CN119306841A - A PB transposase monoclonal antibody and its detection kit - Google Patents

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

PB transposase monoclonal antibody and detection kit thereof

Technical Field

The invention relates to the field of antibodies, in particular to a PB transposase monoclonal antibody and a detection kit thereof.

Background

PB transposase is derived from the PiggyBac DNA transposon gene, originally found in the Trichoplusia ni (Trichoplusia ni) genome. The PiggyBac transposon is a "cut-and-paste" DNA transposon that is capable of cutting DNA and inserting it into a new locus in the genome by means of a "traceless transposition".

PB transposase is a key enzyme to achieve this process of movement. It recognizes the terminal repeat of the PiggyBac transposon (TERMINAL REPEAT ELEMENT, TRE) and catalyzes the process of cleavage and integration of the transposon DNA. PB transposase has the characteristic of carrying larger DNA transgene fragments to move without leaving a transposable blot, and is a transgene tool widely applied to cell and gene therapies.

During cell and gene therapy, researchers typically introduce into cells a PB transposon and a PB transposase separately, and introduce or remove specific gene fragments in the genome by transposase activity. Wherein the PB transposase is normally expressed from a nucleic acid in the form of a DNA plasmid or in the form of mRNA. Cell therapy and gene therapy generally require that these transposases degrade as quickly and/or completely as possible after their transgene function is driven, thereby maximizing the safety of gene therapy. For the above reasons, monitoring the expression and retention of these transposase proteins during the treatment cycle is an important analytical indicator.

Transposase proteins are known to be positively charged amino acid-rich proteins, and expression of a functional native full-length protein is an important challenge in the industry, which results in monoclonal antibodies based on native structural PB proteins also being difficult to obtain. The invention obtains a pair of PB natural structural protein high-specificity antibody pairs in a series of mouse monoclonal antibodies, which shows that the antibody has the characteristics of very high sensitivity and low detection limit.

Disclosure of Invention

Based on the problems in the prior art, the application provides a monoclonal antibody of PB transposase, a detection kit containing the antibody, which is developed based on an ELISA method, and the quantitative detection of PB transposase is successfully completed by using the kit.

In a first aspect, the invention provides a PB transposase binding molecule that is an antibody or antigen binding fragment thereof that targets a PB transposase or a variant that has at least 85% sequence identity to the antibody or antigen binding fragment thereof and retains its PB transposase binding activity.

In some embodiments, the antibody contains any one set of CDRs selected from the group consisting of:

(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, LCDR3 shown in SEQ ID NO. 15.

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.

In some embodiments, each of the FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody is independently selected from the group consisting of FR1, FR2, FR3, and FR4 of the heavy chain variable region shown in SEQ ID No.4 or 12, and/or each of the FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody is independently selected from the group consisting of FR1, FR2, FR3, and FR4 of the light chain variable region shown in SEQ ID No. 8 or 16.

In some embodiments, the heavy chain variable region of the antibody has the sequence set forth 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 set forth in SEQ ID NO. 8 or a sequence having at least 85% sequence identity thereto.

In some embodiments, the heavy chain variable region of the antibody has the sequence set forth 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 set forth in SEQ ID NO. 16 or a sequence having at least 85% sequence identity thereto.

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

In some embodiments, the antibody further comprises a murine IgG Fc fragment, a rabbit IgG Fc fragment, or a human IgG Fc fragment.

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 additionally, the light chain of the antibody comprises a light chain constant region of a human kappa chain, lambda chain, or a sequence having at least 85% sequence identity thereto.

In some 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 thereto, and the light chain comprises a light chain constant region of human kappa chain or a sequence having at least 85% sequence identity thereto.

In some embodiments, the antibody is a monoclonal antibody.

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

The invention also provides a polynucleotide selected from the group consisting of:

(1) The coding sequence of the PB transposase binding molecule of any of the embodiments herein;

(2) The complement of (1).

The invention also provides a nucleic acid construct comprising a polynucleotide as described in any of the embodiments herein.

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

The invention also provides a host cell which:

(1) Expressing and/or secreting a PB transposase binding molecule as described in any one of the embodiments herein;

(2) Comprising a polynucleotide as described herein, and/or

(3) Comprising the nucleic acid construct described herein.

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

In some embodiments, the host cell is a mammalian cell.

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

In a second aspect, 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, 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, LCDR3 shown in SEQ ID NO. 15.

In some embodiments, the first PB transposase binding molecule comprises the heavy chain variable region set forth in SEQ ID NO. 4 and the light chain variable region set forth in SEQ ID NO. 8, and the second PB transposase binding molecule comprises the heavy chain variable region set forth in SEQ ID NO. 12 and the light chain variable region set forth in SEQ ID NO. 16.

In some embodiments, the first PB-transposase binding molecule or the second PB-transposase binding molecule further comprises a label. The label comprises any one selected from fluorescent substances, quantum dots, digoxin label probes, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron dense substances, chemiluminescent labels, ultrasonic contrast agents, photosensitizers or enzymes.

In some embodiments, the fluorescent material comprises 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-carboxytetramethyl rhodamine, 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 Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol purple, light cresol Blue, para-aminobenzoic acid, erythrosine any one of phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptates, tripyridyl diamine europium, europium cryptates or chelates, diamines, bisanthocyanin, la Jolla Blue dye, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), tetramethyl rhodamine and texas red.

In some embodiments, the radioisotope 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 83 Sr.

In some embodiments, the fluorescent microsphere is a polystyrene fluorescent microsphere, and rare earth fluorescent ion europium is wrapped inside the fluorescent microsphere.

In some embodiments, the marker is biotin. The invention adopts biotin-avidin system to develop color, and improves sensitivity. Affinity between avidin and biotin is very strong, affinity constant of the two is at least 1 ten thousand times higher than that of antigen and antibody, therefore the two can be combined rapidly, and the reaction is not interfered by the outside.

The invention also provides a kit for detecting a PB transposase, the kit comprising a PB transposase binding molecule or a pair of PB transposase binding molecules, a polynucleotide, a nucleic acid construct, a phage or a host cell as described in any of the embodiments herein.

In some embodiments, the kit further comprises reagents for detecting binding of the PB transposase to the PB transposase binding molecule or the pair of PB transposase binding molecules. The bound reagent is detected, for example, by an enzyme-linked immunosorbent assay.

In some embodiments, the agent that detects binding is a detectable label, such as biotin, that is capable of linking to a PB transposase binding molecule or a pair of PB transposase binding molecules. The detectable label is attached to the antibody or antigen binding fragment thereof or is separately present in a kit.

In some embodiments, the PB transposase binding molecule is for use in a sandwich assay, and the kit comprises a pair of PB transposase binding molecules as described in any of the embodiments of the second aspect herein. The sandwich method completes detection in the form of a "support-first PB transposase binding molecule-to-be-detected-second PB transposase binding molecule-label" or a "support-second PB transposase binding molecule-to-be-detected-first PB transposase binding molecule-label".

In some embodiments, the first PB transposase binding molecule is coupled to a support. In some embodiments, the kit further comprises an agent that couples the first PB transposase binding molecule to the support.

In some embodiments, the support includes, but is not limited to, an elisa plate, pellet, microparticle, slide, chip, plastic, film, etc. made of polystyrene, polyethylene, cellulose, nitrocellulose, cellulose acetate, silicide, etc.

In some embodiments, the kit further comprises a recognition signal with which the label is visualized by a biotin-avidin system.

In some embodiments, the recognition signal comprises any one of the group consisting of a fluorescent substance, a quantum dot, a digoxin-labeled probe, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, or an enzyme.

In some embodiments, the recognition signal comprises 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. Correspondingly, the substrate is 3,3', 5' -tetramethyl benzidine or o-phenylenediamine. When 3,3', 5' -tetramethylbenzidine is used as the substrate, the time for color development is not constant because of the different batches of the color development liquid, and the standard is generally that the OD value of the standard having the highest concentration is about 2.0.

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

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

In some embodiments, the kit comprises a reaction plate coated with a first PB transposase binding molecule, a biotin-labeled second PB transposase binding molecule, an avidin-labeled catalytic enzyme, and a substrate.

The preferred scheme of the kit is that streptavidin is marked by horseradish peroxidase, and the mark is adopted to identify a biotinylated second PB transposase binding molecule, namely more sensitive detection is realized by a biotin-avidin amplification system.

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

The present invention also provides a non-diagnostic method of detecting the presence of a PB transposase in a sample, the method comprising incubating the sample with a PB transposase binding molecule or a PB transposase binding molecule pair as described in any of the embodiments herein, and detecting the binding of the PB transposase to the PB transposase binding molecule or the PB transposase binding molecule pair, thereby determining the presence of the PB transposase in the sample. The detection is an enzyme-linked immunosorbent assay.

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

(1) Coupling the first PB transposase binding molecule to the support, optionally washing, optionally blocking,

(2) Contacting a first PB transposase binding molecule coupled to a support with a sample, the PB transposase in the sample binding to the first PB transposase binding molecule to form a complex, optionally washing,

(3) Contacting a second PB transposase binding molecule with the label attached to the complex, allowing the PB transposase in the complex to bind to the second PB transposase binding molecule, optionally washing,

(4) Detecting the PB transposase by a detectable reaction mediated by the label.

In some embodiments, the method comprises the steps of:

(1) Coating a first PB transposase binding molecule onto a support and washing,

(2) Blocking, namely blocking the vacancy points of the support, which are not coated with the first PB transposase binding molecule, by using inert proteins so as to avoid nonspecific adsorption in subsequent reactions,

(3) The first PB transposase binding molecule captures antigen, after being added into a sample to be detected, the first PB transposase binding molecule captures antigen in the sample,

(4) The antigen captures the second binding molecule, the second PB transposase binding molecule is added and then incubated, the antigen captures the second PB transposase binding molecule,

Detection is performed by introducing the label in one or more steps.

In some embodiments, the label comprises any one selected from a fluorescent substance, a quantum dot, a digoxin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, or an enzyme.

In some embodiments, the recognition signal comprises any one selected from the group consisting of a fluorescent substance, a quantum dot, a digoxin-labeled probe, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, or an enzyme.

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 chromogenic reaction of the catalytic enzyme-catalyzed substrate. Further, the catalytic enzyme is selected from horseradish peroxidase or alkaline phosphatase. Correspondingly, the substrate is 3,3', 5' -tetramethyl benzidine or o-phenylenediamine.

The PB transposase detection kit provided by the invention has the advantages of strong detection specificity, high sensitivity, good stability and accurate quantification.

Drawings

FIG. 1 is a standard ELISA curve for native PB transposase.

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are well explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989), oligonucleotide Synthesis (M.J. Gait edit, 1984), ANIMAL CELL Culture (R.I. Freshney edit ,1987）;Methods in Enzymology（Academic Press,Inc.）;Current Protocols in Molecular Biology（F.M.Ausubel , et al, 1987 edition and periodically updated versions thereof), PCR: the Polymerase Chain Reaction (Mullis et al, edit ,1994）;A Practical Guide to Molecular Cloning（Perbal Bernard V.,1988）;Phage Display：A Laboratory Manual（Barbas, et al, 2001).

Herein, a "PB transposase binding molecule" is a protein that specifically binds to a 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.

Herein, the term "antibody" includes monoclonal antibodies (including full length antibodies, which have an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies and single chain molecules, and antibody fragments, particularly antigen binding fragments, such as Fab, F (ab') 2, and Fv. The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein.

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

The term "variable" refers to the situation where certain segments in the variable domain differ widely in antibody sequences, HCDR1, HCDR2, HCDR3 for the heavy chain variable region and LCDR1, LCDR2 and LCDR3 for the light chain variable region, respectively. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions (FR 1, FR2, FR3 and FR4. Typically, the light chain variable region has the structure FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4 and the heavy chain variable region has the structure FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. For chimeric antibodies, the FR regions may be of non-human (e.g., murine) origin.

Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, abM, chothia, IMGT, a combination of Kabat/Chothia et al. Such coding systems are known in the art. For example, the amino acid sequence numbering of the antigen binding proteins may be according to the IMGT numbering scheme.

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

In some embodiments, the antibodies described herein further comprise a heavy chain constant region, e.g., a heavy chain constant region derived from IgA, igD, igE, igG and IgM or a heavy chain constant region derived from IgG1, igG2A, igG2B, igG, igG4, igA1, and IgA2 or a sequence thereof having at least 85% sequence identity, and/or a light chain constant region, e.g., a light chain constant region of a kappa or lambda chain or a sequence having at least 85% sequence identity thereto.

Antibodies of the application may be prepared using methods conventional in the art, such as hybridoma technology, phage display technology, cell line expression, as are well known in the art. The application also provides fusion proteins comprising an antibody or antigen binding fragment of the application as an expression target, active molecule or targeting molecule. For example, a fusion protein formed by adding a tag (e.g., his6 tag) that facilitates functions such as protein expression and purification to both ends of an antibody. The tag does not affect the function of the target protein and can be easily excised.

The invention also provides polynucleotides encoding the binding molecules, binding molecule pairs, fusion proteins described herein. Provided herein are polynucleotides encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains, and CDRs. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The invention also includes degenerate variants of the polynucleotide sequence encoding an antibody or antigen-binding fragment thereof, fusion protein or antibody conjugate, i.e., nucleotide sequences encoding the same amino acid sequence but differing in nucleotide sequence.

Based on the amino acid sequence and the codons, the coding sequence of the antibody or fragment thereof can be readily obtained by a person skilled in the art. The art can also alter the expression of polypeptides in different species by codon optimization, with codon bias for different species belonging to routine skill in the art.

The antibodies of the invention are useful in assays, such as binding assays, to detect and/or quantify PB transposase due to their high affinity for PB transposase. The invention also provides a detection kit for detecting PB transposase level, which comprises 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 markers, detection substrates and the like.

The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and materials used in the examples are those conventional in the art, unless otherwise indicated.

Examples

EXAMPLE 1 PB transposase monoclonal antibody preparation

1. Immunogen preparation, namely preparing a His-tagged inclusion body variegated PB transposase full-length protein and an MBP-tagged active natural PB transposase full-length protein respectively, wherein the amino acid sequence of the PB transposase is shown as SEQ ID NO. 17.

2. Animal immunization 5 Balb/c mice aged 6-8 weeks were selected using conventional immunization protocols. The method comprises the steps of taking inclusion body renaturation PB transposase full-length protein as an immunogen, uniformly mixing the inclusion body renaturation PB transposase full-length protein with Freund complete adjuvant according to a ratio of 1:1, performing primary immunization according to a dose of 50 mug/dose, performing subcutaneous multipoint injection on the abdomen at intervals of 2-3 weeks, uniformly mixing the inclusion body renaturation PB transposase full-length protein with Freund complete adjuvant according to a ratio of 1:1, performing immunization according to a dose of 50 mug/dose after uniformly mixing the inclusion body renaturation PB transposase full-length protein with Freund complete adjuvant at intervals of 2 weeks, taking blood for one week after three times of immunization, determining serum titer, waiting for fusion, selecting mice for boosting immunization, taking spleens of the mice after 3 days of boosting immunization, performing hybridoma fusion, determining death of the animals by cervical dislocation after carbon dioxide asphyxiation, and performing cell fusion. The immune response level is determined by detecting multiple antibody titers by an indirect ELISA method, respectively coating inclusion body variegated PB transposase full-length protein and PB transposase full-length protein with natural activity, and detecting immune animal serum by an indirect ELISA method.

3. Blood sampling and titer detection, namely taking 50-60 mu L of blood through the orbital venous plexus of a mouse one week after each immunization, standing overnight at 4 ℃, and centrifuging to separate upper serum for standby detection;

diluting the protein for detection into 5 mu g/mL with coating buffer solution, adding 100 mu L of coating buffer solution into each well of a 96-well plate, tapping the plates to mix the samples uniformly, sealing the plates with preservative film, coating the plates at 4 ℃ for overnight, using washing solution to dry the ELISA plate 1 time at 200 mu L/Kong Xi plates, using sealing solution to seal the ELISA plate at 300 mu L/well for 1 hour at room temperature, using washing solution to sample after 400 mu L/Kong Xiban times (adding the samples subjected to gradient dilution and sample diluent at 100 mu L/well), adding detection antibody into a 96-well plate at 100 mu L/Kong Jiazhi, reacting for 2 hours at room temperature, using washing solution to add the chromogenic solution at 400 mu L/Kong Xi plates 5 times at 200 mu L/well, standing at room temperature for 12 minutes, using 50 mu L/well to add stop solution, and using the ELISA to carry out detection, wherein the detection wavelength is 450nm.

4. Cell fusion and screening pairing, namely mixing all spleen cells of the immunized mice with myeloma SP2/0 cells of the mice according to a ratio of 1:1, and fusing by using an electrofusion method to obtain hybridoma cells. Coating by using inclusion body variegated PB transposase full-length protein and natural active PB transposase full-length protein, measuring cell supernatant by ELISA method, selecting positive holes combined with antigen and natural PB protein, cloning by limiting dilution until hybridoma cell strain capable of stably secreting monoclonal antibody is obtained. 25 hybridoma cells are obtained through fusion screening, the number of clones with positive binding of the supernatant and the immunogen protein is 25, and the number of positive clones combined with the immunogen protein and the natural PB protein is 24.

5. Subcloning, expanding culturing and low-temperature preserving, namely transferring 1mL of hybridoma cells into a 100mL culture bottle, adding a certain amount of culture medium periodically to amplify the cells, and culturing for 10-12 days. The selected subclones were amplified and stored in liquid nitrogen.

6. Antibody production and screening selected monoclonal antibodies were produced, purified by protein A affinity chromatography and stored in Phosphate Buffer (PBS) by dialysis. QC detection is carried out by polyacrylamide gel electrophoresis (SDS-PAGE) and indirect ELISA, and the concentration of the antibody is measured at Nanodrop2000, and the murine monoclonal antibody is purified, so that the purity and activity result of the antibody are obtained.

7. Antibody sequencing the optimal subclone selected was sequenced by Sanger/NGS and the optimal mab was MM04H, MM H, heavy chain, light chain and their CDR sequences are shown in Table 1.

TABLE 1 antibody sequences

Example 2 Indirect ELISA results for purified murine monoclonal antibodies

The binding of the supernatant purified murine monoclonal antibody to the antigen was detected by indirect ELISA:

(1) Coating inclusion body variegated PB transposase and natural PB transposase 0.1 ug/ml, 1 ug/ml, 5 ug/ml, 100 uL/hole, coating at 4 ℃ overnight;

(2) Blocking, namely, liquid in the plate is thrown and beaten to dryness, 2% BSA is added, 300 mu L/hole is formed, and the plate is incubated for 1h at room temperature after being sealed;

(3) Washing the plate, namely washing the plate for 2 times by 300 mu L/hole washing liquid, and finally beating the plate to dryness;

(4) Diluting the mouse monoclonal antibody to 1 ug/ml, adding each 100: 100 ul into a corresponding pore plate, uniformly mixing, and reacting at room temperature for 2: 2 h;

(5) Washing the plate, namely washing the plate for 3 times by 300 mu L/hole washing liquid, and finally beating the plate to dryness;

(6) Adding secondary antibody, namely diluting Rabbit Anti-Mouse IgG F (ab) 2/HRP secondary antibody to the use concentration of 100 mu L/hole, uniformly mixing, and incubating for 1h at room temperature;

(7) Washing the plate, namely, a step 5;

(8) Color development, namely uniformly mixing the solution A and the solution B according to a ratio of 1:1, adding 200 mu L of the mixture into each hole, and incubating the mixture at room temperature and in a dark place for 20 min;

(9) Stop 50. Mu.L of stop solution was added to each well and OD was measured immediately at a wavelength of 450 nm.

The results of the indirect antibody ELISA are shown in Table 2 and Table 3.

TABLE 2 ELISA binding assays for inclusion body variegated-renaturation PB transposase coating

TABLE 3 ELISA binding assay for naturally active PB transposase coating

Example 3 double antibody Sandwich ELISA pairing

(1) Coating, namely coating the monoclonal antibody 2 ug/ml,100 uL/hole and 4 ℃ overnight;

(2) Blocking, namely, liquid in the plate is thrown and beaten to dryness, 2% BSA is added, 300 mu L/hole is formed, and the plate is incubated at room temperature for 1 h after being sealed;

(3) Washing the plate, namely washing the plate for 2 times by 300 mu L/hole washing liquid, and finally beating the plate to dryness;

(4) Protein dilution, namely diluting PB protein and natural PB enzyme protein to 10 ng/ml, respectively adding into corresponding holes, adding blank control into sample diluent, respectively adding into 100 uL/holes, and reacting at room temperature for 2 h;

(5) Washing the plate, namely washing the plate for 3 times by 300 mu L/hole washing liquid, and finally beating the plate to dryness;

(6) Adding detection antibodies, namely respectively diluting the detection antibodies to 0.5 ug/ml and 100 mu L/hole, uniformly mixing, and incubating at room temperature for 1 h;

(7) Washing the plate, namely, a step 5;

(8) Color development, namely uniformly mixing the solution A and the solution B according to a ratio of 1:1, adding 200 mu L of the mixture into each hole, and incubating the mixture at room temperature and in a dark place for 20 min;

(9) Stop 50. Mu.L of stop solution was added to each well and OD was measured immediately at a wavelength of 450 nm.

ELISA results of the antibody sandwich method are shown in Table 4, the SXZ2 MM15H/HRP and monoclonal antibody have obvious positive values, the blank background is low, and the screening antibody pair is finally determined to be SXZ2-MM 15H and SXZ2-MM04H by combining the antibody purity and activity results.

TABLE 4 ELISA results by double antibody sandwich method

Example 4 PB transposase detection kit Sandwich ELISA results

This example uses the antibodies described above to prepare a sandwich ELISA kit and to prepare a standard curve. The PB transposase detection kit comprises a reaction plate, SXZ2-MM04H antibody for coating, HRP-labeled SXZ2-MM15H antibody, ELISA kit supplementing solution sets (Beijing Yiqiao SEKCR02-15, containing diluent, sealing liquid, washing liquid, termination liquid and AB developing liquid), and standard reference natural PB transposase.

4.1 PB transposase ELISA detection plate coating

(A) Taking out the coated antibody, melting at room temperature, vibrating, centrifuging and mixing uniformly.

(B) The coated antibodies were diluted to 2.5. Mu.g/ml using a Coating buffer in the kit, then 100. Mu.l/Kong Jiazhi in a 96 well plate, sealed with a sealing plate membrane and placed in a 4℃refrigerator for Coating overnight.

4.2 Preparing a natural PB transposase standard, namely taking out the natural PB transposase standard and a cell total protein sample, melting at room temperature, vortex centrifuging and mixing uniformly, and preparing according to the table 5 by taking natural PB transposase zymogen liquid as ST and taking a Dilution buffer as diluent:

TABLE 5 preparation of Natural PB transposase standard curve

4.3 ELISA test of PB transposase standard sample

(A) The reagent was prepared by diluting 20 XWash buffer, 20 Xpartition buffer, and 20 XBlock buffer to 1 XWash buffer, 1 Xpartition buffer, and 1 XBlock buffer with ultrapure water, respectively, for use.

(B) And (3) washing the plate, namely taking out the 96-well plate coated overnight, carefully uncovering the sealing plate film, discarding the liquid in the hole, gently beating the liquid on absorbent paper, adding 300 mu l/hole of washing liquid (wash buffer), discarding the washing liquid, filling the absorbent paper, gently beating the plate reversely, repeatedly washing for 3 times according to the same method, and beating the plate as much as possible.

(C) Sealing, namely adding 300 mu L of 1 XBlock buffer into each hole after the liquid in the plate is dried, and placing the sealing plate film at 37 ℃ for incubation for 1h.

(D) During the blocked time, the PB enzyme reference can be formulated and sample prepared.

(E) And (3) washing the plate, namely taking out the 96-well plate sealed for 1h, carefully uncovering the sealing plate film, discarding the liquid in the hole, gently beating the liquid on water-absorbing paper, adding 300 mu l/hole of washing liquid (wash buffer), discarding the washing liquid, filling the water-absorbing paper, gently beating the plate reversely, repeatedly washing for 3 times according to the same method, and beating the plate as much as possible.

(F) STD1-STD14 was added to 96-well plates at 100. Mu.L per well, and according to the assay layout plan, 95. Mu.L of sample dilution was added to the cell sample assay wells, followed by 5. Mu.L of cell protein lysate samples per well. Sealing the sealing plate film, and placing the sealing plate film in a micro-pore plate low-speed oscillator to oscillate the room temperature for incubation for 2 hours.

(G) And (3) washing the plate, namely taking the 96-well plate off the oscillator, carefully uncovering the sealing plate film, discarding the liquid in the hole, gently beating the liquid on absorbent paper, adding 300 mu l/hole of washing liquid (wash buffer), discarding the washing liquid, filling the absorbent paper, slightly beating the plate reversely, repeatedly washing for 3 times according to the same method, and beating the plate as much as possible.

(H) Preparing detection antibody, namely taking out the detection antibody, melting at room temperature, vortex centrifugation and uniform mixing, diluting to 0.125ug/ml by using a Dilution buffer, sealing a sealing plate film, and then placing the sealing plate film in a micro-pore plate oscillator for shaking at room temperature for incubation for 1h.

(I) And (3) washing the plate, namely taking the 96-well plate off the oscillator, carefully uncovering the sealing plate film, discarding the liquid in the hole, gently beating the liquid on absorbent paper, adding 300 mu l/hole of washing liquid (wash buffer), discarding the washing liquid, filling the absorbent paper, slightly beating the plate reversely, repeatedly washing for 3 times according to the same method, and beating the plate as much as possible.

(J) Color development, in which Color reagent A and Color reagent B substrate solutions are restored to room temperature in advance, color development solutions are prepared by the A and B Color development reagents according to the ratio of 1:1, 200 mu L/well is added into a 96-well plate, and the solution is incubated for 5-15min at room temperature in a dark place (see the Color condition of the solution).

(K) Stop 50. Mu.L/well stop solution was added and mixed by gentle shaking to allow the reaction to be stopped sufficiently and the color to stabilize to orange.

(L) Plate reading, namely, reading absorbance values at wavelengths of 450nm and 630nm by using an enzyme-labeled instrument within 15 minutes after termination. And (5) after the detection is finished, deriving experimental data.

As shown in FIG. 1 and Table 6, the ELISA standard curve of the native PB transposase shows that when the protein concentration is 20 ng/ml, the absorbance reaches the upper platform of the experimental signal, linear fitting is carried out from the point with the concentration of 10ng/ml and below, the linear R2 = 0.9988 is better, and the linear range from STD2 (10 ng/ml) to STD8 (0.156 ng/ml) is better, wherein the linear range is 10ng/ml to 0.156ng/ml.

TABLE 6 ELISA Standard curve for PB transposase

4.4 ELISA experiment for detecting residual PB transposase of CD19 CAR-T cells

The CD19 CAR-T cell sample electrically transformed by using the PB transposon system is collected 48 hours after the electric transformation, the cell sample is divided into 5E+06 cells per tube, 6 tubes are total, two people 3 tubes per person, and the total protein of the cells is extracted. Cell total protein quantification and PB transposase ELISA detection were then performed. The test results are shown in Table 7. The same cell sample is extracted by two persons, the same plate is detected for 6 times, the CV% is 17.70%, and the CV% is less than or equal to 30%, thereby conforming to the method repeatability acceptance range.

TABLE 7 PB enzyme ELISA detection of CD19 CAR-T cell samples

Partial sequences herein

SEQ ID NO:17:

MGPAAKRVKLDGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF.

Claims (10)

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 has the sequence shown in SEQ ID NO:4, and the light chain variable region of the antibody has the sequence shown in SEQ ID NO:8; or the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:12, and the light chain variable region of the antibody has the sequence 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 as described in 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 as described in claim 4 is characterized in that the second PB transposase binding molecule further comprises a marker, and the marker 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 markers, 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 as claimed in 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: incubating the sample with the PB transposase binding molecule described in any one of claims 1 to 3 or the PB transposase binding molecule pair described in 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, thereby determining the presence of PB transposase in the sample.