Jl transposase monoclonal antibody and its detection kit

Abstract

The present application relates to a kind of JL transposase monoclonal antibody and its detection kit. Specifically provide a kind of JL transposase binding molecule, comprising antibody or its antigen-binding fragment targeted to JL transposase, the antibody contains any one group CDR described herein. The detection kit provided by the present application has high specificity, high sensitivity and good stability.

Description

Technical Field

[0001] This invention relates to the field of biology; more specifically, this invention relates to a JL transposase monoclonal antibody and its detection kit. Background Technology

[0002] DNA transposons are a class of mobile DNA sequences that can freely jump (replicate or translocate) within the genome. They can be transposed from one location to another through a series of processes such as cutting and reintegration. They were first discovered by Mc. Clintock in the 1940s in maize chromosomes, and were subsequently found in various organisms, including bacteria, fungi, and insects.

[0003] Besides significantly improving the efficiency of transgenic animal production based on microinjection technology, transposons can also be used in gene therapy. In gene therapy, transposon-mediated gene transfer offers advantages such as safety and high efficiency. Over the past decade, transposons have become the most commonly used non-viral vector in gene therapy. Furthermore, transposons can be used to create mutants, conduct gene capture, and apply to functional genomics research. This technology has made significant progress in recent years in human gene therapy, transgenic animal production in model organisms such as mice and zebrafish, and in functional genomics research.

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

[0005] During gene writing using the JL transposon system, JL transposase residues may remain. Therefore, a method for detecting JL transposase is needed for quality control. Summary of the Invention

[0006] In view of the problems existing in the prior art, this application provides a monoclonal antibody of JL transposase, a detection kit containing the antibody based on the ELISA method, and successfully completed the quantitative detection of JL transposase using the kit.

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

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

[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 contains: 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 contains: 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 contains: 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, and 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, 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 mouse or human light chain FR region. In one or more embodiments, the heavy chain variable region of the antibody comprises a mouse or human heavy chain FR region.

[0015] In one or more embodiments, the heavy chain variable regions FR1, FR2, FR3 and FR4 of the antibody are each independently selected from the heavy chain variable regions FR1, FR2, FR3 and FR4 shown in any one of the sequences SEQ ID NO:33, 35, 37, 39, 41, and / or, the light chain variable regions FR1, FR2, FR3 and FR4 of the antibody are each independently selected from the light chain variable regions FR1, FR2, FR3 and FR4 shown in any one of the sequences SEQ ID NO:34, 36, 38, 40, 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 with it, 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 with it.

[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 with it, 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 with it.

[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 with it, 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 with it.

[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 with it, 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 with it.

[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 with it, 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 with it.

[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 includes a mouse IgG Fc fragment, a rabbit IgG Fc fragment, or a human IgG Fc fragment.

[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 with it. Alternatively or additionally, the light chain of the antibody comprises a light chain constant region of human κ or λ chains, or a sequence having at least 85% sequence identity with it.

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

[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] This invention also provides polynucleotides selected from:

[0029] (1) The coding sequence of the JL transposase binding molecule described in any of the embodiments herein;

[0030] (2)(1) complementary sequences.

[0031] The present invention also provides a nucleic acid construct comprising the polynucleotides described in any of the embodiments 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, wherein:

[0034] (1) Expressing and / or secreting the JL transposase-binding molecule as described in any of the embodiments herein;

[0035] (2) Contains the polynucleotides described herein; and / or

[0036] (3) Includes the nucleic acid constructs described in this article.

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

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

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

[0040] A 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 any one of the CDRs represented by group A1-A5, and the second JL transposase binding molecule comprises any one of the CDRs represented by group A1-A5. The CDRs in group A1-A5 are as described in the first aspect herein.

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

[0043] In one or more embodiments, the first JL transposase binding molecule and the second JL transposase binding molecule each comprise any one of the following CDRs: 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: the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, the heavy chain variable region shown in SEQ ID NO:35 and the light chain variable region shown in SEQ ID NO:36, the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38, the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO:40, or the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:42; the second JL transposase-binding molecule comprises: the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, the heavy chain variable region shown in SEQ ID NO:35 and the light chain variable region shown in SEQ ID NO:36, the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38, the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO:40, or the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:42; The heavy chain variable region shown in NO:41 and the light chain variable region shown in SEQ ID NO:42 are different from the heavy chain variable region and light chain variable region of the first JL transposase binding molecule and the second JL transposase binding molecule.

[0045] In one or more embodiments, the first JL transposase-binding molecule comprises: the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, the heavy chain variable region shown in SEQ ID NO:35 and the light chain variable region shown in SEQ ID NO:36, the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38, the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO:40, or the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:42; the second JL transposase-binding molecule comprises: the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38, or the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO:40, or the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:42. 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 the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, and the second JL transposase-binding molecule comprises the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38.

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

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

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

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

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

[0052] In one or more embodiments, the second JL transposase binding molecule further comprises a label. The label includes any one of the following: fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense materials, chemiluminescent labels, ultrasound contrast agents, photosensitizers, or enzymes.

[0053] In one or more embodiments, the fluorescent material 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, and 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, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitation compounds, Tribispyridyldiamine europium, europium cavitation compounds or chelates, Diamine, Dianthocyanin, La Jolla Blue dye, Allococyanin B. Phycocyanin C. Phycocyanin R. Thiamine, Phycoerythrin, Phycoerythrin R. REG. Rhodamine Green. Rhodamine Isothiocyanate. Rhodamine Red. ROX. TAMRA. TET. TRIT (Tetramethylrhodamine isothiol). Tetramethylrhodamine. 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 internally encapsulated with rare-earth fluorescent ions europium.

[0056] In one or more embodiments, the marker is biotin. This invention employs a biotin-avidin system for colorimetric development, improving sensitivity. Avidin and biotin have extremely strong affinity; their binding affinity constant is at least 10,000 times higher than that of antigen and antibody. Therefore, they can bind rapidly, and the reaction is unaffected by external interference.

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

[0058] In one or more embodiments, the kit further includes reagents for detecting the binding of JL transposase to a JL transposase-binding molecule or a pair of JL transposase-binding molecules. For example, reagents for detecting the binding by an enzyme-linked immunosorbent assay (ELISA).

[0059] In one or more embodiments, the detection binding reagent is a detectable marker, such as biotin, that can be linked to a JL transposase-binding molecule or a JL transposase-binding molecule pair. The detectable marker is either linked to the antibody or its antigen-binding fragment or is present separately in the kit.

[0060] In one or more embodiments, the JL transposase binding molecule is used for sandwich assay, and the kit comprises the JL transposase binding molecule pair described in any embodiment of the second aspect herein. The sandwich assay is performed in the form of “support-first JL transposase binding molecule-analyte-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 the support.

[0062] In one or more embodiments, the support material includes, but is not limited to, enzyme-linked immunosorbent assay (ELISA) plates, microspheres, microparticles, glass slides, chips, plastics, films, etc., made of materials such as polystyrene, polyethylene, cellulose, nitrocellulose, cellulose acetate, and silicon compounds.

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

[0064] In one or more embodiments, the kit further includes an identification signal, the marker and the identification signal being colorimetrically expressed via a biotin-avidin system.

[0065] In one or more embodiments, the identification signal includes an avidin-labeled material selected from any of the following: fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense materials, chemiluminescent labels, ultrasound contrast agents, photosensitizers, or enzymes.

[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 colorimetric solution vary; generally, the standard is that the OD value of the highest concentration standard is around 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 biotin-labeled second JL transposase binding molecule, an avidin-labeled catalytic enzyme, and a substrate.

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

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

[0072] This invention also provides a non-diagnostic method for detecting the presence of JL transposase in a sample. The method includes: incubating the sample with a JL transposase binding molecule or a JL transposase binding molecule pair as described in any embodiment herein, and detecting the binding of the JL transposase to the JL transposase binding molecule or the JL transposase binding molecule pair, thereby determining the presence of JL transposase in the sample. The detection is performed using an enzyme-linked immunosorbent assay (ELISA).

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

[0074] (1) Couple the first JL transposase-binding molecule to the support, wash, and block as desired.

[0075] (2) The first JL transposase binding molecule coupled to the support is brought into contact with the sample. The JL transposase in the sample binds to the first JL transposase binding molecule to form a complex. Washing is optional.

[0076] (3) Contact the labeled second JL transposase binding molecule with the complex, so that the JL transposase in the complex binds to the second JL transposase binding molecule, and optionally wash.

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

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

[0079] (1) Coating: The first JL transposase binding molecule is coated onto the support and then washed.

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

[0081] (3) First JL transposase binding molecule captures 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 includes any one selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, 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 identification signal includes any of 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 colorimetric reaction of the substrate catalyzed by 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.

[0087] Compared with existing technologies, the JL transposase detection kit provided by this invention has high detection specificity, high sensitivity, and good stability. Attached Figure Description

[0088] Figures 1-6 The standard curve for the sandwich ELISA of the antibody pairs in Table 5 is shown. Detailed Implementation

[0089] Unless otherwise defined, the present invention will be practiced using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of the art. These techniques are well explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Animal Cell Culture (edited by R.R. Freshney, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., 1987 edition and its periodically updated editions); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0090] In this article, "JL transposase-binding molecule" refers to a protein that specifically binds to JL transposase, including but not limited to antibodies, antigen-binding fragments of antibodies, nanobodies, microbodies, affinity molecules, target-binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

[0091] In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, and Fv. In this document, 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 either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0093] The term "variable" refers to the situation where certain segments within a variable domain exhibit significant differences within the antibody sequence, specifically the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the light chain variable regions LCDR1, LCDR2, and LCDR3. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4). Typically, the structure of the variable region in the light chain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the variable region in the heavy chain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. For chimeric antibodies, the FR regions can be non-human (e.g., murine). The antibody CDR can be determined using various coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence numbering of the antigen-binding protein can follow the IMGT numbering scheme.

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

[0095] In one or more embodiments, the antibody described herein further includes a heavy chain constant region and / or a light chain constant region, the heavy chain constant region being, for example, 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 the light chain constant region being, for example, a light chain constant region of the κ or λ chain, or a sequence thereof having at least 85% sequence identity.

[0096] The antibodies of this invention can be prepared using methods conventional in the art, such as hybridoma technology, phage display technology, and cell line expression well-known in the art. This application also provides fusion proteins that contain the antibody or antigen-binding fragment described in this application as an expression target, active molecule, or targeting molecule. For example, fusion proteins formed by adding tags (e.g., His6 tags) to both ends of an antibody to facilitate protein expression or purification. The tags do not affect the function of the target protein and can be easily cleaved.

[0097] This invention also provides polynucleotides encoding the binding molecules, binding molecule pairs, and fusion proteins described herein. This invention provides polynucleotides encoding the heavy chain variable region, the light chain variable region, the heavy chain, the light chain, and each CDR. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. DNA can be single-stranded or double-stranded. This invention also includes degenerate variants of polynucleotide sequences encoding antibodies or their antigen-binding fragments, fusion proteins, or antibody conjugates, i.e., nucleotide sequences encoding the same amino acid sequence but different nucleotide sequences.

[0098] Based on the amino acid sequence and codons, those skilled in the art can readily obtain the coding sequence of an antibody or its fragments. Furthermore, codon optimization can be used to alter the expression of peptides in different species; codon preferences for different species are a conventional technique in this field.

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

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

[0101] Example

[0102] Example 1: Preparation of JL transposase monoclonal antibody

[0103] 1. Immunogen Preparation: Two polypeptide antigens were prepared as primary immunogens, with sequences SEQ ID NO:31 and SEQ ID NO:32, respectively. The synthesized polypeptides were conjugated with KLH carrier proteins. A truncated JL transposase (wtPS1-130aa) was prepared as a booster immunization protein for a third immunization. The sequence of the truncated JL transposase is shown in SEQ ID NO:44.

[0104] 2. Animal Immunization: A standard immunization protocol was used, selecting 5 Balb / c mice aged 6-8 weeks. Two peptides were used as immunogens, mixed with Freund's complete adjuvant at a 1:1 ratio, and administered as the first immunization at a dose of 50 μg / mouse. Two weeks later, a second immunization was performed, with the peptides mixed with Freund's incomplete adjuvant at a 1:1 ratio and administered at a dose of 25 μg / mouse. Two weeks later, a third immunization was performed, with the peptides mixed with Freund's incomplete adjuvant at a 1:1 ratio and administered at a dose of 25 μg / mouse. A final immunization was performed on day 50±7. Four days after the final immunization, the animals were confirmed to have died by carbon dioxide asphyxiation followed by cervical dislocation, and cell fusion was performed. The titer of the polyclonal antibodies was detected using an indirect ELISA method. The antibody-coated peptide immunogens and the full-length protein were detected separately using an indirect ELISA method, and the level of immune response was determined by detecting the serum of the immunized animals.

[0105] 3. Cell Fusion and Screening: Two groups of animals immunized with the peptides underwent one round of cell fusion using an electrofusion method. All cells from each fusion round were seeded into 96-well plates. After fusion, initial screening was performed using indirect ELISA to select supernatants from the fused cells that were positive for the peptide antigen. Confirmatory screening was then conducted, using indirect ELISA to select positive progenitor clones from the positive clones obtained in the initial screening stage that were positive for the full-length protein. Mouse positive serum and unfused cell wells were used as control groups, resulting in the selection of 5 positive clones.

[0106] 4. Subcloning, Expansion Culture, and Cryopreservation: Using the limiting dilution method, cells in 96-well plates were diluted with DMEM medium. Five clones were subcloned into one 96-well plate at a density of 3 cells / well and cultured in a CO2 incubator. Subcloning was performed on the double-positive parent clones (peptide and protein), and subcloning was screened using indirect ELISA. The selected daughter clones were then amplified and stored in liquid nitrogen.

[0107] 5. Antibody Production: Selected monoclonal antibodies were used for antibody production. The antibodies were purified using Protein A affinity chromatography and then stored in phosphate-buffered saline (PBS) via dialysis. Quality control was performed using polyacrylamide gel electrophoresis (SDS-PAGE) and indirect ELISA, and antibody concentration was determined using a NanoDrop 2000.

[0108] 6. Antibody sequencing: Selected subclones are sequenced using Sanger / NGS sequencing.

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

[0110] The heavy chain CDR sequences of the antibody are shown in SEQ ID NO:1-15. The light chain CDR sequences are shown in SEQ ID NO:16-30.

[0111] Table 1: Antibody Sequences

[0112]

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

[0114] The binding of purified mouse monoclonal antibody to antigen in the supernatant was detected using an indirect ELISA method:

[0115] 1. The antigen (A, PS transposase-peptide 1, B, truncated JL transposase (wtPS1-130aa), C, MBP-JL (wt-ps) full-length protein, D, MBP-irrelevant protein; sequences as shown in SEQ ID NO: 43-46) was immobilized on a solid-phase support and blocked 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 the substrate chromogenic solution, incubate in the dark, then add the stop solution, and measure the OD value using a microplate reader with a wavelength of 450 nm.

[0119] In addition, antigens A, B, C, and D were obtained through expression and purification using the pMBP-His vector.

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

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

[0122] 1. The antigens (PS transposase-peptide 1, MBP-JL (wt-ps) full-length protein, JL transposase, MBP irrelevant protein) were immobilized onto a solid-phase carrier and then coated and blocked.

[0123] 2. Biotin-labeled antibodies 18B4H8, 11D1F1, 5D5D7, 14E6A9, and 10D8F1 were added as primary antibodies and incubated at 37°C for 60 minutes to allow them to react with the coated antigen.

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

[0125] 4. Add the substrate chromogenic solution, incubate in the dark, then add the stop solution, and measure the OD value using a microplate reader with a wavelength of 450 nm.

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

[0127]

[0128]

[0129] Example 4: JL transposase detection kit sandwich ELISA results

[0130] This embodiment uses the above-described antibody to prepare a sandwich ELISA kit and to generate 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, streptavidin-horseradish peroxidase coupler (Streptavidin-HRP), TMB chromogenic solution, standard sample JL transposase (wt-ps, 1-130aa), sample dilution solution, second JL transposase binding molecule and streptavidin-HRP dilution solution, washing solution and stop solution;

[0132] Coating solution: Dilute 20×PBS to 1×PBS, purchased from Sangon Biotech, catalog number: B548117-0500;

[0133] Termination solution: purchased from Solarbio, product number: C1058;

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

[0135] Blocking fluid: Thermo Blocker™ Casein, purchased from Thermo, catalog number: 37528;

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

[0137] Second dilution: one-third Thermo Blocker™ Casein + two-thirds PBST;

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

[0139] Streptavidin-HRP: Purchased from Biolegend, item number: 405210;

[0140] The reaction plate coated with antibody against the first JL transposase was prepared using the following method:

[0141] 1) Dilute the antibody to 2.5 μg / ml with coating buffer, coat 100 μl / well of enzyme-labeled reaction plate, and incubate overnight at 4°C.

[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] The standard curve was prepared using the kit provided above, and the steps are as follows:

[0146] 1) Dilute the standard with 20 ng / ml of the first solution, set 10 gradients (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 standard to each well, seal the plate with sealing film, and incubate at room temperature for 2 hours with a shaker 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 biotin-labeled JL transposase antibody to a final concentration of 0.375 ng / ml using the second dilution buffer. Add 100 μl of standard to each well, seal the plate with sealing film, and incubate at room temperature for 2 hours using a shaker 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 for 45 minutes with a shaker at 750±50 rpm.

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

[0152] 7) Add 100 μl of TMB developer to each well and incubate at room temperature in the dark for 15-20 minutes.

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

[0154] Standard curve compliance screening was performed to identify antibody pairs that met the standard curve requirements. The results are shown in Tables 4 and 5. Figure 1-6 As shown in the table. Table 4 shows the results for all antibody pairs, and Table 5 shows the further experimental results for 6 antibody pairs. Figure 1-6 The standard curves for the six pairs of antibodies are shown separately.

[0155] Table 4

[0156]

[0157] Table 5

[0158]

[0159]

[0160] Based on the results in Table 5 and the standard curve, six pairs of antibodies were identified, as shown in Table 6. Using these six pairs of antibodies as antibody pairs in sandwich ELISA can achieve excellent results.

[0161] Table 6

[0162] 1 11D1F1 18B4H8-biotin: 1ug / ml 0-60 pg / ml 2 5D5D7 18B4H8-biotin: 0.5ug / ml 0-80 pg / 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 of the present invention:

[0164] SEQ ID NO: 31-Peptide Antigen 1

[0165] KANSKGWMDEEKMSC

[0166] SEQ ID NO: 32-Peptide Antigen 2

[0167] SFRGNKARWPQLEDC

[0168] SEQ ID NO: 33

[0169] EVLLQQSGAELVKPGASVKLSCTVSGFNIKDSYIHWVKQRPGQGLEWIGRIFPANGNTKYDPKF QVKATLTADTSSNTASLQLSSLTSEDTAVYYCARPYDYLFDYWGQGTTLTVSS

[0170] SEQ ID NO: 34

[0171] DIKMTQSPSSMYASLGERVTITCKASQDIKSYLSWYQQKPWKSPKTLIYYATDLADGVPSRFSG SGSGQDYSLTISSLESDDIAIYYCLQHRESPLTFGAGTKLELK

[0172] SEQ ID NO: 35

[0173] QVQLKESGPGLVAPSQSLSITCTVSGFSLTSFGVHWVRQPPGKGLEWLGVIWPGGNTNYNSAL MSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAREDDGYYDAMDYWGQGTSVTVSS

[0174] SEQ ID NO: 36

[0175] DIVMTQSHKFMSTSVGDRVSITCKASQDVDSAVAWYQQNPGQSPKLVIYSASYRYTGVPDRFT GSGSGTDFTLTISSVQAGDLAVYYCQQHYSPPPTFGGGTKLEIK

[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 the JL transposase, said antibody containing any one of the following CDRs: (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, and 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, and 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, and 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, and LCDR3 shown in SEQ ID NO:

30.

2. The JL transposase-binding molecule as described in claim 1, characterized in that, The heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:33, and 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 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 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 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 the light chain variable region of the antibody has the sequence shown in SEQ ID NO:

42.

3. The JL transposase-binding molecule as described in claim 1, characterized in that, The JL transposase binding molecule also includes mouse IgG Fc fragment, rabbit IgG Fc fragment, or human IgG Fc fragment.

4. A JL transposase binding molecule pair, wherein the first JL transposase binding molecule is as described in any one of claims 1-3, the second JL transposase binding molecule is as described in any one of claims 1-3, and the CDR groups of the first JL transposase binding molecule and the second JL transposase binding molecule are different.

5. The JL transposase binding molecule pair as described in claim 4, characterized in that, The first JL transposase binding molecule contains any of the CDRs shown in A1-A5, and the second JL transposase binding molecule contains 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.

6. The JL transposase binding molecule pair as described in claim 5, characterized in that, The first JL transposase binding molecule and the second JL transposase binding molecule each contain any of the following sets of CDRs: A1 and A3, A2 and A3, A3 and A1, A4 and A5, A5 and A1, A4 and A3.

7. The JL transposase binding molecule pair according to any one of claims 4-6, characterized in that, The first JL transposase-binding molecule comprises: the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, the heavy chain variable region shown in SEQ ID NO:35 and the light chain variable region shown in SEQ ID NO:36, the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38, the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO:40, or the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:42; the second JL transposase-binding molecule comprises: the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38, or the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:42, 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.

8. The JL transposase binding molecule pair as described in claim 7, characterized in that, The first JL transposase-binding molecule contains the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, and the second JL transposase-binding molecule contains the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:

38. The first JL transposase-binding molecule contains the heavy chain variable region shown in SEQ ID NO:35 and the light chain variable region shown in SEQ ID NO:36, and the second JL transposase-binding molecule contains the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:

38. The first JL transposase-binding molecule contains the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:38, and the second JL transposase-binding molecule contains the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:

34. The first JL transposase-binding molecule contains the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO:40, and the second JL transposase-binding molecule contains the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:

42. The first JL transposase binding molecule contains the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:42, and the second JL transposase binding molecule contains the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:34, or The first JL transposase binding molecule contains the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO:40, and the second JL transposase binding molecule contains the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:

38.

9. The JL transposase binding molecule pair according to any one of claims 4-6, characterized in that, The second JL transposase binding molecule also contains a marker.

10. The JL transposase binding molecule pair as described in claim 9, characterized in that, The marker includes any one selected from fluorescent substances, radioactive contrast agents, electron-dense substances, or chemiluminescent markers.

11. The JL transposase binding molecule pair as described in claim 9, characterized in that, The label includes any one selected from quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, paramagnetic ion fluorescent microspheres, ultrasound contrast agents, photosensitizers, or enzymes.

12. A kit for detecting JL transposase, comprising the JL transposase binding molecule according to any one of claims 1-3 or the JL transposase binding molecule pair according to any one of claims 4-11, The kit also includes reagents for detecting the binding of JL transposase to JL transposase-binding molecules or JL transposase-binding molecule pairs.

13. The kit according to claim 12, characterized in that, The JL transposase binding molecule is used for sandwich assay, and the kit contains the JL transposase binding molecule pair.

14. The kit according to claim 13, characterized in that, The kit also includes one or more of the following features: The first JL transposase binding molecule is coupled to the support. The second JL transposase binding molecule contains a marker. The weight of the first JL transposase binding molecule is greater than the weight of the second JL transposase binding molecule. The weight ratio of the first JL transposase binding molecule to the second JL transposase binding molecule is 5:1-4.

15. The kit according to claim 14, characterized in that, The weight ratio of the first JL transposase binding molecule to the second JL transposase binding molecule is 5:2-3.

16. The kit according to any one of claims 12-15, characterized in that, The kit includes a reaction plate coated with a first JL transposase binding molecule, a biotin-labeled second JL transposase binding molecule, an avidin-labeled catalytic enzyme, and a substrate.

17. The kit according to claim 16, characterized in that, The kit also includes one or more of the following: JL transposase standard, sample diluent, washing solution, and stop solution.

18. A non-diagnostic method for detecting the presence of JL transposase in a sample, the method comprising: The presence of JL transposase in the sample is determined by incubating the sample with the JL transposase binding molecule or the JL transposase binding molecule pair as described in any one of claims 1-3 or any one of claims 4-11, and detecting the binding of JL transposase to the JL transposase binding molecule or the JL transposase binding molecule pair.

Images