen was placed in a 15 mL sterile centrifuge tube containing 10 mL of DMEM medium, and after the spleen was moistened, the excess medium was carefully discarded. 10 mL of DMEM medium was aspirated into a sterile flat dish, and the spleen was ground with a glass slide to prepare a single cell suspension and filtered through a 200 mesh nylon mesh into a sterile centrifuge tube. 30 mL of DMEM was added to a 50 mL sterile centrifuge tube, and the nylon mesh was rinsed with a pipette. The centrifuge tube containing the spleen cell suspension was centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cell pellet was gently dispersed by hand, and 30 mL of DMEM medium was added to resuspend it, and the mixture was centrifuged again. The cell pellet was gently dispersed by hand, and 10 mL of DMEM medium was added to resuspend it.

[0068] 3.3 Cell fusion

[0069] The well-grown SP2 / 0 myeloma cells were collected by centrifugation at 1000 rpm for 5 min, resuspended in 30 mL of DMEM medium, and after the second centrifugation, 10 mL of DMEM medium was added to resuspend it. The B lymphocyte suspension was mixed with the SP2 / 0 myeloma cell suspension, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was gently dispersed. It was placed in a 37°C water bath environment, and 1 mL of PEG fusion agent was added dropwise within 1 min. At this time, the cell state was a red homogeneous sand-like state.

[0070] 1 mL of preheated DMEM medium was added dropwise within 1 min, and 1 mL of preheated DMEM medium was added dropwise again within 1 min. Then, 7 mL of preheated DMEM medium was added dropwise within 3 min, and the mixture was allowed to stand stably in a 37°C water bath for 5 min. The cell fusion was terminated by centrifugation at 800 rpm for 5 min.

[0071] The supernatant was discarded, HAT medium was added, and the cells were mixed. The fusion cell suspension was evenly spread on the 96-well cell plate with feeder cells, and the plate was placed in a CO2 cell culture incubator and cultured at 37°C.

[0072] 3.4 Screening of positive hybridoma cells

[0073] Seven days after cell fusion, the cell pellets were observed to be relatively large, and the supernatant was detected by indirect ELISA. The coating antigen (prepared according to the method and steps of the present embodiment 1.2) was used as the detection antigen at a concentration of 1 μg / mL. The positive control was the serum of the immunized mouse (i.e., the serum of the selected mouse collected before cell fusion), and the negative control was the serum of the PBS immunized mouse. The inhibition rate of the positive well against the gentamicin standard was retested.

[0074] Table 2 Screening results of hybridoma cells (cell supernatant within 24 h)

[0075]

[0076] 3.5 Cloning culture

[0077] The positive hybridoma cells screened were subcloned by limiting dilution method.

[0078] Table 3. Results of limiting dilution test (cell supernatant within 24h)

[0079]

[0080] The hybridoma cell strain 3A3-G3 identified after subcloning and capable of stably secreting monoclonal antibody was transferred to cell bottles for expansion culture and preservation in liquid nitrogen.

[0081] 3.6 Collection of antibody

[0082] When the cell number of the cloned culture reached about 80%, 10 mL of sterile 1xPBS was added to blow off the cell layer, which was then transferred to a 15 mL centrifuge tube after resuspension. Centrifugation was performed at 1000 r / min for 10 min, and the supernatant was discarded. The precipitate was resuspended in 1 mL of sterile 1xPBS and mixed well. Each mouse was injected with 500 μL of cell suspension, and the growth state of the mouse was observed. After one week, when the mouse's abdomen was swollen, the ascites was collected in a centrifuge tube. Centrifugation was performed at 8000 r / min for 20 min, and the middle ascites layer was aspirated. Crude purification was performed by the caprylic acid-ammonium sulfate method, and the crude purified monoclonal antibody was purified again using a Protein G pre-packed column to obtain the anti- gentamicin monoclonal antibody 3A3-G3, i.e., the anti-GM monoclonal antibody 3A3-G3.

[0083] 4. Identification and performance test of the anti-GM monoclonal antibody 3A3-G3

[0084] 4.1 SDS-PAGE identification

[0085] The concentration of the anti-GM monoclonal antibody 3A3-G3 was determined using a microspectrophotometer, and the concentration was 8.3 mg / mL.

[0086] SDS-PAGE electrophoresis analysis was performed on the anti-GM monoclonal antibody 3A3-G3, and as shown in FIG. 2, two clear bands appeared near 25 kDa and 50 kDa, with the band at 25 kDa being the light chain of the antibody and the band at 50 kDa being the heavy chain of the antibody, and no other bands. Figure 1 Fiji software analysis showed that the purity of the anti-GM monoclonal antibody 3A3-G3 reached 95%.

[0087] 4.2 Sensitivity test of the anti-GM monoclonal antibody 3A3-G3

[0088] The sensitivity of anti-GM monoclonal antibody 3A3-G3 was tested by indirect ELISA. The coating antigen (prepared according to the method and steps of the present embodiment 1.2) was coated at 1 μg / mL; anti-GM monoclonal antibody 3A3-G3 was diluted to 10 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, and 0.03125 μg / mL from the starting concentration of 1 mg / mL, and the sensitivity of anti-GM monoclonal antibody 3A3-G3 was determined by indirect ELISA. The results are shown in Table 4, and the inhibition rate of anti-GM monoclonal antibody 3A3-G3 was as high as 88.73% at 0.0625 μg / mL.

[0089] Table 4. Results of sensitivity test of anti-GM monoclonal antibody 3A3-G3

[0090]

[0091] 4.3 Specificity test of anti-GM monoclonal antibody 3A3-G3

[0092] The specificity of anti-GM monoclonal antibody 3A3-G3 was tested by indirect ELISA. The coating antigen prepared according to the method and steps of the present embodiment 1.2 was used as the gentamicin complete antigen, and the gentamicin, kanamycin, streptomycin, and lincomycin complete antigens were coated at 1 μg / mL, and the specificity of anti-GM monoclonal antibody 3A3-G3 was verified by diluting the starting concentration of 1 mg / mL to 0.0625 μg / mL. The results are shown in Table 5, and anti-GM monoclonal antibody 3A3-G3 can specifically react with the gentamicin complete antigen, but not with the kanamycin, streptomycin, lincomycin, and erythromycin complete antigens, indicating that the specificity is good and no cross-reaction occurs.

[0093] Table 5. Results of specificity test of anti-GM monoclonal antibody 3A3-G3

[0094]

[0095] 4.4 Sequencing of anti-GM monoclonal antibody 3A3-G3

[0096] The subclone cell strain of anti-GM monoclonal antibody 3A3-G3 was sent to GenScript for sequencing; the antibody was sequenced by Sanger / NGS sequencing, and the V region amino acid sequence and related nucleotide sequence of anti-GM monoclonal antibody 3A3-G3 were obtained.

[0097] The present embodiment uses V(D)J-IMGT to describe the V region gene recombination of antibodies and their amino acid sequences. "V(D)J-IMGT" is a standardized expression in the field of immunogenetics, which specifically refers to a systematic framework established by the International Immunogenetics Database (IMGT) for describing and analyzing the variable region (V region) gene recombination (V(D)J rearrangement) of antibodies and the amino acid sequences encoded thereby.

[0098] The variable region (V region) amino acid sequence of anti-GM monoclonal antibody 3A3-G3 and its corresponding nucleotide sequence are as follows:

[0099] Heavy chain variable region (VH):

[0100] HCDR1 amino acid sequence (SEQ ID No. 1):

[0101] GFTFSHYA;

[0102] HCDR2 amino acid sequence (SEQ ID No. 2):

[0103] ITAAGTYT;

[0104] HCDR3 amino acid sequence (SEQ ID No. 3):

[0105] VRQGGFYSHYFAY;

[0106] Full-length amino acid sequence of heavy chain variable region (SEQ ID No. 7):

[0107] QVQLQESGGGLVKPGGSLKLSCAASGFTFSHYAMSWVRQTPEKRLEWVATITAAGTYTYYLDSVRGRFTISRDNGNNTLFLKMSSLRSEDTAMYYCVRQGGFYSHYFAYWGQGTALTVSS;

[0108] and its corresponding nucleotide sequence (SEQ ID No. 9):

[0109] CAGGTCCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTCACTATGCCATGTCCTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATCACTGCTGCTGGTACTTACACCTACTATCTAGACAGTGTGAGGGGTCGATTCACCATCTCCAGAGACAATGGCAATAATACCCTGTTCCTGAAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGTAAGACAAGGGGGTTTCTATTCACACTACTTTGCCTATTGGGGCCAAGGCACCGCTCTCACAGTCTCCTCA;

[0110] Heavy chain variable region (VH):

[0111] HCDR1 amino acid sequence (SEQ ID No. 3):

[0112] SSYMH;

[0113] HCDR2 amino acid sequence (SEQ ID No. 4):

[0114] YIDP;

[0115] HCDR3 amino acid sequence (SEQ ID No. 5):

[0116] QSYDSSSTVALWIHY

[0117] Full-length heavy chain variable region amino acid sequence (SEQ ID No. 7):

[0118] SSYMH YIDP YDQSYDSSSTVALWIHYGGTTTCTVTCRAVRSYGPPRFFDSWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALWCYSASGFTSYGLSWFRQAPGKGLEWVSAISSSGSSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCYADPRTFGQGTKLEIK

[0119] and its corresponding nucleotide sequence (SEQ ID No. 9):

[0120] GACATTGTGATCACACAAACTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA.

[0121] II. Example 2: Preparation of gentamicin (GM) residue detection kit

[0122] To further illustrate the application prospect and key role of the anti-GM antibody provided by the present application in the field of immunoassay technology, the current example is based on the anti-GM monoclonal antibody 3A3-G3 provided in Example 1, and a gentamicin (GM) residue rapid detection kit (colloidal gold immunochromatography method) is constructed for rapid detection of gentamicin residues in the sample to be tested.

[0123] For the above-mentioned gentamicin (GM) residue rapid detection kit (colloidal gold immunochromatography method), it will be referred to as a gentamicin (GM) rapid detection kit for convenience, and its preparation process is as follows.

[0124] 1. Preparation of gentamicin (GM) rapid detection kit

[0125] 1.1 Preparation of colloidal gold label

[0126] The anti-GM monoclonal antibody 3A3-G3 of Example 1 is selected as the colloidal gold label required for the preparation of the gentamicin (GM) rapid detection kit in the current example, and its preparation process is as follows:

[0127] Add chloroauric acid (2 mL, 1%) to ultrapure water, and after the water boils, quickly stir and add the reducing agent trisodium citrate (4 mL, 1.2%) for reaction to prepare colloidal gold;

[0128] Anti-GM monoclonal antibody 3A3-G3 was added to colloidal gold at a ratio of 20 μg antibody / mL colloidal gold (antibody was added dropwise, room temperature, 200 rpm, pH 7.5), blocked and stabilized (blocking agent (1% bovine serum albumin or 0.05% casein) was added to a final concentration; stirring was continued for 30 minutes to avoid aggregation), and centrifuged to purify the colloidal gold label of anti-GM monoclonal antibody 3A3-G3 (4°C, 12,000 g centrifugation for 30 minutes, supernatant was discarded) and resuspended for storage (the precipitate was resuspended to 1 / 10 of the original volume with 0.01 mol / L PBS (pH 7.4) containing 1% BSA and 5% sucrose) for later use.

[0129] 1.2 Preparation of an immunochromatographic test strip

[0130] (1) Preparation of a sample pad

[0131] The high water absorption material used to prepare the sample pad was soaked in a 0.5% BSA, pH 7.2, 0.1 mol / L phosphate buffer for 2 h and dried at 37°C for 2 h to obtain a sample pad assembly.

[0132] (2) Preparation of a gold label pad

[0133] A glass fiber membrane was used to prepare the conjugate pad (i.e., the gold label pad), which was pretreated by soaking in a buffer (containing 1% BSA, 0.5% Triton X-100, and 1% sucrose) and drying (37°C, 12 h) before preparation to reduce non-specific adsorption.

[0134] During the process of coating the colloidal gold label on the conjugate pad, the colloidal gold label of anti-GM monoclonal antibody 3A3-G3 was uniformly sprayed at a linear flow rate of 10 μL / cm using a dot membrane instrument, and the gold label pad assembly was obtained after drying at 37°C for 24 h.

[0135] (3) Preparation of a chromatographic membrane

[0136] The coating agent (prepared according to the method and steps of 1.2 in Example 1) was coated on the test line (T line) of the chromatographic membrane using a dot membrane instrument, and the secondary antibody adapted to anti-GM monoclonal antibody 3A3-G3 (considered as a primary antibody) was coated on the control line (C line) of the chromatographic membrane using a dot membrane instrument. After coating, the chromatographic membrane was dried at 37°C for 2 h to obtain a chromatographic membrane assembly.

[0137] (4) Assembly of an immunochromatographic test strip

[0138] The sample pad assembly, the gold label pad assembly, the chromatography membrane assembly, and the water absorption pad are sequentially adhered to the bottom plate made of PVC; wherein the initial end of the sample pad assembly is aligned with the initial end of the bottom plate, the terminal end of the sample pad assembly is overlaid with the initial end of the gold label pad assembly by 1-3 mm, the terminal end of the gold label pad assembly is overlaid with the initial end of the chromatography membrane assembly by 1-3 mm, the terminal end of the chromatography membrane assembly is overlaid with the initial end of the water absorption pad by 1-3 mm, and the terminal end of the water absorption pad is aligned with the terminal end of the bottom plate; the assembled immunochromatographic test strip template is cut into a proper width, and an immunochromatographic test strip for detection is obtained.

[0139] 1.3 Assembling gentamicin (GM) rapid test kit

[0140] The above immunochromatographic test strip is packaged and encapsulated, and a gentamicin (GM) rapid test kit is obtained.

[0141] 2. Performance verification of gentamicin (GM) rapid test kit

[0142] The basic performance verification of the gentamicin (GM) rapid test kit of the current embodiment is performed mainly by using the results of a gold label instrument (also known as an immunochromatographic reading instrument or a colloidal gold card reading instrument) and supplemented by naked eye interpretation.

[0143] 2.1 Sensitivity test of gentamicin (GM) rapid test kit

[0144] The concentration of the gentamicin standard is diluted to 5 ppb (ppb is a dimensionless unit commonly used in the field of food safety residue detection technology; when the medium type is an aqueous solution, 1 ppb corresponds to 1 ng / mL) and 10 ppb, and some gentamicin (GM) rapid test kits prepared in the embodiment are used for the standard addition detection of the milk sample to verify the sensitivity, and the results are shown in Table 6. The results show that the gentamicin (GM) rapid test kit and the same batch product thereof used in the current sensitivity test have high sensitivity, and the minimum detection limit of the gentamicin standard can reach 10 ppb.

[0145] Table 6 Sensitivity test results of gentamicin (GM) rapid test kit

[0146]

[0147] 2.2 Specificity test of gentamicin (GM) rapid test kit

[0148] The standard addition milk samples of 500 ppb of gentamicin, kanamycin, streptomycin, lincomycin, and erythromycin are detected by using some gentamicin (GM) rapid test kits prepared in the embodiment, and the specificity is verified, and the results are shown in Table 7. Figure 2The results show that the gentamicin (GM) rapid detection kit for the current specificity test and its same batch product not only do not have cross-reaction with lincomycin and erythromycin which are non-amino glycoside antibiotics, but also do not have cross-reaction with kanamycin and streptomycin which are amino glycoside antibiotics, and the specificity is good, and it is suitable for the specificity detection of gentamicin (GM) residue.

[0149] III. Example 3: Application of gentamicin (GM) residue detection kit

[0150] Considering the urgent need for gentamicin residue detection in the field of food safety monitoring, environmental pollution monitoring and other fields of antibiotic residue analysis, the gentamicin (GM) rapid detection kit in Example 2 can be applied to related detection. Therefore, the current example applies the gentamicin (GM) rapid detection kit in Example 2 to the detection of gentamicin (GM) residue in food safety monitoring / environmental pollution monitoring field samples.

[0151] 1. Matrix verification and sample processing

[0152] The matrix verification and sample processing of the gentamicin (GM) rapid detection kit is a necessary prerequisite to ensure the accuracy and reliability of the detection results in the application process. The so-called "matrix" refers to all background substances in the sample except the target substance; its core influence is matrix effect, that is, the matrix other than the target substance in the sample interferes with the detection results, causing the measured value to deviate from the true value. Spiking experiment is the most direct and effective way of matrix verification.

[0153] For unverified matrix, before using the gentamicin (GM) rapid detection kit for gentamicin residue detection, according to its sensitivity, the concentration of gentamicin standard is diluted to 5ppb, 10ppb (i.e. the detection limit of gentamicin (GM) rapid detection kit), and the sample is detected by spiking to verify the matrix; for samples without matrix interference, the gentamicin (GM) rapid detection kit can be used for gentamicin residue detection.

[0154] Sample processing aims to adapt the original sample collected to the detection conditions of the gentamicin (GM) rapid detection kit in the current example. For the gentamicin (GM) rapid detection kit, it mainly adapts to liquid samples; therefore, the sample processing in the current example includes but is not limited to: for solid or other non-liquid original samples, it is necessary to prepare them into liquid samples that can be detected by the gentamicin (GM) rapid detection kit; for samples with too high gentamicin (GM) residue concentration, it is appropriate to dilute them; and for original samples with interfering substances, the interfering substances can be removed by, for example, filtration, centrifugation, digestion, etc.

[0155] 2. Rapid detection of samples

[0156] Bring the sample to be tested and reagents (the required number of portions) in the gentamicin (GM) rapid test kit, such as the immunochromatographic test strip, to room temperature (20~25℃); take 200 μL of the sample to be tested and place it on the sample pad. Through chromatography and incubation with the colloidal gold label, determine the detection result according to the color development of the immunochromatographic test strip.

[0157] 3. Detection result determination

[0158] As a rapid detection product based on the specific binding of antigen-antibody, the result of the gentamicin (GM) rapid test kit supports naked-eye rapid interpretation, that is, the detection result is determined by naked-eye reading the color development of the test line (T line) and the quality control line (C line) on the chromatography membrane. The identification criteria are as follows.

[0159] Negative (color development characteristics): both the C line and the T line develop color, and the T line develops color stronger than the C line;

[0160] Negative result, indicating that the residual concentration of gentamicin in the sample is lower than the detection limit;

[0161] Positive (color development characteristics): the C line develops color, and the C line develops color the same as the T line, the T line develops color weaker than the C line, or the T line does not develop color;

[0162] Positive result, indicating that the residual concentration of gentamicin in the sample is equal to or higher than the detection limit.

[0163] Invalid (color development characteristics): no C line appears;

[0164] Invalid result, indicating incorrect operation process or that the immunochromatographic test strip has deteriorated and is no longer valid.

Claims

1. An anti-GM antibody, targeting a gentamicin antigen epitope, characterized in that, It comprises: HCDR1, HCDR2, HCDR3; and, LCDR1, LCDR2, LCDR3; Wherein, the amino acid sequence of the HCDR1 is shown as SEQ ID No. 1; the amino acid sequence of the HCDR2 is shown as SEQ ID No. 2; the amino acid sequence of the HCDR3 is shown as SEQ ID No. 3; the amino acid sequence of the LCDR1 is shown as SEQ ID No. 4; the amino acid sequence of the LCDR2 is shown as SEQ ID No. 5; the amino acid sequence of the LCDR3 is shown as SEQ ID No.

6.

2. The anti-gentamicin antibody of claim 1, wherein, the heavy chain variable region of the anti- gentamicin antibody comprises a heavy chain variable region framework region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 7; and / or, the light chain variable region of the anti- gentamicin antibody comprises a light chain variable region framework region; the amino acid sequence of the light chain variable region is shown as SEQ ID No.

8.

3. The anti- gentamicin antibody of claim 1, wherein, the anti- gentamicin antibody further comprises a heavy chain constant region and / or a light chain constant region; and / or, the anti- gentamicin antibody is a murine antibody.

4. A polynucleotide encoding the anti- gentamicin antibody of any one of claims 1-3.

5. The polynucleotide of claim 4, wherein, It comprises: a nucleotide fragment with the sequence shown as SEQ ID No. 9 encoding the heavy chain variable region of the anti- gentamicin antibody of claim 2; and / or, a nucleotide fragment with the sequence shown as SEQ ID No. 10 encoding the light chain variable region of the anti- gentamicin antibody of claim 2.

6. A kit for detecting gentamicin residues, characterized in that, It comprises: the anti- gentamicin antibody of any one of claims 1-3; and a reagent for detecting the binding of free gentamicin to the anti- gentamicin antibody.

7. The kit for detecting gentamicin residues of claim 6, wherein, the kit for detecting gentamicin residues is an ELISA kit; the ELISA kit comprises: a solid phase carrier pre-coated with the anti- gentamicin antibody; or, an enzyme-labeled anti- gentamicin antibody; or, the anti- gentamicin antibody and an enzyme-labeled secondary antibody adapted thereto.

8. The kit for detecting gentamicin residues of claim 6, wherein, the kit for detecting gentamicin residues is an immunochromatographic kit; the immunochromatographic kit comprises an immunochromatographic test strip; wherein, the immunochromatographic kit is coated with a label or independently loaded with a label through the conjugate pad / binding zone of the immunochromatographic test strip; the label is an anti- gentamicin antibody labeled by a chromogenic or fluorescent nanoparticle; wherein, the chromogenic nanoparticle is colloidal gold, colloidal carbon or latex microspheres; the fluorescent nanoparticle is quantum dots or fluorescent microspheres.

9. A non-diagnostic method for detecting gentamicin residues, characterized in that, It comprises: The anti- gentamicin antibody according to any one of claims 1 to 3 or the anti- gentamicin antibody in the kit for detecting gentamicin residue according to any one of claims 6 to 8 is incubated with a sample to be tested, and the binding of free gentamicin in the sample to be tested to the anti- gentamicin antibody is detected, thereby determining the gentamicin residue in the sample to be tested.

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