An NAD + Antibodies and uses thereof

Abstract

The application discloses a kind of NAD + Antibody and its application, relate to the field of biological pharmaceutical technology.The NAD + Provided by the application has strong affinity to NAD + The antibody is high in specificity, uses it to detect NAD + , high sensitivity and simple operation.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more particularly to an NAD+. + Antibodies and their applications. Background Technology

[0002] Traditional detection of nicotinamide adenine dinucleotide (NAD) + The methods are mainly based on the principles of spectroscopy, chromatography, or enzyme-catalyzed reactions. They are classic detection methods in biochemistry and molecular biology research. However, due to limitations in technical principles and operational design, they have obvious shortcomings in terms of sensitivity, specificity, ease of operation, detection throughput, time efficiency, and cost.

[0003] To meet the practical application needs of the in vitro diagnostics (IVD) field, we will develop a simple-to-operate, instrument-independent, and accurate NAD reading method. + There is an urgent need for detection methods. Summary of the Invention

[0004] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a NAD + Antibodies and their applications. The NAD provided by this invention... + Antibody against NAD + It has strong affinity and high specificity, and its use for NAD+ + It is highly sensitive and easy to operate for testing.

[0005] (II) Technical Solution To achieve the above objectives, the present invention provides a NAD + Antibody, the NAD + The antibody contains heavy chain variable regions CDR1, CDR2, and CDR3. The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85 or 91; The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86 or 92; The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87 or 93.

[0006] In one embodiment, the NAD +The antibody also contains light chain variable regions CDR1, CDR2, and CDR3. The amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88 or 94; The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89 or 95; The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90 or 96.

[0007] In one embodiment, the amino acid sequence of the heavy chain variable region CDR1 is as shown in SEQ ID NO: 1, 7, 13 or 19; The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 2, 8, 14 or 20; The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 3, 9, 15 or 21; The amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 4, 10, 16 or 22; The amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 5, 11, 17 or 23; The amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 6, 12, 18 or 24.

[0008] In one embodiment, the NAD + The antibody (8R2-A7) comprises a heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 2, a heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 3, a light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and / or a light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0009] In one embodiment, the NAD +The antibody (8R2-B8) comprises a heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 7, a heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 8, a heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 9, a light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 10, a light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 11, and / or a light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 12.

[0010] In one embodiment, the NAD + The antibody (8R2-C12) comprises a heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 13, a heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 14, a heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 15, a light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 16, a light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and / or a light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 18.

[0011] In one embodiment, the NAD + The antibody (8R2-D15) comprises the heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 19, the heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 20, the heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 21, the light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 22, the light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 23, and / or the light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 24.

[0012] In one embodiment, the NAD + The antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 25 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 26.

[0013] In one embodiment, the NAD + The antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 27 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 28.

[0014] In one embodiment, the NAD +The antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 29 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0015] In one embodiment, the NAD + The antibody also contains an Fc domain, which includes one or a combination of IgG1 Fc domain, IgG2 Fc domain, IgG3 Fc domain or IgG4 Fc domain.

[0016] In one embodiment, the Fc domain is the IgG1 Fc domain.

[0017] In one embodiment, the IgG1 Fc domain is a human IgG1 Fc (hIgG1) domain.

[0018] In one embodiment, the aforementioned NAD + The antibody also contains an immunomarker, which includes one or a combination of enzyme markers, fluorescein markers, isotope markers, or biotin markers.

[0019] In another aspect, the present invention provides an encoding of the aforementioned NAD. + The DNA molecule of an antibody.

[0020] In another aspect, the present invention also provides an expression vector comprising the aforementioned DNA molecule.

[0021] In another aspect, the present invention provides a cell comprising the aforementioned NAD+. + Antibodies, DNA molecules, and / or expression vectors.

[0022] In another aspect, the present invention also provides a method for generating an antibody or an antigen-binding fragment thereof, comprising: The cells were cultured under conditions sufficient to induce them to produce antibodies or antigen-binding fragments; and Collect antibodies or antigen-binding fragments produced by the cells.

[0023] In another aspect, the present invention also provides the aforementioned NAD + Antibodies in the detection of NAD + Applications in [the context of the text].

[0024] This invention relates only to detection for non-diagnostic purposes.

[0025] In another aspect, the present invention also provides NAD + The detection reagent contains the above-mentioned NAD. + Antibodies, DNA molecules, expression vectors, and / or cells.

[0026] In another aspect, the present invention also provides NAD + The detection kit contains the aforementioned detection reagents.

[0027] In another aspect, the present invention provides a NAD + The testing products include the aforementioned testing reagents or testing kits.

[0028] (III) Beneficial Effects This invention provides an NAD + Antibodies and their applications. Compared with existing technologies, they have the following advantages: The NAD provided by this invention + Antibody against NAD + It has strong affinity and high specificity, and its use for NAD+ + It is highly sensitive and easy to operate for testing. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Terms and Definitions As used herein, the term "expression vector" can be a viral vector or a non-viral vector, and the viral vector can be an adeno-associated virus (AAV) vector, adenovirus vector, alphavirus vector, herpes simplex virus vector or vaccinia virus vector, Sendai virus vector, flavivirus vector, rod-shaped virus vector, retrovirus vector, herpesvirus vector, poxvirus vector, or lentivirus vector. In this method, the non-viral vector can be a DNA vector, nanoparticles, cationic polymers, exosomes, extracellular vesicles, or liposomes, and the DNA vector can be a plasmid vector, granular vector, phage particle vector, or human artificial chromosome.

[0031] Expression vectors may contain links to encoding NAD + Antibody polynucleotides enable NAD + Antibodies can be overexpressed in host cells with appropriate regulatory sequences, wherein the construct sequence is operatively linked to the polynucleotide. In this case, the regulatory sequence and the NAD-encoding sequence operatively linked thereto are integrated. + Antibody polynucleotides can be referred to as "gene constructs". Gene constructs may contain appropriate restriction enzyme recognition sites at both ends for cloning in expression vectors.

[0032] As used herein, the term "operably linked" refers to the ligation of a nucleic acid sequence of interest (e.g., in an in vitro transcription / translation system or in a host cell) to a regulatory sequence in a manner that enables its expression. The term "regulatory sequence" includes promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include constitutive elements that guide the constitutive expression of the target nucleic acid in a variety of host cells, and inducible elements that guide the expression of the target nucleic acid only in specific tissues or cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of expression vectors can vary depending on factors such as the choice of host cells to be transformed and the desired level of protein expression. Regulatory sequences that enable expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, they typically contain a regulatory sequence responsible for the initiation of transcription, and optionally a polyadenylation signal responsible for the termination and stabilization of transcription. In addition to transcriptional regulators, additional regulatory sequences may include translational enhancers and / or naturally bound or heterologous promoter regions. Possible regulatory sequences enabling expression in, for example, mammalian host cells include the CMV-HSV thymidine kinase promoter, the SV40 promoter, the RSV promoter (Rouse sarcoma virus), the human kidney element 1α promoter, the glucocorticoid-inducible MMTV (mouse mammary tumor virus) promoter, the metallothionein-inducible promoter, or the tetracycline-inducible promoter, or enhancers such as the CMV enhancer or the SV40 enhancer. For expression in neurons, neurofilament promoters, PGDF promoters, NSE promoters, PrP promoters, or thy-1 promoters can be considered. These promoters are known in the art and described in the literature (Charron et al., J. Biol. Chem. 270: 25739-25745, 1995, etc.). For expression in prokaryotic cells, numerous promoters have been disclosed, including the lac promoter, the tac promoter, or the trp promoter. In addition to factors capable of initiating transcription, regulatory sequences include transcription termination signals, such as SV40 polyA sites or TK polyA sites downstream of a polynucleotide according to one embodiment of the invention. Suitable expression vectors used herein are known in the art, such as the Okaya-Berg cDNA expression vectors pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pX (Pagano et al., Science 255:1144-1147, 1992), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris et al., Cell 75:791-803, 1995), or prokaryotic expression vectors such as λgt11 or pGEX (Amersham-Pharmacia).In addition to the nucleic acid molecules disclosed in this invention, the vector may further comprise a polynucleotide encoding a secretion signal. Secretion signals are well known to those skilled in the art. Furthermore, depending on the expression system used, a leader sequence capable of guiding the translated protein to a cellular compartment (e.g., the nucleus) is combined with a coding sequence of a polynucleotide according to an embodiment of the invention, wherein the leader sequence is preferably a leader sequence capable of secreting the translated protein into the pericytoplasm or extracellular space.

[0033] Furthermore, the expression vectors used in this invention can be prepared, for example, by standard recombinant DNA techniques, which include, for example, ligation of blunt and sticky ends, treatment with restriction enzymes to provide suitable ends, removal of phosphate groups by alkaline phosphatase treatment to prevent inappropriate binding, and enzymatic ligation by T4 DNA ligase, etc. The vectors of this invention can be prepared by using DNA encoding a signal peptide obtained through chemical synthesis or genetic recombination techniques, or encoding NAD according to an embodiment of the invention. + Antibody DNA is prepared by recombining it into a vector containing appropriate regulatory sequences. Vectors containing regulatory sequences can be purchased or commercially prepared.

[0034] Furthermore, in this method, the gene construct may further contain polynucleotides encoding one or more immunostimulatory peptides. In this case, the immunostimulatory peptide is in the form of a separate regulatory sequence, i.e., a bicistronic form. It is contained in an expression vector or linked to a regulatory sequence, but an internal ribosome entry site (IRES) may be inserted between the polynucleotides encoding two proteins, which are transcribed into a single mRNA and then translated into the respective proteins. The immunostimulatory peptide may be CD28, ICOS (inducible costimulatory factor), CTLA4 (cytotoxic T lymphocyte-associated protein 4), PD1 (programmed cell death protein 1), BTLA (B and T lymphocyte-associated protein), DR3 (death receptor 3), 4-1BB, CD2, CD40, CD40L, CD30, CD27, signal transduction lymphocyte activation molecule (SLAM), 2B4 (CD244), NKG2D (natural killer cell family 2, member D) / DAP12 (DNAX activator protein 12). TIM1 (protein 1 containing T cell immunoglobulin and mucin domains), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3), B7-1, B7-H1, GITR (glucocorticoid-induced TNFR family-associated protein), Flt3 ligand (fms-like tyrosine kinase 3 ligand), flagellin, herpesvirus entry mediator (HVEM), or cytoplasmic domains of OX40L [ligands of CD134 (OX40), CD252], or combinations of two or more thereof.

[0035] The polynucleotides disclosed in this invention, when expressed in higher eukaryotic cells, will enhance transcription when an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0036] As used herein, the term "antibody" refers to a polypeptide that comprises components sufficient to confer antibodies against a specific target antigen (e.g., NAD+). + This refers to the specific binding of canonical immunoglobulin sequence elements. As is known in the art, naturally occurring intact antibodies are tetrameric reagents of about 150 kDa, comprising two identical heavy chain polypeptides (each about 50 kDa) and two identical light chain polypeptides (each about 25 kDa), which associate with each other to form a structure commonly referred to as a “Y-shape.” Each heavy chain contains at least four domains (each about 110 amino acids long): an N-terminal variable (VH) domain (located at the tip of the Y-shape), followed by three constant domains: CHI, CH2, and C-terminal CH3 (located at the base of the Y-stem). A short region called a “switch” connects the heavy chain variable and constant regions. A “hinge” connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region link the two heavy chain polypeptides to each other in the intact antibody. Each light chain contains two domains: an N-terminal variable (VL) domain, followed by a C-terminal constant (CL) domain, separated from each other by another “switch.” Those skilled in the art are familiar with the structural and sequence elements of antibodies, can identify “variable” and “constant” regions in the provided sequence, and understand that there may be some flexibility in defining the “boundaries” between these domains, thereby allowing different representations of the same antibody chain sequence to indicate, for example, the location of such boundaries shifted by one or more residues relative to different representations of the same antibody chain sequence.

[0037] A complete antibody tetramer consists of two heavy-chain-light-chain dimers, where the heavy and light chains are linked together by a single disulfide bond; two additional disulfide bonds link the heavy-chain hinge regions together, thus linking the dimers together to form a tetramer. Naturally occurring antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold," formed by two β-sheets (e.g., 3, 4, or 5 folds) stacked on top of each other in compressed antiparallel β-barrels. Each variable domain contains three hypervariable loops called "complementarity-determining regions" (CDR1, CDR2, and CDR3) and four nearly invariant "framework" regions (FR1, FR2, FR3, and FR4). When the natural antibody folds, the FR regions form β-sheets to provide the structural framework of the domain, and the CDR loop regions from both the heavy and light chains aggregate in three-dimensional space, thus forming a single hypervariable antigen-binding site located at the tip of the Y-shaped structure. Naturally occurring antibodies have Fc regions that bind to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity of the Fc region for Fc receptors and / or other binding properties can be modulated by glycosylation or other modifications. In some embodiments, antibodies generated and / or utilized according to the invention comprise glycosylated Fc domains, including modified or engineered such glycosylated Fc domains.

[0038] For the purposes of this invention, in some embodiments, any polypeptide or polypeptide complex including sufficient immunoglobulin domain sequences found in natural antibodies may be referred to as and / or used as an "antibody," regardless of whether the polypeptide is naturally occurring (e.g., produced by an organism reacting with an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences that are specific to mouse, rabbit, primate, or human antibodies. In some embodiments, as known in the art, the antibody sequence elements are humanized, primate-derived, chimeric, etc.

[0039] As used herein, the term "detection reagent" refers to a reagent that specifically binds to a particular antigen. In some embodiments, the term covers any polypeptide or polypeptide complex that includes sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody reagents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, antibody reagents may include one or more constant region sequences specific to mouse, rabbit, primate, or human antibodies. In some embodiments, as known in the art, antibody reagents may include one or more humanized, primate-like, chimeric, etc., sequence elements. In many embodiments, the term "detection reagent" is used to refer to one or more constructs or forms known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative representations.

[0040] As used in this article, the term "specific binding" refers to NAD. + Antibodies can preferably bind to binding couplers, such as NAD+, in competitive binding assays. + The evaluation may be conducted using recombinant forms of proteins, epitopes therein, or native proteins present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

[0041] As used herein, the term "affinity" refers to the strength of binding between an antibody or protein and an epitope. The affinity of an antibody is given by the dissociation constant KD, defined as [Ab] × [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant KA is defined by 1 / KD. Preferred methods for determining protein affinity can be found in Har low et al., “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988); Coligan et al. (eds.), “Current Protocols in Immunology,” Greene Publishing Assoc and Wiley Interscience, New York (1992, 1993); and Muller, Meth. Enzymol., Vol. 92: pp. 589–601 (1983). A preferred standard method well known in the art for determining protein affinity is the use of surface plasmon resonance (SPR) screening (such as by analysis with the BIAcore™ SPR analysis device).

[0042] As used herein, the terms “identity” or “similarity” when used in relation to the sequences of two or more polypeptides refer to the degree of sequence correlation between the polypeptides, as determined by the number of matches between chains of two or more amino acid residues. “Identity” measures the percentage of identical matches between smaller sequences of two or more sequences with vacancy alignments (if any), resolved by a specific mathematical model or computer program (i.e., an “algorithm”). The identity of related polypeptides can be readily calculated using known methods. Such methods include, but are not limited to, those described in the following literature: “Computational Molecular Biology,” edited by Lesk, AM, Oxford University Press, New York, 1988; “Biocomputing: Informatics and Genome Projects,” edited by Smith, DW, Academic Press, New York, 1993; “Computer Analysis of Sequence Data, Part 1,” edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; “Sequence Analysis in Molecular Biology,” von Heinje, G., Academic Press, 1987; “Sequence Analysis Primers,” edited by Gribskov, M. and Devereux, J., M. Stockton Press. Press), New York, 1991; and Carillo et al., Journal of Industrial and Applied Mathematics (SIAM J. Applied Math.), Vol. 48, p. 1073 (1988).

[0043] The preferred method for determining identity is designed to give the maximum match between the sequences being tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG package, including GAP (Devereux et al., Nucl. Acid. Res.), Vol. 12, p. 387 (1984); the Genetics Computer Group, University of Wisconsin, Madison, Wisconsin; BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., Vol. 215, pp. 403-410 (1990)). The BLASTX procedure is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (“BLAST Manual”, Altschul et al., NCB / NLM / NIH, Bethesda, MD, 20894; Altschul et al., ibid.). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0044] As used herein, the term "antigen" refers to a structure that an antibody can selectively bind to. In some embodiments, the target antigen is NAD+. + In some implementations, the antigen is associated with a cell, for example, it is present on or within the cell.

[0045] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0047] Example 1 Antibody screening: 1. Antibody screening: The amino acid sequences are shown in Table 1 (H represents the heavy chain and K represents the light chain).

[0048] Table 1 Amino acid sequence 2. Recombinant plasmid construction: The heavy chain variable region DNA molecule was seamlessly cloned into the expression vector hIgG1 to obtain the heavy chain expression plasmid. The light chain variable region DNA molecule was seamlessly cloned into the expression vector hIgκ or hIgλ to obtain the light chain expression plasmid. The coding regions of both the heavy and light chains consist of three parts: a signal peptide (enzyme marker), a variable region, and a constant region (the constant region of human IgG antibody).

[0049] 3. Antibody screening: Paired heavy chain expression plasmids and light chain expression plasmids were co-transfected into HEK-293T cells and cultured for 48 hours to express antibodies, yielding the antibody supernatant. The antibody supernatant was used for initial screening of antibody affinity and neutralizing activity.

[0050] 3.1. Screening of positive clones using ELISA combined with experimental methods: 3.1.1. Antigen coating: BSA-NAD diluted with PBS + The antigen was prepared to a final concentration of 1 μg / ml. 100 µl was coated per well of the ELISA plate and incubated overnight at 4°C.

[0051] 3.1.2. Blocking: Discard the solution and wash each well three times with 200 µl of PBST. Add 200 µl of blocking buffer to each well and block at 37°C for 2 h.

[0052] 3.1.3. Washing: Discard the supernatant, add 200 µl PBST to each well and wash 3 times, then pat dry.

[0053] 3.1.4. Add cell culture antibody supernatant: Dilute the antibody supernatant 3-fold serially, add 100 µl of antibody solution to each well, with the first well containing undiluted stock solution, and PBS as the negative control. Incubate at 37°C for 1 h.

[0054] 3.1.5. Add secondary antibody: Discard the supernatant, add 200 µl of PBST to each well and wash 3 times, then blot dry. Add 100 µl of HRP-labeled goat anti-human IgG secondary antibody to each well. Incubate at 37°C for 1 h.

[0055] 3.1.6. Color Development: Discard the supernatant, add 200 µl of PBST to each well and wash 4 times, then blot dry. Add 100 µl of TMB color development solution. After the color changes, add 50 µl of 10% sulfuric acid stop solution to each well to terminate the reaction.

[0056] 3.1.7. Reading: Read the absorbance at 450 nm using a microplate reader. One replicate of the antibody supernatant from each cell culture line was prepared, and each experiment was independently repeated once. Antibodies with an average absorbance higher than 2.0 were selected as positive antibodies.

[0057] The experimental results are shown in Table 2. Four NAD plants were obtained. + Positive antibody.

[0058] Table 2 Antibody strains against NAD + Affinity test results Example 2 Antibody strain NAD + Specific detection: The steps are as follows: 1. Antigen coating: NAD+ diluted with PBS + antigen (BSA-NAD) + The final concentration was set to 1 μg / ml. 100 µl was coated per well of the ELISA plate and incubated overnight at 4°C.

[0059] 2. Blocking: Discard the solution and wash each well three times with 200 µl of PBST. Add 200 µl of blocking buffer to each well and block at 37°C for 2 hours.

[0060] 3. Washing: Discard the supernatant, add 200 µl PBST to each well and wash 3 times, then blot dry.

[0061] 4. Add cell culture antibody supernatant: Add 100 µl of undiluted antibody solution to each well, then add 100 µl of NAD. + The analogue dilutions were all at a competing concentration of 1 μg / ml, with PBS as a negative control, and incubated at 37°C for 1 h.

[0062] 5. Add secondary antibody: Discard the supernatant, add 200 µl of PBST to each well and wash 3 times, then blot dry. Add 100 µl of HRP-labeled goat anti-human IgG secondary antibody to each well and incubate at 37°C for 1 h.

[0063] 6. Color Development: Discard the supernatant, add 200 µl of PBST to each well and wash 4 times, then blot dry. Add 100 µl of TMB color development solution. After the color changes, add 50 µl of 10% sulfuric acid stop solution to each well to terminate the reaction.

[0064] 7. Reading: Read the absorbance at 450 nm using a microplate reader. Set up one replicate for the antibody supernatant from each cell culture line, and repeat each experiment independently. Select antibodies with an average absorbance higher than 2.0 as positive antibodies.

[0065] The experimental results are shown in Table 3.

[0066] Table 3 Antibody strains against NAD + Specific detection results 1. The heavy chain expression plasmids and light chain expression plasmids of the four highly specific antibodies (8R2-A7, 8R2-B8, 8R2-C12, and 8R2-D15) were co-transfected into 293F cells to obtain recombinant cells. The cells were cultured in SMM 293-TII complete medium for 96 h, then centrifuged at 3000 rpm for 30 min at 4 °C, and the supernatant was collected.

[0067] 2. Protein A magnetic bead affinity chromatography purification of antibodies: Affinity chromatography packing material: Protein A magnetic beads; elution buffer: 0.1M glycine, pH 3.0; neutralization buffer: 1M Tris, pH 8.5.

[0068] Mix 50 ml of supernatant with Protein A magnetic beads and incubate at 4°C for 16 h. Separate the solution and beads using a magnetic rack. Add 20 ml of PBS solution to the beads, vortex to mix, wash the beads for 5 min, collect the beads using a magnetic rack and discard the supernatant; repeat the washing step three times. Add 9 ml of elution buffer to the beads, quickly resuspend, mix thoroughly, invert to mix at room temperature, and incubate for 5 min. Collect the beads using a magnetic separator and transfer the elution buffer to a clean centrifuge tube. Add 1 ml of neutralization buffer to the 9 ml of elution buffer to neutralize the pH, in order to maintain the biological activity of the antibody and prevent antibody inactivation.

[0069] 3. Solution displacement: The resulting eluent was concentrated using an ultrafiltration tube, and the solution was replaced with PBS buffer to obtain an antibody solution with a protein concentration of 1 mg / ml.

[0070] The amino acid sequences of the variable regions of the four antibodies are shown in Table 4: Table 4. Amino acid sequence of the variable region Furthermore, the screened antibodies also included light chain constant regions and heavy chain constant regions, and the heavy chain constant regions of all antibody sequences were identical to those of the light chain constant regions: HC (SEQ ID NO: 129): ASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; LC (SEQ ID NO: 130): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0071] Example 3 and "commercially available" NAD + Comparison of test kits: The Wuhan Yunclone NAD kit (kit 1) and Nicotinamide Adenine Dinucleotide (NAD) Polyclonal Antibody (Biomatik, kit 2) were used for detection. Standards were diluted according to the instructions; samples were prepared within the reference curve range; 50 μL of standard and sample were added to each well, along with 50 μL of solution A, and incubated at 37°C for 1 h; the plate was washed 3 times; 100 μL of solution B was added to each well, and incubated at 37°C for 30 min; the plate was washed 5 times; 90 μL of TMB chromogenic solution was added to each well, and incubated at 37°C for 10 min; 50 μL of stop solution was added to each well, and the readings were recorded. The results are shown in Table 5.

[0072] Table 5 Test Results of Commercially Available Test Kits The test results show that the OD values ​​of the two commercially available kits only changed with the different standards, and did not change with NAD. + The concentration of NAD+ changes, therefore, using the two commercially available kits can only detect the standard of that kit, and cannot detect NAD+ in the sample. + content.

[0073] Example 4: Screening antibody detection verification: Two commercially available reagent kit reaction systems were used to detect NAD in samples using the four high-performance antibodies described in this invention. + Content, observe whether the OD value changes with NAD + The content shows a regular variation.

[0074] Table 6. Comparison of detection results between screening antibodies and commercially available kits. Table 7 Comparison of detection results between screening antibodies and commercially available kits As shown in Tables 6 and 7, the OD values ​​of the four high-performance antibodies described in this invention all increased with NAD in different reaction systems. + Regular changes in NAD concentration can be used as an effective method for detecting NAD. + Antibodies.

[0075] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of NAD + Antibody, characterized in that, Include: VH CDR 1, 2, 3 having the selected VH CDR 1, 2, 3 amino acid sequence, and VL CDR 1, 2, 3 having the selected VL CDR 1, 2, 3 amino acid sequence; The selected VH CDR 1, 2, 3 amino acid sequences and the selected VL CDR 1, 2, 3 amino acid sequences are one of the following: (1) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1, 2, and 3, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4, 5, and 6, respectively; (2) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7, 8, and 9, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10, 11, and 12, respectively; (3) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13, 14, and 15, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16, 17, and 18, respectively; (4) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19, 20, and 21, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22, 23, and 24, respectively.

2. The NAD according to claim 1 + Antibody, characterized in that, The NAD + The antibody also contains an Fc domain, which includes one or a combination of IgG1 Fc domain, IgG2 Fc domain, IgG3 Fc domain or IgG4 Fc domain.

3. The NAD according to claim 1 + Antibody, characterized in that, The NAD + The antibody also contains an immunomarker, which includes one or a combination of enzyme markers, fluorescein markers, isotope markers, or biotin markers.

4. A DNA molecule, characterized in that, Encoding the NAD as described in any one of claims 1-3 + Antibody.

5. An expression carrier, characterized in that, The expression vector comprises the DNA molecule of claim 4.

6. A cell, characterized in that, The cells contain NAD according to any one of claims 1-3. + Antibody, the DNA molecule of claim 4 and / or the expression vector of claim 5.

7. A type of NAD + The detection reagent is characterized by, The detection reagent comprises the NAD according to any one of claims 1-3. + Antibody, DNA molecule of claim 4, expression vector of claim 5, and / or cell of claim 6.

8. A type of NAD + The detection kit is characterized in that, The test kit contains the test reagent as described in claim 7.

9. A type of NAD + The testing product is characterized by, The testing product comprises the testing reagent of claim 7 or the testing kit of claim 8.

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