A PB transposase monoclonal antibody and its detection kit

Abstract

The present invention relates to the field of antibodies; more specifically, to a PB transposase monoclonal antibody and a detection kit thereof. A PB transposase binding molecule is specifically provided, comprising an antibody or an antigen-binding fragment thereof targeting PB transposase, wherein the antibody contains any set of CDRs described herein. The detection kit provided by the present invention has strong detection specificity, high sensitivity and good stability.

Description

Technical Field

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

[0002] PB transposase is derived from the PiggyBac DNA transposon gene, which was originally discovered in the genome of the cabbage looper Trichoplusiani. PiggyBac is a "cut-and-paste" DNA transposon that can cut DNA and insert it into a new location in the genome in a "scarless transposition" manner.

[0003] PB transposase is a key enzyme to achieve this movement process. It recognizes the terminal repeat element (TRE) of the PiggyBac transposon and catalyzes the shearing and integration of the transposon DNA. PB transposase has the characteristics of carrying large DNA transgenic fragments without leaving transposition marks, and is a widely used transgenic tool in cell and gene therapy.

[0004] In the process of cell and gene therapy, researchers usually introduce PB transposons and PB transposases into cells separately, and introduce or remove specific gene fragments in the genome through the activity of transposases. Among them, PB transposases are usually expressed by nucleic acids in the form of DNA plasmids or mRNA. Cell therapy and gene therapy generally require that these transposases can be degraded as quickly and / or completely as possible after performing their transgenic functions, so as to maximize the safety of gene therapy. In view of the above reasons, monitoring the expression and residues of these transposase proteins during the treatment cycle is an important analytical indicator.

[0005] It is known that transposase protein is a protein rich in positively charged amino acids. The expression of functional natural full-length protein is an important challenge in the industry, which makes it difficult to obtain monoclonal antibodies based on natural structure PB protein. Antibodies with high specificity for natural structure PB protein with biological enzyme activity are the precursor conditions for achieving extremely low-limit residual detection of transposase in cells or in vivo. The present invention obtains a pair of highly specific antibodies for PB natural structure protein in a series of mouse monoclonal antibodies, indicating that it has very high sensitivity and low detection limit. Summary of the invention

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

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

[0008] In some 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:5, LCDR2 shown in SEQ ID NO:6, LCDR3 shown in SEQ ID NO:7;

[0010] (A2) HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:11, LCDR1 shown in SEQ ID NO:13, LCDR2 shown in SEQ ID NO:14, and LCDR3 shown in SEQ ID NO:15.

[0011] In some embodiments, the light chain variable region of the antibody comprises a murine or human light chain FR region. In some embodiments, the heavy chain variable region of the antibody comprises a murine or human heavy chain FR region.

[0012] In some 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 SEQ ID NO: 4 or 12, 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 SEQ ID NO: 8 or 16.

[0013] In some embodiments, the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:4, 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:8, or a sequence having at least 85% sequence identity thereto.

[0014] In some embodiments, the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:12, 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:16, or a sequence having at least 85% sequence identity thereto.

[0015] In some embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0016] In some embodiments, the antibody further comprises a mouse IgG Fc segment, a rabbit IgG Fc segment, or a human IgG Fc segment.

[0017] In some 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 thereto. 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 thereto.

[0018] In some 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.

[0019] In some embodiments, the antibody is a monoclonal antibody.

[0020] In some embodiments, the antibody is a chimeric antibody or a murine antibody.

[0021] The present invention also provides a polynucleotide selected from:

[0022] (1) a coding sequence of a PB transposase binding molecule according to any embodiment of the present invention;

[0023] (2) The complementary sequence of (1).

[0024] The present invention also provides a nucleic acid construct, which comprises the polynucleotide described in any embodiment herein.

[0025] In some embodiments, the nucleic acid construct is a vector, such as an integration vector, a cloning vector, or an expression vector.

[0026] The present invention also provides a host cell, which:

[0027] (1) expressing and / or secreting the PB transposase binding molecule described in any embodiment of the present invention;

[0028] (2) comprising a polynucleotide as described herein; and / or

[0029] (3) comprising the nucleic acid construct described herein.

[0030] In some embodiments, the host cell is selected from a prokaryotic cell or a eukaryotic cell.

[0031] In some embodiments, the host cell is a mammalian cell.

[0032] The present invention also provides a method for producing a PB transposase binding molecule, comprising: culturing the host cell described herein under conditions suitable for producing the PB transposase binding molecule, and optionally purifying the PB transposase binding molecule from the culture.

[0033] A second aspect of the present invention provides a PB transposase binding molecule pair, wherein the first PB transposase binding molecule comprises the following CDRs: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, and LCDR1 shown in SEQ ID NO: 5, LCDR2 shown in SEQ ID NO: 6, and LCDR3 shown in SEQ ID NO: 7;

[0034] The second PB transposase binding molecule comprises the following CDRs: HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:11, LCDR1 shown in SEQ ID NO:13, LCDR2 shown in SEQ ID NO:14, and LCDR3 shown in SEQ ID NO:15.

[0035] In some embodiments, the first PB transposase binding molecule comprises: a heavy chain variable region shown in SEQ ID NO:4 and a light chain variable region shown in SEQ ID NO:8; the second PB transposase binding molecule comprises: a heavy chain variable region shown in SEQ ID NO:12 and a light chain variable region shown in SEQ ID NO:16.

[0036] In some embodiments, the first PB transposase binding molecule or the second PB transposase binding molecule further comprises a label, wherein 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 labels, ultrasound contrast agents, photosensitizers, or enzymes.

[0037] In some 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.

[0038] In some 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.

[0039] In some embodiments, the fluorescent microspheres are polystyrene fluorescent microspheres, which contain rare earth fluorescent ions europium.

[0040] In some embodiments, the marker 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.

[0041] The present invention also provides a kit for detecting PB transposase, wherein the kit comprises the PB transposase binding molecule or PB transposase binding molecule pair, polynucleotide, nucleic acid construct, bacteriophage or host cell described in any embodiment of the present invention.

[0042] In some embodiments, the kit further comprises a reagent for detecting the binding of PB transposase to a PB transposase binding molecule or a PB transposase binding molecule pair, for example, by detecting the binding by enzyme-linked immunosorbent assay.

[0043] In some embodiments, the reagent for detecting binding is a detectable label, such as biotin, that can be linked to the PB transposase binding molecule or PB transposase binding molecule pair. The detectable label is linked to the antibody or antigen-binding fragment thereof or exists separately in the kit.

[0044] In some embodiments, the PB transposase binding molecule is used for sandwich detection, and the kit comprises the PB 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 PB transposase binding molecule-object to be detected-second PB transposase binding molecule-marker" or "support-second PB transposase binding molecule-object to be detected-first PB transposase binding molecule-marker".

[0045] In some embodiments, the first PB transposase binding molecule is coupled to a support. In some embodiments, the kit further comprises a reagent for coupling the first PB transposase binding molecule to a support.

[0046] In some embodiments, the support includes, but is not limited to, ELISA plates, beads, microparticles, glass slides, chips, plastics, films, etc. made of materials such as polystyrene, polyethylene, cellulose, nitrocellulose, cellulose acetate, and silicate.

[0047] In some embodiments, the kit further comprises a recognition signal, and the label and the recognition signal are visualized by a biotin-avidin system.

[0048] In some embodiments, the recognition signal comprises any one of the following selected from the group consisting of avidin-labeled 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.

[0049] In some 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.

[0050] In some embodiments, the weight of the first PB transposase binding molecule is greater than the weight of the second PB transposase binding molecule.

[0051] In some embodiments, the weight ratio of the first PB transposase binding molecule to the second PB transposase binding molecule is 5:1-4, preferably 5:2-3.

[0052] In some embodiments, the kit includes a reaction plate coated with a first PB transposase binding molecule, a second PB transposase binding molecule labeled with biotin, a catalytic enzyme labeled with avidin, and a substrate.

[0053] 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 PB transposase binding molecule, that is, to achieve more sensitive detection with the help of a biotin-avidin amplification system.

[0054] In some embodiments, the kit further comprises any one or more of a PB transposase standard, a sample diluent, a washing solution, and a stop solution. Further, the washing solution is PBST.

[0055] The present invention also provides a non-diagnostic method for detecting the presence of PB transposase in a sample, the method comprising: incubating the sample with the PB transposase binding molecule or PB transposase binding molecule pair described in any embodiment of the present invention, and detecting the binding of PB transposase to the PB transposase binding molecule or PB transposase binding molecule pair, thereby determining the presence of PB transposase in the sample. The detection is enzyme-linked immunosorbent assay.

[0056] In some embodiments, the method is a sandwich method for detecting using the PB transposase binding molecule pair, the method comprising the steps of:

[0057] (1) coupling the first PB transposase binding molecule to the support, optionally washing, optionally blocking,

[0058] (2) contacting the first PB transposase binding molecule coupled to the support with the sample, so that the PB transposase in the sample binds to the first PB transposase binding molecule to form a complex, and optionally washing,

[0059] (3) contacting the complex with a second PB transposase binding molecule connected to a marker, so that the PB transposase in the complex binds to the second PB transposase binding molecule, and optionally washing.

[0060] (4) Detecting the PB transposase through a detectable reaction mediated by the marker.

[0061] In some embodiments, the method comprises the steps of:

[0062] (1) Coating: Coat the first PB transposase binding molecule onto the support and wash.

[0063] (2) Blocking: The empty sites on the support that are not coated with the first PB transposase binding molecule are blocked with an inert protein to prevent nonspecific adsorption in subsequent reactions.

[0064] (3) The first PB transposase binding molecule captures the antigen: After adding the sample to be tested and incubating, the first PB transposase binding molecule captures the antigen in the sample.

[0065] (4) Antigen capture of the second binding molecule: After adding the second PB transposase binding molecule and incubating, the antigen captures the second PB transposase binding molecule.

[0066] Detection: Detection is performed by introducing markers in one or more steps.

[0067] In some 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.

[0068] In some 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.

[0069] In some 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.

[0070] The PB transposase detection kit provided by the present invention has strong detection specificity, high sensitivity, good stability and accurate quantification. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This is the standard curve of natural PB transposase ELISA. DETAILED DESCRIPTION

[0072] 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 technical scope 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).

[0073] As used herein, "PB transposase binding molecules" are proteins that specifically bind to PB 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.

[0074] 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, such as Fab, F(ab')2 and Fv. Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0075] 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 generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.

[0076] The term "variable" refers to the situation where certain segments in the variable domain vary widely in antibody sequences, 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 domain 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 regions can be non-human (e.g., mouse).

[0077] Antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, comprehensive consideration of Kabat / Chothia, etc. These coding systems are known in the art. For example, the amino acid sequence numbering of the antigen-binding protein can be based on the IMGT numbering scheme.

[0078] "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.

[0079] In some 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.

[0080] 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 tag (such as a His6 tag) that facilitates protein expression or purification at both ends of the antibody. The tag does not affect the function of the target protein and can be easily removed.

[0081] 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.

[0082] 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.

[0083] The antibodies of the present invention can be used for determinations due to their high affinity for PB transposase, such as combined determinations to detect and / or quantify PB transposase. The present invention also provides a detection kit for detecting the level of PB transposase, which includes an anti-PB 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.

[0084] 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.

[0085] Example

[0086] Example 1: Preparation of monoclonal antibodies against PB transposase

[0087] 1. Preparation of immunogens: The inclusion body renatured PB transposase full-length protein with a His tag and the active natural PB transposase full-length protein with an MBP tag were prepared respectively. The amino acid sequence of the PB transposase is shown in SEQ ID NO: 17.

[0088] 2. Animal immunization: A conventional immunization scheme was adopted, and 5 Balb / c mice aged 6-8 weeks were selected. The inclusion body renatured PB transposase full-length protein was used as the immunogen, mixed with Freund's complete adjuvant at a ratio of 1:1, and then immunized at a dose of 50 μg / mouse for the first immunization, and injected subcutaneously at multiple points in the abdomen; after an interval of 2 to 3 weeks, the second immunization was carried out, mixed with Freund's incomplete adjuvant at a ratio of 1:1, and then immunized at a dose of 50 μg / mouse, and injected subcutaneously at multiple points in the abdomen; after an interval of 2 weeks, the third immunization was carried out, mixed with Freund's incomplete adjuvant at a ratio of 1:1, and then immunized at a dose of 50 μg / mouse; blood was collected one week after the third immunization to determine the serum titer, and mice with qualified titers were waiting for fusion, and mice were selected for booster immunization. Three days after the booster immunization, the spleen of the mice was taken for hybridoma fusion, and the animal death was determined by carbon dioxide asphyxiation and cervical dislocation, and cell fusion was performed. The indirect ELISA method was used to detect the titer of the polyclonal antibody. The inclusion body-deformed renatured PB transposase full-length protein and the naturally active PB transposase full-length protein were coated separately. The indirect ELISA method was used to detect the immune animal serum to determine the level of immune response.

[0089] 3. Blood collection and titer detection: One week after each immunization, 50-60 μL of blood was collected from the mouse orbital venous plexus, and after standing at 4°C overnight, the upper serum was separated by centrifugation for testing;

[0090] Take an appropriate amount of detection protein, dilute it to 5μg / mL with coating buffer, then add 100μL to each well of a 96-well plate with a single-channel pipette, tap the plate to mix the sample, seal it with plastic wrap, and coat it overnight at 4°C; wash the plate once with washing solution at 200μL / well, and dry the ELISA plate; then block the ELISA plate with blocking solution at 300μL / well, and block it at room temperature for 1 hour; wash the plate twice with washing solution at 400μL / well, then add the sample (add the gradient diluted sample and sample diluent at 100μL / well), and add the detection antibody at the same time, add it to the 96-well plate at 100μL / well, and let it work at room temperature for 2 hours; wash the plate 5 times with washing solution at 400μL / well, add the color developing solution at 200μL / well and let it stand at room temperature for 12 minutes; add the stop solution at 50μL / well to stop the reaction; detect with an ELISA reader: the measurement wavelength is 450nm.

[0091] 4. Cell fusion and screening pairing: All spleen cells of immunized mice were taken and mixed with mouse myeloma SP2 / 0 cells at a ratio of 1:1. The cells were fused by electrofusion to obtain hybridoma cells. The inclusion body renatured PB transposase full-length protein and the natural active PB transposase full-length protein were used for coating. The cell supernatant was determined by ELISA. The positive wells that bound to both the antigen and the natural PB protein were selected for cloning by limiting dilution until a hybridoma cell line that stably secreted monoclonal antibodies was obtained. A total of 25 hybridoma cells were obtained through fusion screening, and the number of clones whose supernatants bound to the immunogenic protein was 25, and the number of positive clones that bound to both the immunogenic protein and the natural PB protein was 24.

[0092] 5. Subcloning, expansion culture and cryopreservation: Transfer 1 mL of hybridoma cells into a 100 mL culture bottle, add a certain amount of culture medium regularly for cell expansion, and culture for 10-12 days. Expand the selected subclones and store them in liquid nitrogen.

[0093] 6. Antibody production and screening: 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. Polyacrylamide gel electrophoresis (SDS-PAGE) and indirect ELISA were used for QC testing, and the antibody concentration was determined by NanoDrop2000, and the mouse monoclonal antibody was purified to obtain the antibody purity and activity results.

[0094] 7. Antibody sequencing: The best subclones were selected for antibody sequencing by Sanger / NGS sequencing; the best monoclonal antibodies were MM04H and MM15H, and the heavy chain, light chain and CDR sequences were shown in Table 1.

[0095] Table 1: Antibody sequences

[0096]

[0097] Example 2: Indirect ELISA results of purified mouse monoclonal antibodies

[0098] The indirect ELISA method was used to detect the binding of mouse monoclonal antibodies purified from the supernatant to antigens:

[0099] (1) Coating: Coat the inclusion body denatured PB transposase and natural PB transposase at 0.1 ug / ml, 1 ug / ml, 5 ug / ml, 100 uL / well at 4°C overnight;

[0100] (2) Blocking: shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0101] (3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry for the last time;

[0102] (4) Antibody dilution: Mouse monoclonal antibody was diluted to 1 ug / ml, 100 ul of each was added to the corresponding well plate, mixed evenly, and reacted at room temperature for 2 h;

[0103] (5) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry for the last time;

[0104] (6) Add secondary antibody: dilute Rabbit Anti-Mouse IgG F(ab)2 / HRP secondary antibody to the working concentration, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0105] (7) Washing the plate: Same as step 5;

[0106] (8) Color development: Mix Solution A and Solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min.

[0107] (9) Stop: Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0108] The results of indirect antibody ELISA are shown in Tables 2 and 3.

[0109] Table 2: ELISA binding assay of inclusion body refolding PB transposase coating

[0110]

[0111] Table 3: ELISA binding assay for native active PB transposase coating

[0112]

[0113] Example 3: Double Antibody Sandwich ELISA Pairing

[0114] (1) Coating: Coating monoclonal antibody 2 ug / ml, 100 uL / well, coating overnight at 4℃;

[0115] (2) Blocking: shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0116] (3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry for the last time;

[0117] (4) Protein dilution: dilute PB protein and native PB enzyme protein to 10 ng / ml and add them to the corresponding wells respectively. Add sample diluent to the blank control, add 100 uL / well respectively, and react at room temperature for 2 h.

[0118] (5) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry for the last time;

[0119] (6) Add detection antibody: dilute the detection antibody to 0.5 ug / ml, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0120] (7) Washing the plate: Same as step 5;

[0121] (8) Color development: Mix Solution A and Solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min.

[0122] (9) Stop: Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0123] The results of antibody sandwich ELISA are shown in Table 4. The pairing of SXZ2 MM15H / HRP and monoclonal antibody had obvious positive values ​​and low blank background. Based on the antibody purity and activity results, the final screening antibody pair was determined to be SXZ2-MM15H and SXZ2-MM04H.

[0124] Table 4: Double antibody sandwich ELISA results

[0125]

[0126] Example 4: PB Transposase Detection Kit Sandwich ELISA Results

[0127] In this example, the above antibodies are used to prepare a sandwich ELISA kit and a standard curve. The PB transposase detection kit includes the following materials: a reaction plate, an SXZ2-MM04H antibody for coating, an HRP-labeled SXZ2-MM15H antibody, an ELISA kit supplement solution set (SEKCR02-15 from Beijing SinoBio, containing a diluent, a blocking solution, a washing solution, a stop solution, and an AB color development solution), and a standard reference natural PB transposase.

[0128] 4.1 PB transposase ELISA plate coating

[0129] (a) Take out the coated antibody and melt it at room temperature, then shake and centrifuge to mix.

[0130] (b) Dilute the coating antibody to 2.5 μg / ml using the coating buffer in the kit, then add 100 μl / well to a 96-well plate, seal the plate with sealing film, and place in a 4°C refrigerator for overnight coating.

[0131] 4.2 Preparation of natural PB transposase standard: Take out the natural PB transposase standard and the total cell protein sample, thaw at room temperature, and vortex centrifuge to mix. Use the natural PB transposase stock solution as ST and use the dilution buffer as the diluent to prepare according to Table 5:

[0132] Table 5: Preparation of standard curve of natural PB transposase

[0133]

[0134] 4.3 PB transposase standard sample ELISA experiment

[0135] (a) Prepare reagents: Dilute 20× Wash buffer, 20× Dilution buffer, and 20× Block buffer to 1× Wash buffer, 1× Dilution buffer, and 1× Block buffer using ultrapure water.

[0136] (b) Washing: Take out the 96-well plate that has been coated overnight, carefully peel off the sealing film, discard the liquid in the wells, pat dry on absorbent paper, add 300 μl / well of wash buffer, then discard the wash buffer, pad with absorbent paper, turn the plate upside down and pat dry, repeat the washing method 3 times, pat dry as much as possible the last time.

[0137] (c) Blocking: After the liquid in the plate is patted dry, 300 μL of 1× Block buffer is added to each well, and the plate is sealed with a sealing film and incubated at 37°C for 1 h.

[0138] (d) During the blocking period, the PB enzyme reference material and sample preparation can be carried out.

[0139] (e) Washing the plate: Take out the 96-well plate that has been sealed for 1 hour, carefully peel off the sealing film, discard the liquid in the wells, pat dry on absorbent paper, add 300 μl / well of wash buffer, then discard the wash buffer, pad with absorbent paper, turn the plate upside down and pat dry, repeat the washing method for 3 times, and pat dry as much as possible the last time.

[0140] (f) Add 100 μL of STD1-STD14 to each well of a 96-well plate. According to the detection layout plan, add 95 μL of sample diluent to the cell sample detection well, and then add 5 μL of cell protein lysate sample to each well. Seal the plate with a sealing film and place it on a microplate low-speed shaker for incubation at room temperature for 2 h.

[0141] (g) Washing: Remove the 96-well plate from the shaker, carefully peel off the sealing film, discard the liquid in the wells, pat dry on absorbent paper, add 300 μl / well of wash buffer, discard the wash buffer, pad with absorbent paper, turn the plate upside down and pat dry, repeat the same washing method for 3 times, pat dry as much as possible the last time.

[0142] (h) Preparation of detection antibody: After the detection antibody is taken out and thawed at room temperature, it is vortexed and centrifuged to mix well, and then diluted to 0.125 ug / ml with Dilution buffer. 100 μL / well is added to a 96-well plate, and the plate is sealed with a sealing film and placed on a microplate shaker for incubation at room temperature for 1 hour.

[0143] (i) Washing: Remove the 96-well plate from the shaker, carefully remove the sealing film, discard the liquid in the wells, pat dry on absorbent paper, add 300 μl / well of wash buffer, discard the wash buffer, pad with absorbent paper, turn the plate upside down and pat dry. Repeat the washing method for 3 times, patting as dry as possible the last time.

[0144] (j) Color development: restore the Color reagent A and Color reagent B substrate solutions to room temperature in advance, prepare the color development solution with color reagent A and B in a 1:1 ratio, add 200 μL / well to a 96-well plate, and incubate at room temperature in the dark for 5-15 min (depending on the color of the solution).

[0145] (k) Termination: Add 50 μL / well of stop solution and gently shake to mix until the reaction is fully terminated and the color stabilizes to orange-yellow.

[0146] (l) Plate reading: Within 15 minutes after termination, use an ELISA reader to read the absorbance at 450nm and 630nm. Export the experimental data after the test is completed.

[0147] The results of the standard curve of the natural PB transposase ELISA are as follows Figure 1 As shown in Table 6, when the protein concentration of the homemade natural active PB enzyme was 20 ng / ml, the absorbance value reached the upper platform of the experimental signal. Linear fitting was performed from the points at and below 10 ng / ml, with a good linearity R2=0.9988. There was good linearity in the range of STD2 (10 ng / ml) ~ STD8 (0.156 ng / ml), and the range was: 10 ng / ml ~ 0.156 ng / ml.

[0148] Table 6: PB transposase ELISA standard curve

[0149]

[0150] 4.4 ELISA assay for detecting residual PB transposase in CD19 CAR-T cells

[0151] CD19 CAR-T cell samples electroporated with the PB transposon system were collected 48 hours after electroporation, and the cell samples were divided into 5E+06 cells per tube, a total of 6 tubes, 3 tubes per person for two people, and total cell protein was extracted. Then total cell protein quantification and PB transposase ELISA were performed. The test results are shown in Table 7. For the same cell sample, two people extracted the cell sample, and the same plate was tested 6 times, with a CV% of 17.70%, which is within the range of CV%≤30%, and meets the acceptable range of method repeatability.

[0152] Table 7: PB enzyme ELISA detection of CD19 CAR-T cell samples

[0153]

[0154] Part of this article

[0155] SEQ ID NO:17:

[0156] MGPAAKRVKLDGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF。

Claims

1. A PB transposase binding molecule, comprising an antibody or an antigen-binding fragment thereof targeting PB transposase, wherein the antibody comprises 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:5, LCDR2 shown in SEQ ID NO:6, LCDR3 shown in SEQ ID NO:7; (A2) HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:11, LCDR1 shown in SEQ ID NO:13, LCDR2 shown in SEQ ID NO:14, and LCDR3 shown in SEQ ID NO:

15.

2. The PB transposase binding molecule according to claim 1, characterized in that The heavy chain variable region of the antibody is shown in SEQ ID NO:4, and the light chain variable region of the antibody is shown in SEQ ID NO:8; or the heavy chain variable region of the antibody is shown in SEQ ID NO:12, and the light chain variable region of the antibody is shown in SEQ ID NO:

16.

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

4. A PB transposase binding molecule pair, characterized in that: The first PB transposase binding molecule comprises the following CDRs: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, and LCDR1 shown in SEQ ID NO: 5, LCDR2 shown in SEQ ID NO: 6, and LCDR3 shown in SEQ ID NO: 7; The second PB transposase binding molecule comprises the following CDRs: HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:11, LCDR1 shown in SEQ ID NO:13, LCDR2 shown in SEQ ID NO:14, and LCDR3 shown in SEQ ID NO:

15.

5. The PB transposase binding molecule pair according to claim 4, characterized in that: The first PB transposase binding molecule comprises: a heavy chain variable region shown in SEQ ID NO:4 and a light chain variable region shown in SEQ ID NO:8; the second PB transposase binding molecule comprises: a heavy chain variable region shown in SEQ ID NO:12 and a light chain variable region shown in SEQ ID NO:

16.

6. The PB transposase binding molecule pair according to claim 4, characterized in that: The second PB transposase binding molecule further comprises a label, which is selected from any one of 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 PB transposase, comprising the PB transposase binding molecule according to any one of claims 1 to 3 or the PB transposase binding molecule pair according to any one of claims 4 to 6, The kit also includes reagents for detecting binding of the PB transposase to a PB transposase binding molecule or a PB transposase binding molecule pair.

8. The kit according to claim 7, characterized in that The PB transposase binding molecule is used for sandwich detection. The kit comprises the PB transposase binding molecule pair. The first PB transposase binding molecule is coupled to a support, and the second PB transposase binding molecule comprises a label.

9. The kit according to claim 7, characterized in that The kit comprises a reaction plate coated with a first PB transposase binding molecule, a second PB transposase binding molecule labeled with biotin, a catalytic enzyme labeled with avidin, and a substrate.

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

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