JL transposase monoclonal antibody and detection kit thereof

Abstract

The invention relates to a JL transposase monoclonal antibody and a detection kit thereof. In particular, provided is a JL transposase binding molecule comprising an antibody targeting a JL transposase, or an antigen-binding fragment thereof, the antibody comprising any group of CDRs described herein. The detection kit provided by the invention is strong in detection specificity, high in sensitivity and good in stability.

Description

Technical Field

[0001] The present invention relates to the field of biology; more specifically, the present invention relates to a JL transposase monoclonal antibody and a detection kit thereof. Background Art

[0002] DNA transposons are a type of mobile DNA sequence that can freely jump (copy or shift) on the genome and can be transferred from one location in the genome to another through a series of processes such as cutting and reintegration. They were first discovered by McCintock in corn chromosomes in the 1940s, and later found in various organisms such as bacteria, fungi and insects.

[0003] In addition to effectively improving the efficiency of preparing transgenic animals based on microinjection technology, transposons can also be used in gene therapy. In gene therapy, transposon-mediated gene transfer has the advantages of safety and high efficiency. In the past decade, transposons have developed into the most commonly used non-viral vectors in gene therapy. In addition, transposons can also prepare mutants, carry out gene capture, and be applied to functional genomics research. In recent years, this technology has made important progress in human gene therapy, transgenic preparation of model animals such as mice and zebrafish, and functional genomics research.

[0004] Tc1 / Mariner transposons are the most widely distributed class of DNA transposon superfamily in nature, including bacteria, invertebrates and vertebrates. Among vertebrates, Tc1 / Mariner transposons are most widely distributed in bony fish. The prior application disclosed a PS transposon with a unique transposition catalytic domain "D35D", which belongs to the pogo transposon superfamily, and can exhibit certain transposition activity and cleavage activity in mammalian cells. Although the above-mentioned PS transposon system already has a certain transgenic application value, because the PS transposase sequence is a wild-type natural amino acid sequence, its transposition efficiency is relatively low. CN202310081106.8 discloses amino acid / DNA mutation and / or engineering modification of the PS transposon system to obtain a JL transposon system.

[0005] During the gene writing process using the JL transposon system, residual JL transposase will remain, and a method for detecting the JL transposase needs to be provided for quality control. Summary of the invention

[0006] Based on the above problems existing in the prior art, the present application provides a monoclonal antibody of JL transposase, a detection kit developed based on the ELISA method including the antibody, and the quantitative detection of JL transposase is successfully completed using the kit.

[0007] The first aspect of the present invention provides a JL transposase binding molecule, which is an antibody or an antigen-binding fragment thereof targeting JL transposase, or a variant thereof having at least 85% sequence identity with the antibody or the antigen-binding fragment thereof and retaining its JL transposase binding activity.

[0008] In one or more embodiments, the antibody contains any one set of CDRs selected from:

[0009] (A1) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, LCDR3 shown in SEQ ID NO: 18;

[0010] (A2) the antibody comprises: HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:5, HCDR3 shown in SEQ ID NO:6, LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20, and LCDR3 shown in SEQ ID NO:21;

[0011] (A3) the antibody comprises: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24;

[0012] (A4) the antibody comprises: HCDR1 shown in SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:25, LCDR2 shown in SEQ ID NO:26, LCDR3 shown in SEQ ID NO:27;

[0013] (A5) The antibody contains: HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15 and LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30.

[0014] In one or more embodiments, the light chain variable region of the antibody comprises a murine or human light chain FR region. In one or more embodiments, the heavy chain variable region of the antibody comprises a murine or human heavy chain FR region.

[0015] In one or more embodiments, FR1, FR2, FR3 and FR4 of the heavy chain variable region of the antibody are each independently selected from FR1, FR2, FR3 and FR4 of the heavy chain variable region shown in any one of SEQ ID NOs: 33, 35, 37, 39, and 41, and / or, FR1, FR2, FR3 and FR4 of the light chain variable region of the antibody are each independently selected from FR1, FR2, FR3 and FR4 of the light chain variable region shown in any one of SEQ ID NOs: 34, 36, 38, 40, and 42.

[0016] In one or more embodiments, the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:33, or a sequence having at least 85% sequence identity thereto, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO:34, or a sequence having at least 85% sequence identity thereto.

[0017] In one or more embodiments, the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:35, or a sequence having at least 85% sequence identity thereto, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO:36, or a sequence having at least 85% sequence identity thereto.

[0018] In one or more embodiments, the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:37, or a sequence having at least 85% sequence identity thereto, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO:38, or a sequence having at least 85% sequence identity thereto.

[0019] In one or more embodiments, the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:39, or a sequence having at least 85% sequence identity thereto, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO:40, or a sequence having at least 85% sequence identity thereto.

[0020] In one or more embodiments, the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:41, or a sequence having at least 85% sequence identity thereto, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO:42, or a sequence having at least 85% sequence identity thereto.

[0021] In one or more embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0022] In one or more embodiments, the antibody further comprises a mouse IgG Fc segment, a rabbit IgG Fc segment, or a human IgG Fc segment.

[0023] In one or more embodiments, the heavy chain of the antibody comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, or a sequence having at least 85% sequence identity therewith. Alternatively or in addition, the light chain of the antibody comprises a light chain constant region of a human κ chain, λ chain, or a sequence having at least 85% sequence identity therewith.

[0024] In one or more embodiments, the heavy chain of the antibody comprises the heavy chain constant region of human IgG1 or a sequence having at least 85% sequence identity thereto, and the light chain comprises the light chain constant region of human κ chain or a sequence having at least 85% sequence identity thereto.

[0025] In one or more embodiments, the antibody is a multispecific antibody, preferably a bispecific antibody.

[0026] In one or more embodiments, the antibody is a monoclonal antibody.

[0027] In one or more embodiments, the antibody is a chimeric antibody or a murine antibody.

[0028] The present invention also provides a polynucleotide selected from:

[0029] (1) the coding sequence of the JL transposase binding molecule described in any embodiment of the present invention;

[0030] (2) The complementary sequence of (1).

[0031] The present invention also provides a nucleic acid construct, which comprises the polynucleotide described in any embodiment herein.

[0032] In one or more embodiments, the nucleic acid construct is a vector, such as an integration vector, a cloning vector, or an expression vector.

[0033] The present invention also provides a host cell, which:

[0034] (1) expressing and / or secreting the JL transposase binding molecule described in any embodiment of the present invention;

[0035] (2) comprising a polynucleotide as described herein; and / or

[0036] (3) comprising the nucleic acid construct described herein.

[0037] In one or more embodiments, the host cell is selected from a prokaryotic cell or a eukaryotic cell.

[0038] In one or more embodiments, the host cell is a mammalian cell.

[0039] The present invention also provides a method for producing a JL transposase binding molecule, comprising: culturing the host cell described herein under conditions suitable for producing the JL transposase binding molecule, and optionally purifying the JL transposase binding molecule from the culture.

[0040] The second aspect of the present invention provides a JL transposase binding molecule pair, wherein the first JL transposase binding molecule is as described in any embodiment of the first aspect of the present invention, the second JL transposase binding molecule is as described in any embodiment of the first aspect of the present invention, and the CDR of the first JL transposase binding molecule is different from the CDR of the second JL transposase binding molecule.

[0041] In one or more embodiments, the first JL transposase binding molecule comprises the CDRs shown in any group A1-A5, and the second JL transposase binding molecule comprises the CDRs shown in any group A1-A5. The A1-A5 group of CDRs are as described in the first aspect of this invention.

[0042] In one or more embodiments, the first JL transposase binding molecule comprises the CDRs shown in any one group A1-A5, and the second JL transposase binding molecule comprises the CDRs shown in group A1, A3 or A5.

[0043] In one or more embodiments, the first JL transposase binding molecule and the second JL transposase binding molecule respectively comprise CDRs shown in any of the following groups: A1 and A3, A2 and A3, A3 and A1, A4 and A5, A5 and A4, A4 and A3.

[0044] In one or more embodiments, the first JL transposase binding molecule comprises: a heavy chain variable region set forth in SEQ ID NO:33 and a light chain variable region set forth in SEQ ID NO:34, a heavy chain variable region set forth in SEQ ID NO:35 and a light chain variable region set forth in SEQ ID NO:36, a heavy chain variable region set forth in SEQ ID NO:37 and a light chain variable region set forth in SEQ ID NO:38, a heavy chain variable region set forth in SEQ ID NO:39 and a light chain variable region set forth in SEQ ID NO:40, or a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42; and the second JL transposase binding molecule comprises: a heavy chain variable region set forth in SEQ ID NO:33 and a light chain variable region set forth in SEQ ID NO:34, a heavy chain variable region set forth in SEQ ID NO:35 and a light chain variable region set forth in SEQ ID NO:36, a heavy chain variable region set forth in SEQ ID NO:37 and a light chain variable region set forth in SEQ ID NO:38, a heavy chain variable region set forth in SEQ ID NO:39 and a light chain variable region set forth in SEQ ID NO:40, or a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42. NO:41 and the light chain variable region shown in SEQ ID NO:42, and the heavy chain variable region and light chain variable region of the first JL transposase binding molecule are different from those of the second JL transposase binding molecule.

[0045] In one or more embodiments, the first JL transposase binding molecule comprises: a heavy chain variable region set forth in SEQ ID NO:33 and a light chain variable region set forth in SEQ ID NO:34, a heavy chain variable region set forth in SEQ ID NO:35 and a light chain variable region set forth in SEQ ID NO:36, a heavy chain variable region set forth in SEQ ID NO:37 and a light chain variable region set forth in SEQ ID NO:38, a heavy chain variable region set forth in SEQ ID NO:39 and a light chain variable region set forth in SEQ ID NO:40, or a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42; the second JL transposase binding molecule comprises: a heavy chain variable region set forth in SEQ ID NO:33 and a light chain variable region set forth in SEQ ID NO:34, a heavy chain variable region set forth in SEQ ID NO:37 and a light chain variable region set forth in SEQ ID NO:38, or a heavy chain variable region set forth in SEQ ID NO:39 and a light chain variable region set forth in SEQ ID NO:40. The light chain variable region is the light chain variable region shown in IDNO:40, and the heavy chain variable region and light chain variable region of the first JL transposase binding molecule are different from those of the second JL transposase binding molecule.

[0046] In one or more embodiments, the first JL transposase binding molecule comprises a heavy chain variable region as set forth in SEQ ID NO:33 and a light chain variable region as set forth in SEQ ID NO:34, and the second JL transposase binding molecule comprises a heavy chain variable region as set forth in SEQ ID NO:37 and a light chain variable region as set forth in SEQ ID NO:38,

[0047] The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO: 35 and a light chain variable region as shown in SEQ ID NO: 36, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO: 37 and a light chain variable region as shown in SEQ ID NO: 38,

[0048] The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO: 37 and a light chain variable region as shown in SEQ ID NO: 38, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO: 33 and a light chain variable region as shown in SEQ ID NO: 34,

[0049] The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:42,

[0050] The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:42, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40, or

[0051] The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:38.

[0052] In one or more embodiments, the second JL transposase binding molecule further comprises a label. The label comprises any one selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, radiocontrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers or enzymes.

[0053] In one or more embodiments, the fluorescent substance includes Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, OregonGreen514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, trisbipyridyl diamine europium, europium cryptates or chelates, diamines, bicyanines, La Jolla blue dye, allophycocyanin, allococyanin B, any one of phycocyanin C, phycocyanin R, thiamine, phycoerythrin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine and Texas Red.

[0054] In one or more embodiments, the radioactive isotope includes any one of 110In, 111In, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y, 90Y, 89Zr, 94mTc, 94Tc, 99mTc, 120I, 123I, 124I, 125I, 131I, 154-158Gd, 32P, 11C, 13N, 15O, 186Re, 188Re, 51Mn, 52mMn, 55Co, 72As, 75Br, 76Br, 82mRb and 83Sr.

[0055] In one or more embodiments, the fluorescent microspheres are polystyrene fluorescent microspheres, which contain rare earth fluorescent ions europium.

[0056] In one or more embodiments, the label is biotin. The present invention uses a biotin-avidin system for color development to improve sensitivity. The affinity between avidin and biotin is extremely strong, and the affinity constant of the combination of the two is at least 10,000 times higher than the affinity constant of the antigen and the antibody, so the two can combine quickly and the reaction is not disturbed by the outside world.

[0057] The present invention also provides a kit for detecting JL transposase, wherein the kit comprises the JL transposase binding molecule or JL transposase binding molecule pair, polynucleotide, nucleic acid construct, bacteriophage or host cell described in any embodiment of the present invention.

[0058] In one or more embodiments, the kit further comprises a reagent for detecting the binding of JL transposase to a JL transposase binding molecule or a JL transposase binding molecule pair, for example, a reagent for detecting the binding by enzyme-linked immunosorbent assay.

[0059] In one or more embodiments, the reagent for detecting binding is a detectable label, such as biotin, that can be linked to the JL transposase binding molecule or JL transposase binding molecule pair. The detectable label is linked to the antibody or antigen-binding fragment thereof or exists separately in the kit.

[0060] In one or more embodiments, the JL transposase binding molecule is used for sandwich detection, and the kit comprises the JL transposase binding molecule pair described in any embodiment of the second aspect of this invention. The sandwich method completes the detection in the form of "support-first JL transposase binding molecule-object to be detected-second JL transposase binding molecule-label".

[0061] In one or more embodiments, the first JL transposase binding molecule is coupled to a support. In one or more embodiments, the kit further comprises a reagent for coupling the first JL transposase binding molecule to a support.

[0062] In one or more embodiments, the support includes, but is not limited to, ELISA plates, beads, microparticles, slides, chips, plastics, films, etc. made of materials such as polystyrene, polyethylene, cellulose, nitrocellulose, cellulose acetate, and silicate.

[0063] In one or more embodiments, the second JL transposase binding molecule further comprises a label. The label comprises any one selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, radiocontrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers or enzymes. Preferably, the label is biotin.

[0064] In one or more embodiments, the kit further comprises a recognition signal, and the label and the recognition signal are visualized by a biotin-avidin system.

[0065] In one or more embodiments, the recognition signal comprises any one selected from the following: fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers or enzymes labeled with avidin.

[0066] In one or more embodiments, the recognition signal includes an avidin-labeled catalytic enzyme and a substrate compatible with the catalytic enzyme. Further, the catalytic enzyme is selected from horseradish peroxidase or alkaline phosphatase. Accordingly, the substrate is 3,3',5,5'-tetramethylbenzidine or o-phenylenediamine. When the substrate is 3,3',5,5'-tetramethylbenzidine, the color development time is not fixed because the batches of the color developing solution are different. The general standard is that the OD value of the standard with the highest concentration is about 2.0.

[0067] In one or more embodiments, the weight of the first JL transposase binding molecule is greater than the weight of the second JL transposase binding molecule.

[0068] In one or more embodiments, the weight ratio of the first JL transposase binding molecule to the second JL transposase binding molecule is 5:1-4, preferably 5:2-3.

[0069] In one or more embodiments, the kit includes a reaction plate coated with a first JL transposase binding molecule, a second JL transposase binding molecule labeled with biotin, a catalytic enzyme labeled with avidin, and a substrate.

[0070] The preferred embodiment of the kit of the present invention is to label streptavidin with horseradish peroxidase, and use the label to identify the biotinylated second JL transposase binding molecule, that is, to achieve more sensitive detection with the help of a biotin-avidin amplification system.

[0071] In one or more embodiments, the kit further comprises any one or more of a JL transposase standard, a sample diluent, a washing solution, and a stop solution. Further, the washing solution is PBST.

[0072] The present invention also provides a non-diagnostic method for detecting the presence of JL transposase in a sample, the method comprising: incubating the sample with the JL transposase binding molecule or JL transposase binding molecule pair described in any embodiment of the present invention, and detecting the binding of JL transposase to the JL transposase binding molecule or JL transposase binding molecule pair, thereby determining the presence of JL transposase in the sample. The detection is enzyme-linked immunosorbent assay.

[0073] In one or more embodiments, the method is a sandwich method for detecting using the JL transposase binding molecule pair, the method comprising the steps of:

[0074] (1) coupling the first JL transposase binding molecule to the support, optionally washing, optionally blocking,

[0075] (2) contacting a first JL transposase binding molecule coupled to a support with a sample, allowing the JL transposase in the sample to bind to the first JL transposase binding molecule to form a complex, and optionally washing,

[0076] (3) contacting the complex with a second JL transposase binding molecule connected to a label, so that the JL transposase in the complex binds to the second JL transposase binding molecule, and optionally washing,

[0077] (4) Detecting the JL transposase through a detectable reaction mediated by the marker.

[0078] In one or more embodiments, the method comprises the steps of:

[0079] (1) Coating: Coating the first JL transposase binding molecule onto the support and washing.

[0080] (2) Blocking: The empty sites on the support that are not coated with the first JL transposase binding molecule are blocked with an inert protein to prevent nonspecific adsorption in subsequent reactions.

[0081] (3) The first JL transposase binding molecule captures the antigen: After adding the sample to be tested and incubating, the first JL transposase binding molecule captures the antigen in the sample.

[0082] (4) Antigen capture of the second binding molecule: After adding the second JL transposase binding molecule and incubating, the antigen captures the second JL transposase binding molecule.

[0083] Detection: Detection is performed by introducing markers in one or more steps.

[0084] In one or more embodiments, the label comprises any one selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent labels, ultrasound contrast agents, photosensitizers or enzymes.

[0085] In one or more embodiments, the recognition signal comprises any one selected from the following: fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers or enzymes.

[0086] In one or more embodiments, the recognition signal comprises an avidin-labeled catalytic enzyme and a substrate compatible with the catalytic enzyme, and the detectable reaction is a color development reaction of the catalytic enzyme catalyzed substrate. Further, the catalytic enzyme is selected from horseradish peroxidase or alkaline phosphatase. Accordingly, the substrate is 3,3',5,5'-tetramethylbenzidine or o-phenylenediamine.

[0087] Compared with the prior art, the JL transposase detection kit provided by the present invention has strong detection specificity, high sensitivity and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1-Figure 6 The standard curve of sandwich ELISA for the antibody pairs in Table 5. DETAILED DESCRIPTION

[0089] Unless otherwise defined, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., ed., 1987 edition and periodic updates thereof); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0090] As used herein, a "JL transposase binding molecule" is a protein that specifically binds to JL transposase, including but not limited to antibodies, antigen-binding fragments of antibodies, nanobodies, minibodies, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

[0091] Herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, e.g., Fab, F(ab')2 and Fv. Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0092] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.

[0093] The term "variable" refers to the fact that certain segments in the variable domains differ widely in antibody sequence, namely HCDR1, HCDR2, HCDR3 in the heavy chain variable region and LCDR1, LCDR2 and LCDR3 in the light chain variable region. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions (FR1, FR2, FR3 and FR4. Typically, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. For chimeric antibodies, the FR region may be non-human (e.g., mouse). Antibody CDRs may be identified by a variety of coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, Kabat / Chothia, etc. These coding systems are known in the art, and may be specifically found at, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence numbering of the antigen-binding protein may be according to the IMGT numbering scheme.

[0094] "Fc region" or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, the CH2 domain and the CH3 domain from the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

[0095] In one or more embodiments, the antibodies described herein further include a heavy chain constant region and / or a light chain constant region, the heavy chain constant region, such as a heavy chain constant region derived from IgA, IgD, IgE, IgG and IgM, or a heavy chain constant region derived from IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2, or a sequence thereof having at least 85% sequence identity, and a light chain constant region, such as a light chain constant region of a κ or λ chain, or a sequence thereof having at least 85% sequence identity.

[0096] The antibodies of the present invention can be prepared by conventional methods in the art, such as hybridoma technology, phage display technology, and cell line expression well known in the art. The present application also provides a fusion protein, which comprises the antibody or antigen-binding fragment described in the present application as an expression target, an active molecule, or a targeting molecule. For example, a fusion protein formed by adding a label (e.g., a His6 label) that facilitates protein expression or purification at both ends of the antibody. The label does not affect the function of the target protein and can be easily removed.

[0097] The present invention also provides polynucleotides encoding the binding molecules, binding molecule pairs, and fusion proteins described herein. Provided herein are polynucleotides encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains, and each CDR. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. The present invention also includes degenerate variants of polynucleotide sequences encoding antibodies or antigen-binding fragments thereof, fusion proteins, or antibody conjugates, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0098] According to the amino acid sequence and codons, those skilled in the art can easily obtain the coding sequence of the antibody or its fragment. The expression of the polypeptide in different species can also be changed by codon optimization in this field, and the codon preference of different species belongs to the routine technology in this field.

[0099] The antibodies of the present invention can be used for assays due to their high affinity for JL transposase, such as combined assays to detect and / or quantify JL transposase. The present invention also provides a detection kit for detecting the level of JL transposase, the kit comprising an anti-JL transposase antibody, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc.

[0100] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, materials and methods conventional in the art.

[0101] Example

[0102] Example 1: Preparation of monoclonal antibodies against JL transposase

[0103] 1. Preparation of immunogens: Two polypeptide antigens were prepared as primary immunization immunogens, the sequences of which were SEQ ID NO:31 and SEQ ID NO:32, respectively. The synthesized polypeptides were coupled to KLH carrier proteins, respectively. Truncated JL transposase (wtPS1-130aa) was used as a protein for booster immunization, and booster immunization was 3 immunizations. The sequence of the truncated JL transposase was shown in SEQ ID NO:44.

[0104] 2. Animal immunization: A conventional immunization scheme was adopted, and 5 Balb / c mice aged 6-8 weeks were selected. Two polypeptides were used as immunogens, mixed with Freund's complete adjuvant at a ratio of 1:1, and the first immunization was performed at a dose of 50μg / mouse. After an interval of 2 weeks, 2 immunizations were performed, mixed with Freund's incomplete adjuvant at a ratio of 1:1, and immunized at a dose of 25μg / mouse; after an interval of 2 weeks, 3 immunizations were performed, mixed with Freund's incomplete adjuvant at a ratio of 1:1, and immunized at a dose of 25μg / mouse; the final immunization was performed on the 50±7th day, and 4 days after the final immunization, the animal was determined to be dead by cervical dislocation after carbon dioxide asphyxiation, and cell fusion was performed. The indirect ELISA method was used to detect the titer of polyclonal antibodies, and the polypeptide immunogens and full-length proteins were coated respectively. The sera of immunized animals were tested by the indirect ELISA method to determine the level of immune response.

[0105] 3. Cell fusion and screening pairing: For the two groups of peptide-immunized animals, one round of cell fusion was performed using the electrofusion method. All cells fused in each round were plated in a 96-well plate. After fusion, a primary screening was first performed, and the supernatant of the fused cells was screened by the indirect ELISA method to select the supernatant that was positive for the peptide antigen. Then a confirmation screening was performed, and the positive mother clone cells that were positive for the full-length protein were screened by the indirect ELISA method for the positive clones obtained in the primary screening stage. Mouse positive serum and unfused cell wells were set as control groups, and 5 positive clones were screened.

[0106] 4. Subcloning, expanded culture and cryopreservation: Using the limiting dilution method, dilute the cells in the 96-well plate with DMEM medium, and sub-plate each of the five clones into one 96-well plate at a cell density of 3 cells / well, and culture them in a carbon dioxide incubator. Subclone the double-positive mother clones of the polypeptide protein, and screen the subclones using the indirect ELISA method. Amplify the screened subclones and store them in liquid nitrogen.

[0107] 5. Antibody production: The selected monoclonal antibody was used for antibody production, and the antibody was purified by Protein A affinity chromatography, and the purified antibody was stored in phosphate buffered saline (PBS) by dialysis. QC was performed by polyacrylamide gel electrophoresis (SDS-PAGE) and indirect ELISA, and the antibody concentration was determined by NanoDrop2000.

[0108] 6. Antibody sequencing: The selected subclones were sequenced by Sanger / NGS sequencing.

[0109] Monoclonal antibody types: 18B4H8, 11D1F1, 5D5D7, 14E6A9, 10D8F1, sequences see Table 1.

[0110] The heavy chain CDR sequences of the antibodies are shown in SEQ ID NOs: 1 to 15, and the light chain CDR sequences are shown in SEQ ID NOs: 16 to 30.

[0111] Table 1: Antibody sequences

[0112]

[0113] Example 2: Indirect ELISA results of purified mouse monoclonal antibodies

[0114] The indirect ELISA method was used to detect the binding of mouse monoclonal antibodies purified from the supernatant to antigens:

[0115] 1. Immobilize the antigens (A, PS transposase-polypeptide 1, B, truncated JL transposase (wtPS1-130aa), C, MBP-JL (wt-ps) full-length protein, D, MBP-irrelevant protein; the sequences are shown in SEQ ID NOs: 43-46) to the solid phase carrier and coat and block with PBS blocking solution;

[0116] 2. Mouse monoclonal antibodies 18B4H8, 11D1F1, 5D5D7, 14E6A9, and 10D8F1 purified from the culture supernatant were used as primary antibodies and incubated at 37°C for 60 min to react with the coated antigen;

[0117] 3. Add diluted secondary antibody (Peroxidase-AffiniPure Goat Anti-Mouse IgG, FeyFragment Specific) and incubate at 37°C for 30 minutes;

[0118] 4. Add substrate colorimetric solution, incubate in the dark, then add stop solution, read the sample in a 450nm ELISA reader, and measure the OD value.

[0119] In addition, antigens A, B, C and D were expressed and purified using the pMBP-His vector.

[0120] The results of indirect antibody ELISA are shown in Table 2 , where A, B, C, and D refer to: PS transposase-polypeptide 1, truncated JL transposase (1-130aa), MBP-JL full-length protein, and MBP-unrelated protein, respectively.

[0121] Example 3: Biotin-labeled antibody ELISA results

[0122] 1. Immobilize the antigens (PS transposase-peptide 1, MBP-JL (wt-ps) full-length protein, JL transposase, MBP irrelevant protein) to the solid phase carrier for coating and blocking;

[0123] 2. Add biotin-labeled 18B4H8, 11D1F1, 5D5D7, 14E6A9, 10D8F1 antibodies as the primary antibody and incubate at 37°C for 60 min to allow them to react with the coated antigen;

[0124] 3. Add diluted secondary antibody (Streptavidin-HRP) and incubate at 37°C for 30 min;

[0125] 4. Add substrate colorimetric solution, incubate in the dark, then add stop solution, read the sample in a 450nm ELISA reader, and measure the OD value.

[0126] The results of indirect antibody ELISA are shown in Table 3 , where A, B, C, and D refer to: PS transposase-polypeptide 1, truncated JL transposase (1-130aa), MBP-JL full-length protein, and MBP-unrelated protein, respectively.

[0127]

[0128]

[0129] Example 4: JL Transposase Detection Kit Sandwich ELISA Results

[0130] In this example, the above antibodies are used to prepare a sandwich ELISA kit and a standard curve. The JL transposase detection kit includes the following materials:

[0131] A reaction plate coated with a first JL transposase antibody, a biotin-labeled second JL transposase antibody, a streptavidin-horseradish peroxidase conjugate (Streptavidin-HRP), a TMB colorimetric solution, a standard sample JL transposase (wt-ps, 1-130aa), a sample diluent, a second JL transposase binding molecule and a Streptavidin-HRP diluent, a washing solution, and a stop solution;

[0132] Coating solution: dilute 20×PBS to 1×PBS, purchased from Bio-Tech, catalog number: B548117-0500;

[0133] Stop solution: purchased from Solebol, product number: C1058;

[0134] PBST: 1×PBS+0.05% Tween (500 ml PBS+250 μl Tween);

[0135] Blocking solution: Thermo BlockerTMCasein, purchased from Thermo, catalog number: 37528;

[0136] First dilution: one-third Thermo BlockerTMCasein + two-thirds 1×PBS;

[0137] Second dilution: one-third Thermo BlockerTMCasein + two-thirds PBST;

[0138] TMB (3,3',5,5'-tetramethylbenzidine): purchased from Abcam, catalog number: ab171524;

[0139] Streptavidin-HRP: purchased from Biolegend, catalog number: 405210;

[0140] The reaction plate coated with the primary JL transposase antibody was prepared as follows:

[0141] 1) Dilute the antibody to 2.5 μg / ml with coating solution, coat the ELISA plate with 100 μl / well, and incubate at 4°C overnight.

[0142] 2) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0143] 3) Add 200 μl of blocking solution to each well and incubate in a 37°C biochemical incubator for 2 hours.

[0144] 4) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0145] Use the kit provided above to prepare the standard curve as follows:

[0146] 1) Dilute the standard with 20 ng / ml of the first solution, set 10 gradients downward (10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml...) and 0 ng / ml, add to the reaction plate coated with the first JL transposase binding molecule, add 100 μl of the standard to each well, seal the plate with a sealing film, and incubate at room temperature for 2 hours on an oscillator at 750±50 rpm.

[0147] 2) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0148] 3) Dilute the biotinylated second JL transposase antibody to a final concentration of 0.375 ng / ml with the second diluent, add 100 μl of the standard to each well, seal the plate with a sealing film, and incubate at room temperature for 2 hours on an oscillator at 750 ± 50 rpm

[0149] 4) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0150] 5) Dilute Streptavidin (HRP) with the second diluent at a ratio of 1:4000 and incubate at room temperature with an oscillator at 750±50 rpm for 45 minutes.

[0151] 6) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0152] 7) Add TMB colorimetric solution, 100 μl / well, and color development at room temperature in the dark for 15-20 minutes.

[0153] 8) Add 50 μl / well of stop solution to terminate the reaction.

[0154] The standard curve conformity screening was performed to screen out antibody pairs that conformed to the standard curve. The results are shown in Tables 4, 5 and Figure 1-6 Table 4 shows the results of all antibody pairs, and Table 5 shows further experimental results of 6 antibody pairs. Figure 1-6 The standard curves of 6 pairs of antibodies are shown separately.

[0155] Table 4

[0156]

[0157] Table 5

[0158]

[0159]

[0160] According to the results of Table 5 and the standard curve, 6 pairs of antibodies were determined. As shown in Table 6, excellent effects can be obtained by using these 6 pairs of antibodies as antibody pairs in sandwich ELISA.

[0161] Table 6

[0162] Number pair Capture Antibody Detection antibodies Sensitivity 1 11D1F1 18B4H8-biotin: 1ug / ml 0-60pg / ml 2 5D5D7 18B4H8-biotin: 0.5ug / ml 0-80pg / ml 3 18B4H8 11D1F1-biotin:0.5ug / ml 0-120pg / ml 4 14E6A9 10D8F1-biotin:0.5ug / ml 0-120pg / ml 5 10D8F1 11D1F1-biotin:0.5ug / ml 0-160pg / ml 6 14E6A9 18B4H8-biotin: 1ug / ml 0-240pg / ml

[0163] Partial sequences herein:

[0164] SEQ ID NO:31 - Polypeptide antigen 1

[0165] KANSKGWMDEEKMSC

[0166] SEQ ID NO:32 - Polypeptide antigen 2

[0167] SFRGNKARWPQLEDC

[0168] SEQ ID NO:33

[0169] EVLLQQSGAELVKPGASVKLSCTVSGFNIKDSYIHWVKQRPGQGLEWIGRIFPANGNTKYDPKFQVKATLTADTSSNTASLQLSSLTSEDTAVYYCARPYDYLFDYWGQGTTLTVSS

[0170] SEQ ID NO:34

[0171] DIKMTQSPSSMYASLGERVTITCKASQDIKSYLSWYQQKPWKSPKTLIYYATDLADGVPSRFSGSGSGQDYSLTISSLESDDIAIYYCLQHRESPLTFGAGTKLELK

[0172] SEQ ID NO:35

[0173] QVQLKESGPGLVAPSQSLSITCTVSGFSLTSFGVHWVRQPPGKGLEWLGVIWPGGNTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAREDDGYYDAMDYWGQGTSVTVSS

[0174] SEQ ID NO:36

[0175] DIVMTQSHKFMSTSVGDRVSITCKASQDVDSAVAWYQQNPGQSPKLVIYSASYRYTGVPDRFTGSGSGTDFTLTISSVQAGDLAVYYCQQHYSPPPTFGGGTKLEIK

[0176] SEQ ID NO:37

[0177] DVQLQESGPGLVKPSQSLSLTCTVTGHSITSDYAWNWIRQFPGNKLEWMGYINYSGTTTYNPS LKSRISITRDTFKNQFFLQLNSVTSEDTATYYCARREDYRYGDAMDFWGQGTSVTVSS

[0178] SEQ ID NO:38

[0179] DIVLTQSPASLTVSLGQRATISCRASQSVSTSTYNYMHWYQQKPGQPPKLLINYASNLESGVPA RFSGSGSGTDFTLNIHPVEEEDTATYYCQHTWEIPWTFGGGTKLEIQ

[0180] SEQ ID NO:39

[0181] QVQLKESGPGLVAPSQSLSITCTVSGFSLTSFGVHWVRQPPGKGLEWLGVIWPGGSTNYNSAL MSRLSISKDNSKSQVFLKMSSLQTDDTAMYYCAREDDGYYDAMDYWGQGTSVTVSS

[0182] SEQ ID NO:40

[0183] DIVMTQSHKFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPELLIYSASYRYTGVPDRFT GSGSETDFTFTISSVQAEDLAVYYCQQHYSPPPTFGGGTKLEIK

[0184] SEQ ID NO:41

[0185] EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRVDPANGDSRYDPK FQGKATVTADTSSNTAYLHLSSLTSADTGVYYCARGVLRSYYTLDYWGQGTSVTVSS

[0186] SEQ ID NO:42

[0187] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYVATNLADGVPSRFSG SGSGTQYSLKINSLQSEDFGTYYCQHFWDTPFTIGSGTKLEIK

[0188] SEQ ID NO:43

[0189] MAPTKRHAYNAEFKLKAISHAQEHGNRAAAREFNINESMVRKWRKYEDELRQVKKTKKAFRGNKARWPQLEDKVEQWVAEQRAASRSVSTVTIRMKAIALAREHNISEFRGGPSWCFRFMKRRHLSIRTRTTVSQQLPADYQEKLATFRTYCRNKITEKKIQPEHIINMDEVPLTFDIPVNRTVDKTGARTVNIRTTGNEKTSFTVVLACQANGHKLPPMVIFKRKTLPKENFPAGIVIKANSKGWMDEEKMSEWLREIYVKRPGGFFHTAPSLLIYDSMRAHITEHVKKQVKHTNSVLAVIPGGLTKELQPLDVGVNRAFKARLRTAWEQWMTEGEHTFTKTGRQRRTTYANICKWIVNAWAGISVTTVVRAFRKAGIVTELPDNSSDTDSVNDDFDKTEPGVLDAAIAQLFNSDTEEEVFEGF

[0190] SEQ ID NO:44

[0191] MAPTKRHAYNAEFKLKAISHAQEHGNRAAAREFNINESMVRKWRKYEDELRQVKKTTQSFRGNKARWPQLEDKVEQWVAEQRAASRSVSTVTIRMKAIALAREHNISEFRGGPSWCFRFMKRRHLSIRTR

[0192] SEQ ID NO:45

[0193] MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGASTGSLVPRGSASENLYFQGMGTLEMAPTKRHAYNAEFKLKAISHAQEHGNRAAAREFNINESMVRKWRKYEDELRQVKKTTQSFRGNKARWPQLEDKVEQWVAEQRAASRSVSTVTIRMKAIALAREHNISEFRGGPSWCFRFMKRRHLSIRTRTTVSQQLPADYQEKLATFRTYCRNKITEKKIQPEHIINMDEVPLTFDIPVNRTVDKTGARTVNIRTTGNEKTSFTVVLACQANGHKLPPMVIFKRKTLPKENFPAGIVIKANSKGWMDEEKMSEWLREIYVKRPGGFFHTAPSLLIYDSMRAHITEHVKKQVKHTNSVLAVIPGGLTKELQPLDVGVNRAFKARLRTAWEQWMTEGEHTFTKTGRQRRTTYANICKWIVNAWAGISVTTVVRAFRKAGIVTELPDNSSDTDSVNDDFDKTEPGVLDAAIAQLFNSDTEEEVFEGF

[0194] SEQ ID NO:46

[0195] MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVVLSAGINAASPNKEFLENYLLTDEGLAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINASGRQTVDEALKDAQTGASTGSLVPRGSASENLYFQG

Claims

1. A JL transposase binding molecule, comprising an antibody or an antigen-binding fragment thereof targeting JL transposase, wherein the antibody comprises any one group of CDRs selected from the following: (A1) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, LCDR3 shown in SEQ ID NO: 18; (A2) HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:5, HCDR3 shown in SEQ ID NO:6, LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20, LCDR3 shown in SEQ ID NO:21; (A3) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24; (A4) HCDR1 shown in SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:25, LCDR2 shown in SEQ ID NO:26, LCDR3 shown in SEQ ID NO:27; (A5) HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, LCDR3 shown in SEQ ID NO:

30.

2. The JL transposase binding molecule according to claim 1, characterized in that The heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 33, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 34, The heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 35, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 36, The heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 37, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 38, The heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 39, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 40, or The heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:41, and / or the light chain variable region of the antibody has the sequence shown in SEQ ID NO:

42.

3. The JL transposase binding molecule according to claim 1, characterized in that The JL transposase binding molecule further comprises a mouse IgG Fc segment, a rabbit IgG Fc segment or a human IgG Fc segment.

4. A JL transposase binding molecule pair, wherein the first JL transposase binding molecule is as described in any one of claims 1 to 3, the second JL transposase binding molecule is as described in any one of claims 1 to 3, and the first JL transposase binding molecule and the second JL transposase binding molecule have different CDR groups, Preferably, the first JL transposase binding molecule comprises the CDRs shown in any one of A1-A5, the second JL transposase binding molecule comprises the CDRs shown in A1, A3 or A5, and the CDR groups of the first JL transposase binding molecule and the second JL transposase binding molecule are different, More preferably, the first JL transposase binding molecule and the second JL transposase binding molecule respectively comprise CDRs shown in any one of the following groups: A1 and A3, A2 and A3, A3 and A1, A4 and A5, A5 and A4, A4 and A3.

5. The JL transposase binding molecule pair according to claim 4, characterized in that: The first JL transposase-binding molecule comprises: a heavy chain variable region as shown in SEQ ID NO:33 and a light chain variable region as shown in SEQ ID NO:34, a heavy chain variable region as shown in SEQ ID NO:35 and a light chain variable region as shown in SEQ ID NO:36, a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:38, a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40, or a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:42; the second JL transposase-binding molecule comprises: a heavy chain variable region as shown in SEQ ID NO:33 and a light chain variable region as shown in SEQ ID NO:34, a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:38, or a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40, and the first JL transposase-binding molecule and the second JL transposase-binding molecule have different heavy chain variable regions and light chain variable regions, Preferably, The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:33 and a light chain variable region as shown in SEQ ID NO:34, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:38, The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:35 and a light chain variable region as shown in SEQ ID NO:36, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:38, The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:38, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:33 and a light chain variable region as shown in SEQ ID NO:34, The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:42, The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:42, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40, or The first JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40, and the second JL transposase binding molecule comprises a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:

38.

6. The JL transposase binding molecule pair according to claim 4, characterized in that: The second JL transposase binding molecule further comprises a marker, Preferably, the label comprises any one selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent labels, ultrasound contrast agents, photosensitizers or enzymes.

7. A kit for detecting JL transposase, comprising the JL transposase binding molecule according to any one of claims 1 to 3 or the JL transposase binding molecule pair according to any one of claims 4 to 6, The kit also includes reagents for detecting binding of the JL transposase to a JL transposase binding molecule or a pair of JL transposase binding molecules.

8. The kit according to claim 7, characterized in that The JL transposase binding molecule is used for sandwich detection, and the kit comprises the JL transposase binding molecule pair. Preferably, The first JL transposase binding molecule is coupled to a support, and / or The second JL transposase binding molecule comprises a label, and / or The weight of the first JL transposase binding molecule is greater than the weight of the second JL transposase binding molecule, and / or The weight ratio of the first JL transposase binding molecule to the second JL transposase binding molecule is 5:1-4, preferably 5:2-3.

9. The kit according to claim 7, characterized in that The kit comprises a reaction plate coated with a first JL transposase binding molecule, a second JL transposase binding molecule labeled with biotin, a catalytic enzyme labeled with avidin, and a substrate. Preferably, the kit further comprises any one or more of a JL transposase standard, a sample diluent, a washing solution and a stop solution.

10. A non-diagnostic method for detecting the presence of JL transposase in a sample, the method comprising: The JL transposase binding molecule according to any one of claims 1 to 3 or the JL transposase binding molecule pair according to any one of claims 4 to 6 is incubated with the sample, and the binding of JL transposase to the JL transposase binding molecule or the JL transposase binding molecule pair is detected to determine the presence of JL transposase in the sample.

Images