All-potent nuclease residue detection kit based on immunochemiluminescence method

Abstract

The invention provides an all-round nuclease residue detection kit based on an immunochemiluminescence method. Specifically, a pair of high-affinity totipotent nuclease specific antibodies is obtained through screening, and a totipotent nuclease residue detection kit is developed based on the antibody pair. The all-round nuclease residue detection kit based on the immunochemiluminescence method is high in specificity and sensitivity and has a wider linear range, and a more efficient and convenient tool is provided for all-round nuclease residue detection.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to an all-purpose nuclease residue detection kit based on immunochemiluminescence method. Background Art

[0002] NucA (Uniprot: P13717) is a broad-spectrum nonspecific endonuclease derived from Serratia Marcescen that can degrade various forms of DNA and RNA (double-stranded, single-stranded, linear, circular, native or denatured). As a nucleic acid degradation enzyme, it is widely used in the production of pharmaceutical products in the field of gene and cell therapy. The detection of residual nucA has also become a quality control indicator for pharmaceutical products.

[0003] Conventional ELISA tests use universal nuclease-specific antibodies, which have high specificity, but low sensitivity, with a detection limit of only tens or hundreds of pg / ml, and a narrow linear range (approximately 3ng / ml-0.05ng / ml). The widely used fluorescence residual detection uses fluorescently labeled DNA probes as substrates to detect the fluorescent signal produced after the substrate is digested by universal nucleases. Although it has high sensitivity and a detection limit of 0.3pg / ml, it does not have universal nuclease specificity.

[0004] Therefore, there is an urgent need in the art for a method for detecting residual universal nucleases that has both universal nuclease specificity and high sensitivity. Summary of the invention

[0005] The object of the present invention is to provide a universal nuclease-specific antibody with high affinity, and use of the antibody in preparing a universal nuclease residue detection reagent and kit with high specificity, high sensitivity and wide linear range.

[0006] In a first aspect of the present invention, an anti-totipotent nuclease antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

[0007] (Z1) the heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO: 15; H-CDR2 as shown in SEQ ID NO: 16; H-CDR3 as shown in SEQ ID NO: 17; and

[0008] The light chain variable region comprises the following three CDRs: L-CDR1 as shown in SEQ ID NO: 13; L-CDR2 as shown in YAS; L-CDR3 as shown in SEQ ID NO: 14; or

[0009] (Z2) the heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO:20; H-CDR2 as shown in SEQ ID NO:21; H-CDR3 as shown in SEQ ID NO:22; and

[0010] The light chain variable region comprises the following three CDRs: L-CDR1 shown in SEQ ID NO: 18; L-CDR2 whose sequence is shown in SAK; and L-CDR3 shown in SEQ ID NO: 19.

[0011] In another preferred example, the heavy chain variable region and the light chain variable region further comprise a FR region.

[0012] In another preferred example, the heavy chain variable region comprises a murine or human FR region, and / or the light chain variable region comprises a murine or human FR region.

[0013] In another preferred embodiment, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and / or

[0014] The light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0015] In another preferred example, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 1.

[0016] In another preferred embodiment, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and / or

[0017] The light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0018] In another preferred example, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:4, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO:3.

[0019] In another preferred embodiment, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0020] In another preferred embodiment, the heavy chain constant region and / or light chain constant region is of murine or human origin.

[0021] In another preferred embodiment, the heavy chain constant region is derived from human IgG1.

[0022] In another preferred embodiment, the light chain constant region is derived from human kappa (κ) chain.

[0023] In another preferred embodiment, the heavy chain constant region is derived from mouse IgG1.

[0024] In another preferred embodiment, the light chain constant region is derived from mouse kappa (κ) chain.

[0025] In another preferred example, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 26 or 30.

[0026] In another preferred example, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 24 or 28.

[0027] In another preferred embodiment, the antibody or antigen-binding fragment thereof includes: monoclonal antibody, polyclonal antibody, diabody, single-chain antibody (scFv), Fab, Fab', F(ab')2 antibody.

[0028] In another preferred embodiment, the antibodies include mouse antibodies, human-mouse chimeric antibodies, and humanized antibodies.

[0029] In another preferred example, the antibody is a human-mouse chimeric antibody; preferably, it comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2, and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 26; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 1, and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 24.

[0030] In another preferred embodiment, the antibody is a murine antibody; preferably, it comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:4, and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:30; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:3, and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:28.

[0031] In a second aspect of the present invention, a recombinant antibody is provided, comprising:

[0032] (i) the sequence of the anti-universal nuclease antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0033] (ii) a signal peptide that promotes secretory expression of the antibody and / or a tag sequence for purification and detection.

[0034] In another preferred embodiment, the recombinant antibody has a signal peptide that promotes secretory expression of the antibody.

[0035] In another preferred example, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 23, 25, 27 or 29.

[0036] In another preferred embodiment, the tag sequence is selected from: FLAG, Myc, His tag, etc.

[0037] In another preferred example, the recombinant antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:6, and a light chain with an amino acid sequence as shown in SEQ ID NO:5.

[0038] In another preferred example, the recombinant antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:8, and a light chain with an amino acid sequence as shown in SEQ ID NO:7.

[0039] In the third aspect of the present invention, a polynucleotide molecule is provided, which encodes a polypeptide selected from the group consisting of:

[0040] (1) the anti-universal nuclease antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; or

[0041] (2) The recombinant antibody according to the second aspect of the present invention.

[0042] In another preferred example, the polynucleotide molecule comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 9-12.

[0043] In the fourth aspect of the present invention, an expression vector is provided, which contains the polynucleotide molecule as described in the third aspect of the present invention.

[0044] In another preferred embodiment, the vector includes a eukaryotic cell expression vector and a prokaryotic cell expression vector.

[0045] In another preferred embodiment, the vector is a mammalian cell expression vector.

[0046] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.

[0047] In the fifth aspect of the present invention, a host cell is provided, which contains the vector as described in the fourth aspect of the present invention, or the polynucleotide molecule as described in the third aspect of the present invention is integrated into its genome.

[0048] In another preferred embodiment, the host cells include eukaryotic cells (such as mammalian cells) and prokaryotic cells.

[0049] In a sixth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises:

[0050] (a) the anti-universal nuclease antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or the recombinant antibody as described in the second aspect of the present invention; and

[0051] (b) a conjugated moiety selected from the group consisting of a detectable label, an enzyme, a radionuclide, a nanoparticle / nanorod.

[0052] In another preferred embodiment, the detectable marker includes a fluorescent marker or a chemiluminescent marker.

[0053] In another preferred embodiment, the coupling moiety is biotin or acridinium ester.

[0054] In the seventh aspect of the present invention, there is provided an antibody combination targeting a universal nuclease, the antibody combination comprising:

[0055] (A1) a first antibody, whose heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO: 15; H-CDR2 as shown in SEQ ID NO: 16; H-CDR3 as shown in SEQ ID NO: 17; and

[0056] The light chain variable region comprises the following three CDRs: L-CDR1 as shown in SEQ ID NO: 13; L-CDR2 as shown in the sequence YAS; L-CDR3 as shown in SEQ ID NO: 14; and

[0057] (A2) a second antibody, whose heavy chain variable region comprises the following three CDRs: H-CDR1 shown in SEQ ID NO: 20; H-CDR2 shown in SEQ ID NO: 21; H-CDR3 shown in SEQ ID NO: 22; and

[0058] The light chain variable region comprises the following three CDRs: L-CDR1 as shown in SEQ ID NO: 18; L-CDR2 as shown in SAK; and L-CDR3 as shown in SEQ ID NO: 19.

[0059] In another preferred embodiment, the heavy chain variable region of the first antibody comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and / or

[0060] The light chain variable region of the first antibody comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0061] In another preferred example, the first antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 1.

[0062] In another preferred embodiment, the heavy chain variable region of the second antibody comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and / or

[0063] The light chain variable region of the second antibody comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0064] In another preferred example, the second antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:4, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO:3.

[0065] In another preferred example, the first antibody and the second antibody further comprise a heavy chain constant region and a light chain constant region.

[0066] In another preferred embodiment, the heavy chain constant region and / or light chain constant region is of murine or human origin.

[0067] In another preferred embodiment, the heavy chain constant region is derived from human IgG1.

[0068] In another preferred embodiment, the light chain constant region is derived from human kappa (κ) chain.

[0069] In another preferred embodiment, the heavy chain constant region is derived from mouse IgG1.

[0070] In another preferred embodiment, the light chain constant region is derived from mouse kappa (κ) chain.

[0071] In another preferred example, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 26 or 30.

[0072] In another preferred example, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 24 or 28.

[0073] In another preferred example, the first antibody is a human-mouse chimeric antibody; preferably, it comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:2, and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:26; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:1, and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:24.

[0074] In another preferred example, the second antibody is a murine antibody; preferably, it comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:4, and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:30; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:3, and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:28.

[0075] In another preferred embodiment, the antibody combination is used to prepare a detection reagent or a detection kit for detecting residual omnipotent nuclease.

[0076] In the eighth aspect of the present invention, there is provided the use of the anti-totipotent nuclease antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant antibody as described in the second aspect of the present invention, the antibody conjugate as described in the sixth aspect of the present invention, or the antibody combination as described in the seventh aspect of the present invention in the preparation of a detection reagent, a detection plate or a detection kit for detecting totipotent nuclease.

[0077] In another preferred embodiment, the detection includes ELISA detection, chemiluminescence detection, protein immunoblot (WB) detection, flow cytometry (FACS), and cell immunofluorescence detection.

[0078] In another preferred embodiment, the detection includes detecting whether there are residual omnipotent nucleases in the sample (qualitative detection), or detecting the residual amount of omnipotent nucleases in the sample (quantitative detection).

[0079] In the ninth aspect of the present invention, a detection reagent for detecting residual omnipotent nuclease is provided, which comprises the anti-omnipotent nuclease antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant antibody as described in the second aspect of the present invention, the antibody conjugate as described in the sixth aspect of the present invention, or the antibody combination as described in the seventh aspect of the present invention, and a carrier acceptable in detection.

[0080] In the tenth aspect of the present invention, a kit for detecting residual omnipotent nuclease is provided, which comprises the anti-omnipotent nuclease antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant antibody as described in the second aspect of the present invention, the antibody conjugate as described in the sixth aspect of the present invention, or the antibody combination as described in the seventh aspect of the present invention, or a combination thereof.

[0081] In another preferred embodiment, the kit comprises the antibody combination as described in the seventh aspect of the present invention, wherein the first antibody and the second antibody in the antibody combination, one is used as a capture antibody and the other is used as a detection antibody;

[0082] Preferably, the first antibody serves as a capture antibody and the second antibody serves as a detection antibody.

[0083] In another preferred embodiment, the capture antibody is coupled to biotin to obtain a biotin-labeled capture antibody, which is then coated on streptavidin magnetic beads to form a capture antibody-biotin-streptavidin magnetic bead complex for capturing the universal nuclease in the sample.

[0084] In another preferred embodiment, the detection antibody is coupled to a chemiluminescent marker to obtain a detection antibody labeled with a chemiluminescent marker; preferably, the chemiluminescent marker is an acridinium ester.

[0085] In another preferred embodiment, the kit further comprises an instruction manual, which records a method for using the kit to detect residual omnipotent nuclease: using the capture antibody-biotin-streptavidin magnetic bead complex to specifically bind to and capture the omnipotent nuclease in the sample to be tested, and then forming a "capture antibody-omnipotent nuclease-detection antibody" sandwich complex with the detection antibody labeled with the chemiluminescent marker, and detecting its chemiluminescence intensity.

[0086] In another preferred embodiment, the kit further comprises a universal nuclease standard for drawing a standard curve.

[0087] In the eleventh aspect of the present invention, a method for detecting residual omnipotent nuclease is provided, the method comprising the steps of: using the detection reagent as described in the ninth aspect of the present invention or the kit as described in the tenth aspect of the present invention to detect the sample to be tested.

[0088] In another preferred embodiment, the method comprises using the kit as described in the tenth aspect of the present invention to detect the sample to be tested, specifically comprising the following steps:

[0089] (S1) providing a sample to be tested, and using the capture antibody-biotin-streptavidin magnetic bead complex to specifically bind to and capture the universal nuclease in the sample to be tested;

[0090] (S2) adding the detection antibody labeled with the chemiluminescent marker to the detection system of step (S1), and combining it with the captured universal nuclease to form a "capture antibody-universal nuclease-detection antibody" sandwich complex;

[0091] (S3) After washing the magnetic beads, the chemiluminescence intensity is detected.

[0092] In another preferred embodiment, the method is an in vitro method.

[0093] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0094] In another preferred embodiment, the method is a quality control method.

[0095] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 The results of SPR surface plasmon resonance characterization of the binding kinetics of 2C5 human-mouse chimeric antibody and 79G6 mouse monoclonal antibody to universal nuclease are shown.

[0097] Figure 2 The standard curve drawn by the one-step immunochemiluminescent universal nuclease residue detection kit of the present invention for detecting universal nuclease standards is shown. DETAILED DESCRIPTION

[0098] After extensive and in-depth research, the inventors unexpectedly obtained a pair of high-affinity all-purpose nuclease-specific antibodies for the first time, and developed an all-purpose nuclease residual detection kit based on the antibody pair. The present invention developed a pair of high-affinity all-purpose nuclease-specific antibodies, human-mouse chimeric monoclonal antibody 2C5 and mouse monoclonal antibody 79G6, with SPR determination KD (M) values ​​of 2.9E-11 and 4.4E-11. The all-purpose nuclease residual detection kit based on immunochemiluminescence prepared using this pair of high-affinity antibodies has a linear range of 50-0.012ng / ml and a detection limit of 12pg / ml, with the characteristics of high specificity, high sensitivity and wide linear range. Using one-step immunochemiluminescence detection, biotin-labeled human-mouse chimeric monoclonal antibody 2C5 is bound to streptavidin magnetic beads as the capture antibody to specifically capture the universal nuclease in the sample, and acridinium ester-coupled mouse monoclonal antibody 79G6 is used as the detection antibody to form an antibody-antigen-antibody sandwich complex in the solution. The test results can be obtained directly on the machine, which is simpler and more efficient than ordinary ELISA kits.

[0099] On this basis, the present invention has been completed.

[0100] the term

[0101] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0102] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0103] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0104] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0105] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between the sequences described herein and the sequences with which they are identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0106] Antibody

[0107] As used herein, the term "antibody" or "immunoglobulin" is a heterotetramer formed by two light chains (L) and two heavy chains (H). The N-terminus of each heavy chain is a variable region (VH), connected to the heavy chain constant region. The N-terminus of each light chain is a variable region (VL), connected to the light chain constant region.

[0108] As used herein, the term "variable" means that the variable region in an antibody is different in a specific sequence, forming the affinity and specificity of a specific antibody for binding to a specific antigen. The variable region of an antibody includes a complementary determining region (CDR) or a hypervariable region and a framework region (FR) whose sequence is relatively conservative. The primary sequence of the heavy chain and light chain variable regions is composed of 4 FR sequences and 3 CDR sequences arranged at intervals (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The sequence and spatial structural conformation of the heavy chain and light chain variable regions determine the specific binding of the antibody to the antigen epitope. The constant region of the antibody does not directly participate in the binding of the antibody to the antigen, but has an impact on the performance of the capture antibody and the detection antibody.

[0109] The "light chain" of vertebrate antibodies (immunoglobulins) can be classified into one of the categories of κ and λ according to the amino acid sequence of their constant region. According to the amino acid sequence of their heavy chain constant region, immunoglobulins can be divided into different categories, mainly 5 types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and antibody subtypes (isotypes), such as mouse IgG including IgG1, IgG2a, IgG2b subtypes. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known to those in the art.

[0110] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical. Monoclonal antibodies are highly specific for a single antigenic determinant (epitope). The modifier "monoclonal" indicates the characteristic of the antibody, being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring any particular method to produce the antibody.

[0111] The present invention also includes monoclonal antibodies having the corresponding amino acid sequence of the monoclonal antibodies against the omnipotent nuclease, monoclonal antibodies having the variable region chains of the monoclonal antibodies against the omnipotent nuclease, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing a variable region (complementarity determining region, CDR), as long as the variable region is identical to the variable region of the light chain and heavy chain of the present invention or at least 90% homology, preferably at least 95% homology.

[0112] As known to those skilled in the art, antibody conjugates and fusion expression products include: fluorescent or luminescent markers, enzymes capable of producing detectable products, gold nanoparticles / nanorods and other molecules that can be used for detection are combined with the anti-totipotent nuclease antibody or its antigen-binding fragment to form a conjugate.

[0113] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond on the hinge region; (iii) a scFv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0114] The present invention includes not only complete monoclonal antibodies, but also antibody fragments having binding activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains or scFv.

[0115] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.

[0116] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen.

[0117] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0118] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0119] In the present invention, antibodies include mouse antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies can be prepared using DNA recombination techniques well known in the art. The term "mouse antibody" in the present invention refers to a monoclonal antibody against a universal nuclease prepared according to the knowledge and skills in the art, and its light and heavy chain variable regions and constant regions are all derived from mice.

[0120] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0121] As used herein, the term "heavy chain variable region" and "VH" are used interchangeably. The term "light chain variable region" and "VL" are used interchangeably.

[0122] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. The three CDRs in the heavy chain variable region are H-CDR1, H-CDR2, and H-CDR3, and the three CDRs in the light chain variable region are L-CDR1, L-CDR2, and L-CDR3.

[0123] In one aspect of the present invention, an anti-totipotent nuclease antibody or an antigen-binding fragment thereof is provided.

[0124] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody comprises the following three CDRs:

[0125] H-CDR1: GTFFSSYA (SEQ ID NO:15)

[0126] H-CDR2:ITNDDNT(SEQ ID NO:16)

[0127] H-CDR3: VSFPYFDY(SEQ ID NO:17)

[0128] And, the light chain variable region of the antibody comprises the following three CDRs:

[0129] L-CDR1: QSIDTS (SEQ ID NO: 13)

[0130] L-CDR2: YAS

[0131] L-CDR3:QQSYSWPTT(SEQ ID NO:14)

[0132] In another preferred example, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:1.

[0133] Further, the antibody is a human-mouse chimeric antibody, which comprises a heavy chain variable region and a light chain variable region derived from a mouse as described above, and also comprises a heavy chain and a light chain constant region derived from a human. In a preferred embodiment, the heavy chain constant region of the antibody is derived from human IgG1, preferably having an amino acid sequence as shown in SEQ ID NO: 26. In a preferred embodiment, the light chain constant region of the antibody is derived from a human kappa (κ) chain, preferably having an amino acid sequence as shown in SEQ ID NO: 24.

[0134] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody comprises the following three CDRs:

[0135] H-CDR1:GYTFTDYN(SEQ ID NO:20)

[0136] H-CDR2:INPNSDDT(SEQ ID NO:21)

[0137] H-CDR3:ARSGYAHYTLDY(SEQ ID NO:22)

[0138] And, the light chain variable region of the antibody comprises the following three CDRs:

[0139] L-CDR1: ENIFSY (SEQ ID NO: 18)

[0140] L-CDR2:SAK

[0141] L-CDR3: QHHYLFPWT(SEQ ID NO:19)

[0142] In another preferred example, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:3.

[0143] Further, the antibody is a murine antibody, which comprises a heavy chain variable region and a light chain variable region derived from a mouse as described above, and also comprises a heavy chain and a light chain constant region derived from a mouse. In a preferred embodiment, the heavy chain constant region of the antibody is derived from mouse IgG1, preferably having an amino acid sequence as shown in SEQ ID NO:30. In a preferred embodiment, the light chain constant region of the antibody is derived from a mouse kappa (κ) chain, preferably having an amino acid sequence as shown in SEQ ID NO:28.

[0144] The function of the antibody of the present invention is determined by the antibody light chain and heavy chain variable region sequences and the antibody structural conformation, and can specifically bind to the omnipotent nuclease. Using the antibody variable region gene or complementary determining region (CDR) gene, different forms of genetically engineered antibodies can be transformed and produced in any expression system using prokaryotic and eukaryotic cells.

[0145] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and refer to antibodies that specifically bind to omnipotent nucleases, such as proteins or polypeptides having heavy chains and / or light chains.

[0146] "Fragments", "derivatives" and "analogs" of antibodies refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by coupling a mature polypeptide with another compound (such as a compound for chemiluminescence, such as an acridinium ester), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a tag protein sequence or other fusion protein sequence used to purify or detect the polypeptide). These fragments, derivatives and analogs are well known to those skilled in the art.

[0147] The antibody of the present invention refers to a polypeptide having omnipotent nuclease binding activity and comprising the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and comprising the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0148] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and polypeptides or proteins obtained by using antiserum against the antibody of the invention.

[0149] The present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibodies of the present invention, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0150] Polynucleotide molecules, vectors and host cells

[0151] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as the coding region sequence shown in SEQ ID NO:9-12 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes an amino acid sequence identical to the polypeptide of the present invention, but differs from the coding region sequence shown in SEQ ID NO:9-12.

[0152] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0153] The term "polynucleotide encoding a polypeptide" may be a polynucleotide encoding the polypeptide, or may be a polynucleotide further comprising additional coding and / or non-coding sequences. The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can usually be obtained by PCR amplification, recombination or artificial synthesis. In addition, the heavy chain or light chain can be fused with a protein or tag sequence (such as a fluorescent protein, a Flag tag) to form a fusion protein.

[0154] Once the antibody sequence is obtained, recombinant antibodies can be prepared using bioengineering methods. Usually, the gene encoding the antibody sequence is cloned into a vector, and then the expression vector is transferred into cells for expression, and the cells or expression supernatant are harvested and purified to obtain recombinant antibodies. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0155] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing plasmids (or vectors) known in the art. In addition, mutations can also be introduced into the antibody sequence of the present invention by chemical synthesis.

[0156] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0157] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0158] Transforming host cells with recombinant DNA to recombinantly express antibodies is a conventional technique well known to those skilled in the art. The recombinantly expressed antibodies can be separated and purified by conventional techniques well known to those skilled in the art, which will not be described in detail here.

[0159] Antibody combination targeting universal nuclease

[0160] The present invention also provides an antibody combination (or antibody pair) targeting omnipotent nuclease, including two anti-omnipotent nuclease antibodies screened by the present invention. Among them, the first antibody is a human-mouse chimeric antibody named 2C5 in the present invention, and the second antibody is a mouse monoclonal antibody named 79G6 in the present invention. One of the two antibodies is used as a capture antibody and the other as a detection antibody for the detection of omnipotent nuclease residues. In one embodiment of the present invention, the first antibody (2C5 human-mouse chimeric antibody) is used as a capture antibody, and the second antibody (79G6 mouse monoclonal antibody) is used as a detection antibody, and the "antibody-antigen-antibody" sandwich method is used to detect the omnipotent nuclease residues in the sample.

[0161] Kit and method for detecting residual universal nuclease

[0162] Based on the two anti-all-purpose nuclease antibodies screened by the present invention, the present invention further provides a kit for detecting all-purpose nuclease residues, that is, an all-purpose nuclease residue detection kit based on immunochemiluminescence. In the kit of the present invention, a capture antibody and a detection antibody are included, wherein the capture antibody is labeled with biotin and thus coated on streptavidin magnetic beads; the detection antibody is labeled with a chemiluminescent marker (e.g., acridinium ester). In a specific embodiment of the present invention, the 2C5 human-mouse chimeric antibody is a capture antibody, which is labeled with biotin and then coated on streptavidin magnetic beads; the 79G6 mouse monoclonal antibody is a detection antibody, which is labeled with a chemiluminescent marker acridinium ester.

[0163] Using the kit of the present invention, the method for detecting the residual universal nuclease comprises the following steps:

[0164] (i) providing a sample to be detected, and using a capture antibody coated on streptavidin magnetic beads to specifically bind to and capture the universal nuclease in the sample;

[0165] (ii) adding a detection antibody labeled with a chemiluminescent marker to the detection system to bind to the captured universal nuclease, thereby forming a "capture antibody-universal nuclease-detection antibody" complex;

[0166] (iii) After washing the magnetic beads, the chemiluminescence intensity is detected.

[0167] The kit of the present invention has the advantages of high specificity, higher sensitivity and wider linear range of detection, and can be applied to the residual universal nuclease in pharmaceutical products to perform quality control detection on the pharmaceutical products.

[0168] The main advantages of the present invention include:

[0169] (1) The present invention uses omnipotent nuclease (GMP-NUC-SE101, Kactus Biosystems) for immune screening to obtain high-affinity anti-omnipotent nuclease paired antibodies.

[0170] (2) An immunochemiluminescence-based detection kit for residual universal nucleases was prepared using the anti-universal nuclease paired antibodies obtained by screening in the present invention as capture antibodies and detection antibodies, respectively. The kit can be used for residual universal nucleases. Compared with the widely used fluorescence residual detection method, it has the advantage of high specificity; compared with the common ELISA kit, it has a wider linear range and higher sensitivity, and is convenient and efficient in actual use.

[0171] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. Experiments for which specific conditions are not specified in the embodiments or test examples of the present invention are generally carried out under conventional conditions or under conditions recommended by the raw material / commodity manufacturer; reagents for which specific sources are not specified are conventional reagents purchased on the market.

[0172] Example 1 Development of paired antibodies against omnipotent nuclease

[0173] 1-1. Mouse immunization

[0174] 50ug of universal nuclease (GMP-NUC-SE101, Kactus Biosystems) was mixed with an equal volume of Freund's complete adjuvant CFA, emulsified, and immunized subcutaneously at multiple points in female Balb / c mice of 6-8 weeks. The same dose mixed with CFA adjuvant was injected subcutaneously at multiple points every two weeks for booster immunization. After three booster immunizations, serum titer was tested. If the titer was greater than 1:1000000, 50ug universal nuclease was used for intraperitoneal booster immunization once three days before hybridoma cell fusion to perform myeloma cell SP2 / 0 fusion.

[0175] 1-2. Screening of hybridoma cell lines

[0176] Three days after the mice were boosted, the spleen cells of the mice were fused with myeloma cells SP2 / 0 in the logarithmic growth phase by PEG1500 to prepare hybridoma cells. Ten days later, the plates were coated with 1ug / ml of the universal nuclease antigen, and the indirect ELISA method was used to detect the positive hybridoma polyclonal cell lines with an ELISA OD value greater than 2.5. The positive polyclonal hybridoma supernatant was then gradiently diluted, and the hybridoma cells with a supernatant titer greater than 100 times were selected for subcloning. After limiting dilution to a monoclonal state, the cells were expanded and frozen. The supernatant of the expanded hybridoma monoclonal cells was combined with the universal nuclease, and the specificity of the hybridoma cell line antibody binding to the antigen was verified again by indirect ELISA. At the same time, the hybridoma monoclonal cell lines were expanded and cultured, and the monoclonal antibodies were purified.

[0177] 1-3. Anti-universal nuclease antibody pairing

[0178] The purified 10 anti-omnipotent nuclease hybridoma monoclonal antibodies were biotin-labeled, and the NHS-Biotin and antibody molar ratio was 20:1. The 10 unlabeled antibodies were coated on ELISA plates at 2ug / ml, and the antigen omnipotent nuclease was added to 10 ELISA plates coated with different antibodies at 1ug / ml and 3-fold gradient dilution samples. After one hour of binding, another 9 different biotin-labeled detection antibodies (2ug / ml) were added to each ELISA plate. The 10 antibodies were paired in pairs by sandwich ELISA, and SA-HRP was used for color development to select a pair of antibodies 2C5 and 79G6 that could be paired and had the strongest ELISA signal, and sent for hybridoma monoclonal antibody sequencing (Suzhou Hongxun Biotechnology Co., Ltd.).

[0179] The amino acid sequence of the light chain variable region of the 2C5 antibody is shown below (SEQ ID NO: 1):

[0180] DIQLTQSPAILSVSPGERVSFSCRAS QSIDTSIHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQSYSWPTT FGGGTKLEIK

[0181] The underlined parts are the light chain variable regions CDR1, CDR2 and CDR3 of the antibody, and the amino acid sequences are SEQ ID NO: 13, YAS and SEQ ID NO: 14, respectively, which are divided according to the IMGT rules.

[0182] The amino acid sequence of the heavy chain variable region of the 2C5 antibody is shown below (SEQ ID NO: 2):

[0183] EVKLVESGGGLVKPGGSLKLSCAAS GFTFSSYA MSWVRQTPEKRLEWVAS ITNDDNT YYPGSVKGRFTISRDNARNILYLQMNSLRSEDTAMYYC VSFPYFDY WGQGTTLTVSS

[0184] The underlined parts are the heavy chain variable regions CDR1, CDR2 and CDR3 of the antibody, and the amino acid sequences are SEQ ID NO: 15, 16 and SEQ ID NO: 17, respectively, which are divided according to the IMGT rules.

[0185] The amino acid sequence of the light chain variable region of the 79G6 antibody is shown below (SEQ ID NO: 3):

[0186] DIQLTQSPASLSASVGETVTITCRAS ENIFSY LAWYQQKQGKSPHLLVY SAK TLAEGVPSRFSGSGSGTQFSLTISSLQPEDFGSYYC QHHYLFPWT FGGGSKLEIK

[0187] The underlined parts are the light chain variable regions CDR1, CDR2 and CDR3 of the antibody, and the amino acid sequences are SEQ ID NO: 18, SAK and SEQ ID NO: 19, respectively, which are divided according to the IMGT rules.

[0188] The amino acid sequence of the heavy chain variable region of the 79G6 antibody is shown below (SEQ ID NO: 4):

[0189] EVQLQQSGPELVKPGASVKISCKAS GYTFTDYN MDWVKQNHGKNLEWIGN INPNSDDTMYNRKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYC ARSGYAHYTLDY WGQGTLVTVSS

[0190] The underlined parts are the heavy chain variable regions CDR1, CDR2 and CDR3 of the antibody, and the amino acid sequences are SEQ ID NO: 20, 21 and SEQ ID NO: 22, respectively, which are divided according to the IMGT rules.

[0191] 1-4. Expression and preparation of recombinant antibodies

[0192] Gene synthesis of 2C5 antibody and 79G6 antibody light chain and heavy chain gene sequences encoding signal peptides, variable regions and constant regions (wherein, the 2C5 antibody light chain and heavy chain gene sequences are shown in SEQ ID NOs: 9 and 10, respectively, encoding amino acid sequences of light chain and heavy chain as shown in SEQ ID NOs: 5 and 6; the 79G6 antibody light chain and heavy chain gene sequences are shown in SEQ ID NOs: 11 and 12, respectively, encoding amino acid sequences of light chain and heavy chain as shown in SEQ ID NOs: 7 and 8), respectively, and constructed into mammalian cell expression vectors, and the expression vectors can be any commercial mammalian cell expression vectors such as pTT5 and pCDNA3.1. The recombinant plasmids are transfected into Expi293 mammalian cells, secreting and expressing 2C5 human-mouse chimeric antibody and 79G6 mouse monoclonal antibody, and the cell expression supernatant is purified by protein A affinity to obtain 2C5 human-mouse chimeric antibody and 79G6 mouse monoclonal antibody. It is used for antibody characterization and immunochemiluminescence kit development.

[0193] 2C5 human-mouse chimeric antibody light chain amino acid sequence (SEQ ID NO: 5, where the underline is the signal peptide sequence (SEQ ID NO: 23), and the italics are the light chain constant region human IgKappa (SEQ ID NO: 24))

[0194] METPAQLLFLLLLWLPDTTG DIQLTQSPAILSVSPGERVSFSCRASQSIDTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSYSWPTTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0195] 2C5 human-mouse chimeric antibody heavy chain amino acid sequence (SEQ ID NO: 6, where the underline is the signal peptide sequence (SEQ ID NO: 25), and the italics are the heavy chain constant region human IgG1 (SEQ ID NO: 26))

[0196] MKHLWFFLLLVAAPRWVLS EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASITNDDNTYYPGSVKGRFTISRDNARNILYLQMNSLRSEDTAMYYCVSFPYFDYWGQGTTLT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0197] 79G6 mouse monoclonal antibody light chain amino acid sequence (SEQ ID NO: 7, where the underline is the signal peptide sequence (SEQ ID NO: 27), and the italics are the light chain constant region mouse IgKappa (SEQ ID NO: 28))

[0198] MESQTQVLMSLLFWVSGTCG DIQLTQSPASLSASVGETVTITCRASENIFSYLAWYQQKQGKSPHLLVYSAKTLAEGVPSRFSGSGSGTQFSLTISSLQPEDFGSYYCQHHYLFPWTFGGGSKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0199] 79G6 mouse monoclonal antibody heavy chain amino acid sequence (SEQ ID NO: 8, where the underline is the signal peptide sequence (SEQ ID NO: 29), and the italicized words are the heavy chain constant region mouse IgG1 (SEQ ID NO: 30))

[0200] MEWSWVSLFFLSVTTGVHS EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMDWVKQNHGKNLEWIGNINPNSDDTMYNRKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARSGYAHYTLDYWG QGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0201] 2C5 human-mouse chimeric antibody light chain nucleotide sequence (SEQ ID NO: 9)

[0202] ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGACATACAGCTGACTCAATCCCCTGCAATACTCTCCGTCTCACCCGGCGAGCGGGTTAGTTTTTCCTGTAGAGCCTCTCAGTCCATCGATACTAGTATTCACTGGTACCAGCAGCGGACCAATGGAAGCCCCCGGCTGTTGATCAAGTACGCTTCTGAGTCAATCAGTGGTATCCCAAGTAGATTTTCCGGGTCTGGGTCCGGCACAGACTTCACACTCAGCATTAATTCCGTGGAGAGCGAAGATATCGCAGATTATTACTGCCAGCAAAGCTATAGCTGGCCCACCACCTTCGGAGGCGGGACTAAGCTGGAGATTAAAAGAACAGTGGCCGCTCCTAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGTCCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTATCCTAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAATAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGATTCCACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACAAAGTCCTTCAACAGAGGCGAGTGT

[0203] 2C5 human-mouse chimeric antibody heavy chain nucleotide sequence (SEQ ID NO: 10)

[0204]

[0205] Nucleotide sequence of the light chain of the 79G6 murine monoclonal antibody (SEQ ID NO:11)

[0206] ATGGAGTCTCAGACCCAGGTTCTCATGTCCCTGCTGTTTTGGGTGAGTGGAACCTGCGGGGACATCCAGCTCACCCAGAGTCCCGCAAGCCTGAGCGCCTCCGTCGGCGAAACCGTGACAATAACGTGCAGAGCAAGTGAAAATATCTTTTCCTATCTGGCGTGGTACCAACAGAAACAGGGGAAAAGCCCCCACCTGCTCGTTTACTCAGCCAAGACTCTGGCCGAGGGCGTGCCCAGCCGGTTTTCCGGGTCTGGCTCTGGCACCCAGTTTTCCCTGACCATATCCTCTCTCCAACCTGAGGACTTCGGAAGTTATTACTGTCAGCACCACTATCTGTTTCCATGGACCTTCGGGGGAGGGTCTAAGCTCGAGATCAAACGGGCAGATGCAGCCCCCACCGTGTCCATTTTCCCCCCATCATCTGAGCAACTTACATCAGGAGGAGCCAGCGTCGTATGTTTCCTGAATAATTTTTATCCCAAAGACATTAATGTTAAGTGGAAGATTGATGGCTCCGAAAGACAGAACGGGGTTCTCAATTCATGGACCGATCAGGACTCCAAGGATTCTACCTATTCCATGTCATCTACTCTGACCCTCACTAAAGATGAGTATGAGCGACATAATTCTTACACATGCGAGGCAACCCACAAAACCTCTACAAGCCCCATTGTTAAGTCCTTTAATCGGAATGAGTGC

[0207] Nucleotide sequence of the heavy chain of the 79G6 murine monoclonal antibody (SEQ ID NO:12)

[0208]

[0209] 1-5. Characterization of the binding of universal nuclease and antibody by SPR surface plasmon resonance

[0210] The binding of the universal nuclease to the 2C5 human-mouse chimeric antibody and the 79G6 mouse monoclonal antibody was characterized by a Biacore T200 (Cytiva) instrument. The SPR running buffer was 1×HBS-EP+buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4). First, the CM5 chip was activated, and 50mM NHS and 200mM EDC were freshly mixed in a 1:1 volume ratio, with a flow rate of 10ul / min, flowing through the chip channel, and activated for 420s. Then, the Anti-His tag antibody was coupled to the activated CM5 chip, diluted to 15μg / ml with 10mM Acetate (pH 4.5), flowed through the chip channel at a flow rate of 10μL / min, and the coupling level was about 9000RU, and then 1M ethanolamine (pH 8.5) was used to flow through the chip at a flow rate of 10μL / min for 420s. The universal nuclease containing the His tag (1μg / ml) flowed through the chip at a flow rate of 10μL / min for 20s and was captured on the chip by binding to the Anti-His tag antibody on the chip. The analytes 2C5 human-mouse chimeric antibody and 79G6 mouse monoclonal antibody were respectively bound to the universal nuclease captured on the chip in two channels. The antibodies flowed over the surface of the antigen chip at five concentration gradients of 25nM, 12.5nM, 6.25nM, 3.125nM, and 1.5625nM, with a flow rate of 30μL / min, a binding time of 120s, and a dissociation time of 300s to obtain the kinetic parameters of antigen-antibody binding.

[0211] The results are as follows Figure 1 As shown, the KD value of the 2C5 human-mouse chimeric antibody binding to the universal nuclease is 2.9E-11M, and the KD value of the 79G6 mouse monoclonal antibody binding to the universal nuclease is 4.4E-11M.

[0212] Example 2 Immunochemiluminescent universal nuclease residual detection kit based on 2C5 and 79G6 antibody pair

[0213] 2-1. Biotin labeling of capture antibody 2C5

[0214] Take the required amount of the recombinant human-mouse chimeric antibody 2C5 to be labeled and dialyze overnight at 2-8°C, and adjust the antibody concentration to 2 mg / ml with PBS. Mix NHS-Biotin and 2C5 antibody at a molar ratio of 20:1, incubate on ice for 2 hours, and after labeling, dialyze with PBS buffer at 2-8°C to obtain biotin-labeled capture antibody.

[0215] 2-2. Streptavidin magnetic beads coated with 2C5 capture antibody

[0216] Streptavidin magnetic beads (1um, Sangon Biotechnology) and biotin-labeled 2C5 antibodies were mixed at a mass ratio of 40:1, with a final concentration of no less than 5 mg / ml, and placed on a constant temperature shaker at 37°C for 16-24 hours. After the reaction, the magnetic beads were washed 4 times with 2 volumes of PBST, and the washed 2C5 capture antibody-coated magnetic beads were adjusted to 4 mg / ml with PBST and stored at 2-8°C for later use.

[0217] 2-3. Detection antibody 79G6 acridinium ester labeling

[0218] Take the required amount of 79G6 mouse monoclonal antibody to be labeled, adjust the antibody concentration to 2 mg / ml with PBS, and dialyze with PBS buffer at 2-8°C overnight. Mix NSP-SA-NHS acridinium ester and 79G6 antibody at a molar ratio of 15:1, place in a constant temperature incubator at 25°C, and shake for 2 hours. After labeling, dialyze with PBS to obtain 79G6 acridinium ester labeled detection antibody, and store at 2-8°C for use.

[0219] 2-4. Universal Nuclease Standards

[0220] The universal nuclease (GMP-NUC-SE101, Kactus Biosystems) was diluted with PBST, 1% BSA solution to obtain a 50 ng / ml standard, and then diluted 4-fold to 6 concentration gradients: 12.5 ng / ml, 3.125 ng / ml, 0.781 ng / ml, 0.195 ng / ml, 0.048 ng / ml, 0.012 ng / ml, to obtain 7 concentrations of standard. PBST, 1% BSA solution was used as a blank control.

[0221] 2-5. Standard curve of one-step immunochemiluminescent universal nuclease residual detection kit

[0222] The 2C5 capture antibody-coated magnetic beads were diluted to 10ug / ml with PBST, and the acridinium ester-labeled 79G6 detection antibody was diluted to 0.5ug / ml with PBST. 100ul of each was taken and placed in a fully automatic chemiluminescence immunoassay analyzer (SMART 500S, Chongqing Cosmay Biotechnology Co., Ltd.). Seven concentration standards and blank controls, 100ul each, were added to 8 reaction cups and placed in the analyzer. The instrument flow was set, and 50ul of capture antibody-coated magnetic beads and detection antibody were taken and added to each reaction cup. After mixing and incubating for 30 minutes, an antibody-antigen-antibody sandwich complex was formed, and the antibody was washed 4 times with PBST, and the chemiluminescence detection value was read. The test results are shown in Table 1.

[0223] Table 1 Results of immunochemiluminescence detection of universal nuclease residues

[0224] Universal Nuclease Standard (ng / ml) Chemiluminescence detection value 50 3553099 12.5 795296 3.125 169135 0.781 35595 0.195 9915 0.049 2372 0.012 1971 0 832

[0225] like Figure 2 As shown in the standard curve, the immunochemiluminescence universal nuclease residual detection kit has good linearity in the range of 0.012-50ng / ml. Compared with ordinary ELISA kits, it has higher sensitivity and wider detection range. The experimental operation can be completed in 40 minutes, which is also faster.

[0226] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An anti-universal nuclease antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (Z1) the heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO: 15; H-CDR2 as shown in SEQ ID NO: 16; H-CDR3 as shown in SEQ ID NO: 17; and The light chain variable region comprises the following three CDRs: L-CDR1 as shown in SEQ ID NO: 13; L-CDR2 as shown in YAS; L-CDR3 as shown in SEQ ID NO: 14; or (Z2) the heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO:20; H-CDR2 as shown in SEQ ID NO:21; H-CDR3 as shown in SEQ ID NO:22; and The light chain variable region comprises the following three CDRs: L-CDR1 shown in SEQ ID NO: 18; L-CDR2 whose sequence is shown in SAK; and L-CDR3 shown in SEQ ID NO:

19.

2. A recombinant antibody, characterized in that: The recombinant antibody comprises: (i) the sequence of the anti-universal nuclease antibody or antigen-binding fragment thereof according to claim 1; and (ii) a signal peptide that promotes secretory expression of the antibody and / or a tag sequence for purification and detection.

3. A polynucleotide molecule encoding a polypeptide selected from the group consisting of: (1) the anti-universal nuclease antibody or antigen-binding fragment thereof according to claim 1; or (2) The recombinant antibody according to claim 2.

4. An expression vector comprising the polynucleotide molecule as described in the third aspect of the present invention.

5. An antibody conjugate, characterized in that: The antibody conjugate comprises: (a) the anti-universal nuclease antibody or antigen-binding fragment thereof according to claim 1 or the recombinant antibody according to claim 2; and (b) a conjugated moiety selected from the group consisting of a detectable label, an enzyme, a radionuclide, a nanoparticle / nanorod.

6. An antibody combination targeting a universal nuclease, characterized in that: The antibody combination includes: (A1) a first antibody, whose heavy chain variable region comprises the following three CDRs: H-CDR1 shown in SEQ ID NO: 15; H-CDR2 shown in SEQ ID NO: 16; H-CDR3 shown in SEQ ID NO: 17; and The light chain variable region comprises the following three CDRs: L-CDR1 as shown in SEQ ID NO: 13; L-CDR2 as shown in the sequence YAS; L-CDR3 as shown in SEQ ID NO: 14; and (A2) a second antibody, whose heavy chain variable region comprises the following three CDRs: H-CDR1 shown in SEQ ID NO: 20; H-CDR2 shown in SEQ ID NO: 21; H-CDR3 shown in SEQ ID NO: 22; and The light chain variable region comprises the following three CDRs: L-CDR1 as shown in SEQ ID NO: 18; L-CDR2 as shown in SAK; and L-CDR3 as shown in SEQ ID NO:

19.

7. Use of the anti-totipotent nuclease antibody or antigen-binding fragment thereof according to claim 1, the recombinant antibody according to claim 2, the antibody conjugate according to claim 5, or the antibody combination according to claim 6 in the preparation of a detection reagent, a detection plate or a detection kit for detecting totipotent nuclease.

8. A detection reagent for detecting residual omnipotent nuclease, comprising the anti-omnipotent nuclease antibody or its antigen-binding fragment as claimed in claim 1, the recombinant antibody as claimed in claim 2, the antibody conjugate as claimed in claim 5, or the antibody combination as claimed in claim 6, and a carrier acceptable for detection.

9. A kit for detecting residual omnipotent nuclease, comprising the anti-omnipotent nuclease antibody or antigen-binding fragment thereof according to claim 1, the recombinant antibody according to claim 2, the antibody conjugate according to claim 5, or the antibody combination according to claim 6, or a combination thereof.

10. A method for detecting residual omnipotent nuclease, the method comprising the steps of: using the detection reagent according to claim 8 or the kit according to claim 9 to detect a sample to be tested.

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