A group of monoclonal antibodies to Nova-1 protein and their products and a method for detecting Nova-1 protein

Abstract

The present invention discloses a group of monoclonal antibodies of Nova‑1 protein and its products and a method for detecting Nova‑1 protein, which belongs to the technical field of monoclonal antibodies. The present invention analyzes and obtains the CDR region sequences of the light chain and heavy chain variable regions of the above monoclonal antibodies, and on this basis, it can provide support for the construction of highly specific and high-affinity Nova‑1 protein genetic engineering antibodies. The human-mouse chimeric monoclonal antibodies of Nova‑1 protein prepared by the present invention are basically consistent with the activity of anti-Nova‑1 mouse monoclonal antibodies after modification, and compared with commercial antibodies, the mouse-derived monoclonal antibodies prepared by the present invention do not react with anti-human Fab antibodies, and have better specificity. The modified human-mouse chimeric antibodies can bind to anti-human Fab antibodies, and can be used as a substitute for human serum antibodies to meet the needs of diagnostic kits, with better specificity, higher affinity and a wider range of applications.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibodies and specifically relates to a group of monoclonal antibodies of Nova-1 protein and their products and a method for detecting Nova-1 protein. Background Art

[0002] Immunohistochemistry is the application of the basic principle of immunology - antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody. Through chemical reaction, the color developer (fluorescein, enzyme, metal ion, isotope) of the labeled antibody is used to develop color to determine the antigen (peptide and protein) in the tissue cells, and to conduct localization, qualitative and quantitative research on it. In this process, antibodies are one of the indispensable reagents. There are currently two main types of antibodies: polyclonal antibodies and monoclonal antibodies. Since polyclonal antibodies have the ability to bind to different antigenic epitopes of a specific molecule, polyclonal antibodies are usually used as secondary antibodies in immunoassays; in view of the ability of monoclonal antibodies to specifically bind to a single antigenic epitope of an antigen (lower cross-reactivity), they are usually used as primary antibodies in immunoassays. For applications that require a large number of the same antigenic epitope and small batch differences (such as therapeutic drugs), monoclonal antibodies are a better choice.

[0003] Nova-1 (Neuro-oncological ventral antigen 1, Nova-1) is a protein expressed in tumors and neurons. It is the target antigen of human paraneoplastic opsoclonus-myoclonusataxia (POMA). Detection of autoantibodies to Nova-1 protein in patient samples plays an extremely important role in the diagnosis and treatment of the disease. In recent years, many studies have shown that Nova-1 protein is involved in regulating the occurrence and development of tumors. The upregulation or downregulation of Nova-1 protein in tissues is closely related to the exploration of etiology and the prognosis of the disease (Reference 1: Zhang YA, Liu HN, Zhu JM, et al. RNA binding protein Nova1 promotes tumor growth invivo and its potential mechanism as an oncogene may due to its interactionwith GABAA Receptor-γ2[J]. Journal of Biomedical Science, 2016, 23(1): 1-9. Reference 2: Kim EK, Yoon SO, Jung WY, et al. Implications of NOVA1 suppression within the microenvironment of gastric cancer: association with immune cell dysregulation[J]. Gastric cancer, 2017, 20: 438-447).

[0004] Whether it is to detect autoantibodies to Nova-1 protein or to identify the expression of Nova-1 protein, Nova-1 antibodies must be used as a control or as a primary antibody; another situation where Nova-1 antibodies are needed is when preparing a kit for detecting Nova-1 autoantibodies, a positive control sample is needed to meet the customer's product quality testing. Usually, the patient's positive serum can be used as a positive control, but because the patient's positive serum is relatively precious, it is necessary to provide a positive control sample that can replace the patient's positive serum. Chimeric antibodies or humanized antibodies can meet such needs. At the same time, the existing commercial Nova-1 antibodies are all mouse / rabbit polyclonal antibodies or mouse / rabbit monoclonal antibodies, so they cannot meet the needs of diagnostic kits. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a group of new monoclonal antibodies of Nova-1 protein and their products and a method for detecting Nova-1 protein, so as to solve the technical problem that the current mouse / rabbit mono / polyclonal antibodies cannot meet the detection requirements of diagnostic kits, and at the same time provide a group of new monoclonal antibodies of Nova-1 protein for identifying the expression of Nova-1 protein.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention discloses a group of monoclonal antibodies to Nova-1 protein, including light chain and heavy chain, wherein:

[0008] The variable region of the light chain includes three light chain complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.2, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO.4 or SEQ ID NO.6;

[0009] The variable region of the heavy chain includes three heavy chain complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the heavy chain complementary determining region CDR1 is shown in SEQ ID NO.8, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.9, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.10 or SEQ ID NO.12.

[0010] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.1 or SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.7 or SEQ ID NO.11.

[0011] Further preferably, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody to the Nova-1 protein is as shown in SEQ ID NO.13 or SEQ ID NO.14; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody to the Nova-1 protein is as shown in SEQ ID NO.15 or SEQ ID NO.16.

[0012] There are 4 groups of monoclonal antibodies with binding properties, which contain all 6 CDRs described in any one of the following groups 1 to 4:

[0013] Group 1

[0014] (a-1) comprises the amino acid sequence of the light chain CDR1 region shown in SEQ ID NO: 2,

[0015] (b-1) comprises the amino acid sequence of the light chain CDR2 region shown in SEQ ID NO: 3,

[0016] (c-1) comprises the amino acid sequence of the light chain CDR3 region shown in SEQ ID NO:4,

[0017] (d-1) comprises the amino acid sequence of the heavy chain CDR1 region shown in SEQ ID NO: 8,

[0018] (e-1) comprises the amino acid sequence of the heavy chain CDR2 region shown in SEQ ID NO:9, and

[0019] (f-1) comprises the amino acid sequence of the heavy chain CDR3 region shown in SEQ ID NO: 10;

[0020] Group 2

[0021] (a-2) comprises the amino acid sequence of the light chain CDR1 region shown in SEQ ID NO: 2,

[0022] (b-2) comprises the amino acid sequence of the light chain CDR2 region shown in SEQ ID NO: 3,

[0023] (c-2) comprises the amino acid sequence of the light chain CDR3 region shown in SEQ ID NO:6,

[0024] (d-2) comprises the amino acid sequence of the heavy chain CDR1 region shown in SEQ ID NO: 8,

[0025] (e-2) comprises the amino acid sequence of the heavy chain CDR2 region shown in SEQ ID NO:9, and

[0026] (f-2) an amino acid sequence comprising the heavy chain CDR3 region shown in SEQ ID NO: 10

[0027] Group 3

[0028] (a-3) comprises the amino acid sequence of the light chain CDR1 region shown in SEQ ID NO: 2,

[0029] (b-3) comprises the amino acid sequence of the light chain CDR2 region shown in SEQ ID NO: 3,

[0030] (c-3) comprises the amino acid sequence of the light chain CDR3 region shown in SEQ ID NO:4,

[0031] (d-3) comprises the amino acid sequence of the heavy chain CDR1 region shown in SEQ ID NO: 8,

[0032] (e-3) comprises the amino acid sequence of the heavy chain CDR2 region shown in SEQ ID NO:9, and

[0033] (f-3) an amino acid sequence comprising the heavy chain CDR3 region shown in SEQ ID NO: 12

[0034] Group 4

[0035] (a-4) comprises the amino acid sequence of the light chain CDR1 region shown in SEQ ID NO: 2,

[0036] (b-4) comprises the amino acid sequence of the light chain CDR2 region shown in SEQ ID NO: 3,

[0037] (c-4) comprises the amino acid sequence of the light chain CDR3 region shown in SEQ ID NO:6,

[0038] (d-4) comprises the amino acid sequence of the heavy chain CDR1 region shown in SEQ ID NO: 8,

[0039] (e-4) comprises the amino acid sequence of the heavy chain CDR2 region shown in SEQ ID NO:9, and

[0040] (f-4) comprises the amino acid sequence of the heavy chain CDR3 region shown in SEQ ID NO: 12;

[0041] Furthermore, the four groups of monoclonal antibodies with binding properties of the present invention comprise a combination of a light chain variable region amino acid sequence and a heavy chain variable region amino acid sequence described in any one of the following groups:

[0042] Group 5

[0043] (g-1) comprises the light chain variable region amino acid sequence shown in SEQ ID NO: 1; and

[0044] (h-1) comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:7;

[0045] Group 6

[0046] (g-2) comprises the light chain variable region amino acid sequence shown in SEQ ID NO:5; and

[0047] (h-2) comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:7;

[0048] Group 7

[0049] (g-3) comprises the light chain variable region amino acid sequence shown in SEQ ID NO: 1; and

[0050] (h-3) comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO: 11;

[0051] Group 8

[0052] (g-4) comprises the light chain variable region amino acid sequence shown in SEQ ID NO:5; and

[0053] (h-4) comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO: 11;

[0054] Furthermore, the nucleotide sequences encoding the four groups of monoclonal antibodies having binding properties of the present invention comprise a combination of the nucleotide sequence of the light chain variable region and the nucleotide sequence of the heavy chain variable region described in any one of the following groups:

[0055] Group 9

[0056] (i-1) comprises the nucleotide sequence shown in SEQ ID NO:13 encoding the light chain variable region amino acid sequence of SEQ ID NO:1; and

[0057] (j-1) comprises the nucleotide sequence shown in SEQ ID NO:15 encoding the heavy chain variable region amino acid sequence of SEQ ID NO:7;

[0058] Group 10

[0059] (i-2) comprising the nucleotide sequence shown in SEQ ID NO:14 encoding the light chain variable region amino acid sequence of SEQ ID NO:5; and

[0060] (j-2) comprising the nucleotide sequence shown in SEQ ID NO:15 encoding the heavy chain variable region amino acid sequence of SEQ ID NO:7;

[0061] Group 11

[0062] (i-3) comprising the nucleotide sequence shown in SEQ ID NO:13 encoding the light chain variable region amino acid sequence of SEQ ID NO:1; and

[0063] (j-3) comprising the nucleotide sequence shown in SEQ ID NO:16 encoding the light chain variable region amino acid sequence of SEQ ID NO:11;

[0064] Group 12

[0065] (i-4) comprising the nucleotide sequence shown in SEQ ID NO:14 encoding the light chain variable region amino acid sequence of SEQ ID NO:5; and

[0066] (j-4) comprises the nucleotide sequence shown in SEQ ID NO:16 encoding the light chain variable region amino acid sequence of SEQ ID NO:11.

[0067] Preferably, the monoclonal antibody comprises a light chain having a complementarity determining region of an amino acid sequence that is at least 75% identical to the sequence of the variable region of the light chain, and the complementary domain of the light chain retains the ability to bind to the Nova-1 protein. The monoclonal antibody comprises a heavy chain having a complementarity determining region of an amino acid sequence that is at least 75% identical to the sequence of the variable region of the heavy chain, and the complementary domain of the heavy chain retains the ability to bind to the Nova-1 protein.

[0068] Further preferably, the monoclonal antibody also includes a light chain having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical to the above-mentioned light chain variable region sequence (g-1), (g-2), (g-3) or (g-4); the monoclonal antibody includes a heavy chain having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical to the heavy chain variable region sequence (h-1), (h-2), (h-3) or (h-4), and the sequence of the light chain variable region and the sequence of the heavy chain variable region maintain the ability to bind to the Nova-1 protein.

[0069] The present invention also discloses a vector comprising the nucleotide sequence of the Nova-1 monoclonal antibody.

[0070] The invention also discloses a host cell comprising the above-mentioned vector.

[0071] The present invention discloses the use of the monoclonal antibody, vector or host cell of the Nova-1 protein in preparing a reagent / kit for detecting the Nova-1 protein.

[0072] The present invention discloses the use of the monoclonal antibody, vector or host cell of the Nova-1 protein in preparing a reagent / kit for diagnosing and / or prognosing human paraneoplastic movement disorders.

[0073] The present invention also discloses a method for detecting Nova-1 protein or Nova-1 polypeptide, wherein a sample to be tested is contacted with a monoclonal antibody of the Nova-1 protein to detect the Nova-1 protein or Nova-1 polypeptide in the sample.

[0074] Compared with the prior art, the present invention has the following beneficial effects:

[0075] The monoclonal antibodies of Nova-1 protein disclosed in the present invention are a group of Nova-1 antibodies with strong specificity and high sensitivity. On the one hand, the present invention analyzes and obtains the CDR region sequences of the light chain and heavy chain variable regions of the above monoclonal antibodies, and on this basis, it can provide support for the construction of highly specific and high-affinity Nova-1 protein genetic engineering antibodies. On the other hand, the human-mouse chimeric monoclonal antibodies of Nova-1 protein prepared by the present invention are basically consistent with the activity of anti-Nova-1 mouse monoclonal antibodies after modification, and compared with commercial antibodies, the mouse-derived monoclonal antibodies prepared by the present invention do not react with anti-human Fab antibodies, and have better specificity. The modified human-mouse chimeric antibodies can bind to anti-human Fab antibodies and can be used as a substitute for human serum antibodies to meet the needs of diagnostic kits. It has better specificity, higher affinity and a wider range of applications, and provides a good biological tool for labeling, separation, purification and identification of human paraneoplastic movement disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 For the purification of Nova-1 protein;

[0077] Figure 2A To determine the binding titer of serum antibodies from 6 mice to Nova-1 protein;

[0078] Figure 2B It is the determination of the binding between serum antibodies of 6 mice and control protein;

[0079] Figure 3A The results are for the 5C2 cell line;

[0080] Figure 3B The results are for the 5D2 cell line;

[0081] Figure 3C The results are for the 1F10 cell line;

[0082] Figure 3D The results are for the 2E9 cell line;

[0083] Figure 3E The results are for the 5A2 cell line;

[0084] Figure 4 Dot blot was used to validate the function of anti-Nova-1 antibody. DETAILED DESCRIPTION

[0085] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0086] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0087] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art.

[0088] The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized monoclonal antibodies, fully humanized monoclonal antibodies, single chain antibodies, or portions thereof that are capable of binding to their antigen, such as Fab fragments or fragments produced from a Fab expression library.

[0089] The term "monoclonal antibody (MAb)" refers to an antibody population of only one species consisting of a unique light chain gene product and a unique heavy chain gene product. The complementarity determining region (CDR) of the monoclonal antibody is the same in all molecules of the antibody population and can bind to the antigen binding site of a specific epitope of the antigen.

[0090] Among them, the term "antigen binding site" refers to the part of the antibody molecule that participates in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable (V) region of the heavy chain (H) and the light chain (L). Three highly branched extensions called "hypervariable regions" in the V region of the heavy chain and the light chain are inserted between multiple conservative lateral extensions called "framework regions" or "FRs". Therefore, the term "FR" refers to the amino acid sequence that naturally exists between and is adjacent to the hypervariable regions of immunoglobulins. In the antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three-dimensional space relative to each other to form an antigen binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are called "complementarity determining regions" or "CDRs", and the three "CDRs" are usually called CDR1, CDR2 and CDR3 and exist from the N-terminus of the light chain / heavy chain.

[0091] Among them, the term "chimeric antibody" means an antibody in which the sequences of the H chain variable region and the L chain variable region (VH and VL) are derived from non-human animal species and the constant region sequences (CH and CL) are derived from humans. The sequence of the variable region is preferably derived from, for example, an animal species that allows easy preparation of hybridomas, such as mice, rats, rabbits, etc.

[0092] Among them, the term "humanized monoclonal antibody" refers to replacing the CDR region of a human antibody with the CDR region from a non-human animal species antibody, also known as a CDR transplanted antibody. The CDR sequence of a non-human animal species antibody is preferably derived from, for example, an animal species that allows easy preparation of hybridomas, such as mice, rats, rabbits, etc.

[0093] Wherein, the term "polypeptide" refers to proteins, protein fragments and fragments or analogs of polypeptide sequences. Protein fragments and analogs are considered to be polypeptides. Further, according to the present invention, examples of polypeptides include light chain immunoglobulin molecules represented by SEQ ID NO: 1, SEQ ID NO: 5 and heavy chain immunoglobulin molecules represented by SEQ ID NO: 7, SEQ ID NO: 11 and CDRs represented by SEQ ID NO: 2, 3, 4, 6, 8, 9, 10 and 12, antibody molecules formed by a combination of a heavy chain immunoglobulin molecule and a light chain immunoglobulin molecule, such as a kappa light chain immunoglobulin molecule (or vice versa), and fragments and analogs thereof.

[0094] Among them, Nova-1 (Neuro-oncological ventral antigen 1, Nova-1) protein is a protein expressed in tumors and neurons. The Nova-1 protein mentioned in the present invention refers to the human Nova-1 protein shown by SEQ ID NO.19, or refers to a polypeptide, fragment or analog that retains the Nova-1 binding ability.

[0095] Wherein, the term "amino acid" refers to the 20 conventional amino acids and their abbreviations used in the present invention as conventionally used.

[0096] The Nova-1 protein monoclonal antibody of the present invention is a monoclonal antibody that has binding properties with the Nova-1 protein, or refers to a polypeptide, fragment or analog that retains the Nova-1 binding ability;

[0097] There are 4 groups of monoclonal antibodies with binding properties, which contain all 6 CDRs described in any one of the following groups 1 to 4:

[0098] Group 1

[0099] (a-1) comprises the amino acid sequence of the light chain CDR1 region shown in SEQ ID NO: 2,

[0100] (b-1) comprises the amino acid sequence of the light chain CDR2 region shown in SEQ ID NO: 3,

[0101] (c-1) comprises the amino acid sequence of the light chain CDR3 region shown in SEQ ID NO:4,

[0102] (d-1) comprises the amino acid sequence of the heavy chain CDR1 region shown in SEQ ID NO: 8,

[0103] (e-1) comprises the amino acid sequence of the heavy chain CDR2 region shown in SEQ ID NO:9, and

[0104] (f-1) comprises the amino acid sequence of the heavy chain CDR3 region shown in SEQ ID NO: 10;

[0105] Group 2

[0106] (a-2) comprises the amino acid sequence of the light chain CDR1 region shown in SEQ ID NO: 2,

[0107] (b-2) comprises the amino acid sequence of the light chain CDR2 region shown in SEQ ID NO: 3,

[0108] (c-2) comprises the amino acid sequence of the light chain CDR3 region shown in SEQ ID NO:6,

[0109] (d-2) comprises the amino acid sequence of the heavy chain CDR1 region shown in SEQ ID NO: 8,

[0110] (e-2) comprises the amino acid sequence of the heavy chain CDR2 region shown in SEQ ID NO:9, and

[0111] (f-2) an amino acid sequence comprising the heavy chain CDR3 region shown in SEQ ID NO: 10

[0112] Group 3

[0113] (a-3) comprises the amino acid sequence of the light chain CDR1 region shown in SEQ ID NO: 2,

[0114] (b-3) comprises the amino acid sequence of the light chain CDR2 region shown in SEQ ID NO: 3,

[0115] (c-3) comprises the amino acid sequence of the light chain CDR3 region shown in SEQ ID NO:4,

[0116] (d-3) comprises the amino acid sequence of the heavy chain CDR1 region shown in SEQ ID NO: 8,

[0117] (e-3) comprises the amino acid sequence of the heavy chain CDR2 region shown in SEQ ID NO:9, and

[0118] (f-3) an amino acid sequence comprising the heavy chain CDR3 region shown in SEQ ID NO: 12

[0119] Group 4

[0120] (a-4) comprises the amino acid sequence of the light chain CDR1 region shown in SEQ ID NO: 2,

[0121] (b-4) comprises the amino acid sequence of the light chain CDR2 region shown in SEQ ID NO: 3,

[0122] (c-4) comprises the amino acid sequence of the light chain CDR3 region shown in SEQ ID NO:6,

[0123] (d-4) comprises the amino acid sequence of the heavy chain CDR1 region shown in SEQ ID NO: 8,

[0124] (e-4) comprises the amino acid sequence of the heavy chain CDR2 region shown in SEQ ID NO:9, and

[0125] (f-4) comprises the amino acid sequence of the heavy chain CDR3 region shown in SEQ ID NO: 12;

[0126] Furthermore, the four groups of monoclonal antibodies having binding properties of the present invention comprise a combination of a light chain variable region and a heavy chain variable region described in any one of the following groups:

[0127] Group 5

[0128] (g-1) comprises the light chain variable region amino acid sequence shown in SEQ ID NO: 1; and

[0129] (h-1) comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:7;

[0130] Group 6

[0131] (g-2) comprises the light chain variable region amino acid sequence shown in SEQ ID NO:5; and

[0132] (h-2) comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:7;

[0133] Group 7

[0134] (g-3) comprises the light chain variable region amino acid sequence shown in SEQ ID NO: 1; and

[0135] (h-3) comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO: 11;

[0136] Group 8

[0137] (g-4) comprises the light chain variable region amino acid sequence shown in SEQ ID NO:5; and

[0138] (h-4) comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO: 11;

[0139] Furthermore, the nucleotide sequences encoding the four groups of monoclonal antibodies having binding properties of the present invention comprise a combination of the nucleotide sequence of the light chain variable region and the nucleotide sequence of the heavy chain variable region described in any one of the following groups:

[0140] Group 9

[0141] (i-1) comprises the nucleotide sequence shown in SEQ ID NO:13 encoding the light chain variable region amino acid sequence of SEQ ID NO:1; and

[0142] (j-1) comprises the nucleotide sequence shown in SEQ ID NO:15 encoding the heavy chain variable region amino acid sequence of SEQ ID NO:7;

[0143] Group 10

[0144] (i-2) comprising the nucleotide sequence shown in SEQ ID NO:14 encoding the light chain variable region amino acid sequence of SEQ ID NO:5; and

[0145] (j-2) comprising the nucleotide sequence shown in SEQ ID NO:15 encoding the heavy chain variable region amino acid sequence of SEQ ID NO:7;

[0146] Group 11

[0147] (i-3) comprising the nucleotide sequence shown in SEQ ID NO:13 encoding the light chain variable region amino acid sequence of SEQ ID NO:1; and

[0148] (j-3) comprising the nucleotide sequence shown in SEQ ID NO:16 encoding the light chain variable region amino acid sequence of SEQ ID NO:11;

[0149] Group 12

[0150] (i-4) comprising the nucleotide sequence shown in SEQ ID NO:14 encoding the light chain variable region amino acid sequence of SEQ ID NO:5; and

[0151] (j-4) comprises the nucleotide sequence shown in SEQ ID NO:16 encoding the light chain variable region amino acid sequence of SEQ ID NO:11.

[0152] The monoclonal antibody of Nova-1 protein of the present invention also includes antibodies having "equivalent binding properties" to the antibody, and the antibodies having "equivalent binding properties" can bind to Nova-1 protein or polypeptide.

[0153] Specifically, it is considered that changes in the amino acid sequence of antibodies with "equivalent binding properties" are covered in the present invention, provided that the changes in the amino acid sequence of antibodies with "equivalent binding properties" maintain at least 75%, more preferably at least 80%, 90%, 95% and most preferably 99%. Including certain percentages in between, such as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity. In particular, conservative amino acid substitutions are included.

[0154] Wherein, the conservative amino acid substitution is a substitution that occurs in the amino acid family associated with the side chain. Genetically encoded amino acids are generally divided into the following families: (1) Acidic amino acids are aspartic acid and glutamic acid; (2) Basic amino acids are lysine, arginine, and histidine; (3) Non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; (4) Uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include (i) serine and threonine, which are aliphatic hydroxyl families; (ii) asparagine and glutamine, which are amide-containing families; (iii) alanine, valine, leucine, and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan, and tyrosine, which are aromatic families. For example, it is reasonable to expect that substitution of alanine alone with leucine or valine, substitution of histidine alone with arginine, substitution of glutamic acid alone with serine, or similar substitution of amino acids with structurally related amino acids will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve amino acids within backbone sites. Whether an amino acid change results in a functional peptide can be readily determined by testing the specific activity of a polypeptide derivative. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art. Preferred amino and carboxyl termini of fragments or analogs occur near functional domain boundaries. Structural and functional domains can be identified by comparison of nucleotide and / or amino acid sequence data with public or private sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function.

[0155] Preferred amino acid substitutions are: (1) substitutions that reduce susceptibility to proteolysis; (2) substitutions that reduce susceptibility to oxidation; (3) substitutions that alter binding affinity for forming a protein complex; (4) substitutions that alter binding affinity; and (4) substitutions that confer or modify other physicochemical or functional properties of such analogs. Analogs may include various mutant proteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domain that forms intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the replacement amino acid should not tend to break the helix present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence).

[0156] Among them, the term "gene" or "DNA" or "nucleic acid molecule": includes not only double-stranded DNA, but also corresponding single-stranded DNA, sense strand and antisense strand constituting double-stranded DNA. It is not particularly limited by its length. Unless otherwise specified, the gene (DNA) in this specification includes double-stranded DNA (including human genomic DNA), single-stranded DNA (positive strand) (including cDNA), single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments thereof. According to the present invention, examples of nucleic acid molecules include nucleotide sequences encoding the light chain variable region of the above-mentioned Nova-1 monoclonal antibody (SEQ ID NO.13, SEQ ID NO.14); nucleotide sequences encoding the heavy chain variable region of the above-mentioned Nova-1 monoclonal antibody (SEQ ID NO.15, SEQ ID NO.16); nucleotide sequences encoding human light chain kappa constant region (SEQ ID NO.17); nucleotide sequences encoding human heavy chain IgG1 constant region (SEQ ID NO.18); nucleic acid sequences encoding Nova-1 protein (NCBI sequence number is NM_002515.3). The nucleic acid molecules of the invention can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example by combining chemical synthesis and recombinant methods.

[0157] The invention still further relates to the variant of above-mentioned nucleic acid molecules, and nucleic acid molecule variant can be naturally occurring variant, such as naturally occurring allele variant, or it can be the variant that non-natural exists.By mutagenesis technique, comprise those mutagenesis techniques that are applied to nucleic acid molecules, cell or organism, these non-natural existence variants of nucleic acid molecules can be prepared.

[0158] The vectors of the present invention include the above-mentioned nucleic acid molecules. Many suitable vectors are known to those skilled in the art of molecular biology, and their selection depends on the desired function. The present invention is not particularly limited to the vector, and can be a vector that can replicate and / or express polynucleotides in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster or mouse cells), plant cells, yeast cells, insect cells and bacterial cells (such as Escherichia coli (E. coli)). Non-limiting examples of vectors include pBAD, pQE-12, pGEX, pBluescript, pET-series expression vectors, pCAI-n, pPOW3.0, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, pREP, pCEP4, pMC1neo, pX T1, pSG5, EBO-pSV2neo, pBPV-1, pFUSE, pRSVgpt, pRSVneo, pIZD35, pRc / CMV, pcDNA1, pcDNA3.1, pSPO RT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pEAK-10, pTriEx-Hygro, pCINeo, pAO815, pPIC9K and pPIC3.5K.

[0159] The host cell of the present invention comprises the above-mentioned nucleic acid molecule or vector.

[0160] In an embodiment of the present invention, the host cells used to introduce the vector include prokaryotic cells and eukaryotic cells, including but not limited to bacterial cells, such as Escherichia coli; yeast cells; fungal cells such as Pichia pastoris, Saccharomyces cerevisiae; insect cells such as Sf9 cells, Sf21 cells, Tn-368 cells, High Five cells; animal cells, such as CHO cells, COS, NSO, 293T, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), Expi293F, PERC.6 (human retinal cells); plant cells. Any cell known to those skilled in the art that can be used as a mammalian host cell can be used in the art.

[0161] The present invention provides a method for detecting Nova-1 protein or polypeptide, which comprises contacting a sample with the above monoclonal antibody, thereby detecting the Nova-1 protein or polypeptide in the sample.

[0162] In the present invention, the method for detecting or measuring the amount of a protein or polypeptide can be any known immunological detection technique, including immunoagglutination assay, immunoprecipitation assay, enzyme-linked immunosorbent assay, fluorescent immunoassay, immunoelectron microscopy, immunoblotting, and immunomagnetic bead assay. Immunological detection techniques are methods for detecting or measuring the amount of an antibody or antigen using a labeled antigen or antibody.

[0163] Biological samples that can be tested by the above-described immunological detection or assay include, but are not limited to, plasma, whole blood, dried whole blood, serum, tissues, cells, or extracts of tissues and cells.

[0164] In an embodiment of the present invention, enzyme immunoassay includes but is not limited to indirect immunofluorescence, direct ELISA, dot blot (linear blot). A method is listed, which includes adsorbing human Nova-1 polypeptide (antigen) onto a solid phase support NC membrane, blocking the solid phase with a blocking protein (e.g., skim milk powder, bovine serum albumin, etc.), contacting and incubating the anti-human Nova-1 monoclonal antibody (test antibody) with the solid phase, removing unreacted antibodies, and adding a labeled second antibody that specifically reacts with the test antibody to the solid phase to determine the amount of the label on the solid phase.

[0165] The product of the present invention includes the above-mentioned monoclonal antibody, nucleic acid molecule, vector or host cell.

[0166] As an optional embodiment, the product is a kit; the kit includes the antibody prepared by the present invention. As another optional embodiment, the kit of the present invention includes a diagnostic composition, the diagnostic composition includes at least one detectable labeling substance, as the labeling substance, a radioactive isotope, an enzyme, a fluorescent substance, a luminescent substance, etc. can be used. The enzyme can be β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.; the fluorescent substance can be fluorescamine, fluorescein isothiocyanate, etc.; the luminescent substance can be luminol, luminol derivatives, luciferin, lucigenin, etc.

[0167] As an optional embodiment, the kit includes a protein immunoassay kit; the protein immunoassay kit includes the above-mentioned monoclonal antibody or a fragment thereof.

[0168] The present invention provides the use of the above-mentioned monoclonal antibody, nucleic acid molecule, vector, host cell or product in detecting Nova-1 protein or polypeptide products.

[0169] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0170] Example 1 Preparation of monoclonal antibodies

[0171] 1. Preparation of recombinant proteins Nova-1, Nova-2 and IGFBP2

[0172] The human Nova-1 gene sequence was obtained from the GenBank sequence database, with the NCBI sequence number of NM_002515.3. A 6his tag sequence was added to the C-terminus of the gene, and the gene sequence was synthesized by Jinweizhi Company into the pcDNA3.1 vector with the insertion sites of Nhe I and Not I to obtain the pcDNA3.1-Nova-1 recombinant vector. The pcDNA3.1-Nova-1 recombinant vector was transferred into Expi293F cells (thermo) using transfection reagent PEI (transfection reagent, plasmid dosage and experimental operation process were carried out according to the instructions of thermo company). On the first day after transfection, 1 mL of supplementary feed (OPM-293ProFeed, Cat No. F081918) was added per 100 mL of cells. On the second day, an appropriate amount of glucose was added (the glucose content of the culture medium was measured using a blood glucose meter and supplemented. The calculation formula is: the amount of supplemented glucose in 100 mL (μl) = (33-N) * 0.06, and the concentration of the glucose solution used was 300 mg / mL). On the third day, the culture supernatant was collected by centrifugation and purified using Ni-NTA (QIAGEN). The purification results are shown in the figure. Figure 1 shown.

[0173] The gene sequence of human Nova-2 was found from the GenBank sequence database, with the sequence number NM_002516.4. The gene sequence was synthesized by Jinweizhi Company into the pET-28a vector, with the insertion sites of BamH I and Xho I, to obtain the pET-28a-Nova-2 recombinant vector. The constructed and correctly sequenced recombinant plasmid was transferred into the expression competent cells of Escherichia coli Arctic Express (Agilent); a single clone was picked and inoculated into LB medium, shaken at 37°C to an OD value of 0.5-1, induced with a final concentration of 0.5mM IPTG, and expressed overnight at 16°C; the cells were collected, ultrasonically broken, and the supernatant was collected after centrifugation. The Nova-2 protein expressed by pET-28a was purified using Ni-NTA (QIAGEN), and the purified protein was recorded as Nova-2.

[0174] The gene sequence of human IGFBP2 was obtained from the GenBank sequence database, with the sequence number NM_000597.3. The gene sequence was synthesized by Jinweizhi Company into the pET-28a vector, with the insertion sites of BamH I and Xho I, to obtain the pET-28a-IGFBP2 recombinant vector. The constructed and correctly sequenced recombinant plasmid was transferred into the expression competent cells of Escherichia coli Arctic Express (Agilent); a single clone was picked and inoculated into LB medium, shaken at 37°C to an OD value of 0.5-1, induced with a final concentration of 0.5mM IPTG, and expressed overnight at 16°C; the cells were collected, ultrasonically broken, and the supernatant was collected after centrifugation. The IGFBP2 protein expressed by pET-28a was purified using Ni-NTA (QIAGEN), and the purified protein was recorded as IGFBP2.

[0175] 2. Mouse Antigen Immunization

[0176] The Nova-1 recombinant protein prepared as above was used as an antigen to immunize 6 Balb / c mice. The antigen was mixed with an equal volume of Freund's adjuvant and fully ground into an oil-in-water emulsion. Three immunizations were required, and the amount of antigen used to immunize mice was 40 μg / mouse. The second immunization was two weeks after the initial immunization. 40ug antigen was mixed with incomplete Freund's adjuvant in a 1:1 ratio, and 100 μL (40 μg antigen) of the mixed antigen was injected into the right hind calf muscle. The third immunization was performed two weeks after the second immunization, and the dose, method, and route of antigen injection were the same as the second immunization. After an interval of 2 weeks, the antigen was injected intraperitoneally to strengthen the immunization, and the tail blood was taken 3 days later to detect its titer using ELISA.

[0177] The specific detection process is as follows: use carbonate buffer with a pH value of 9.6 to coat the purified Nova-1 recombinant protein at 4°C overnight, 100 ng / well (IGFBP2 protein with a his tag was used as a control protein to detect the specificity of antibodies in the serum of each mouse); wash with PBST three times the next day, 3 minutes each time, and pat dry; block the wells with 2% BSA and incubate at 37°C for 1 hour; wash with PBST three times, 3 minutes each time, and pat dry; dilute the sera of 6 mice with PBS in multiple ratios, at 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, and 1:109350, add 100 μL to each well, incubate at 37°C for 1 hour, wash with PBST three times, 3 minutes each time, and pat dry; dilute goat anti-mouse IgG-HRP 1:5000 and incubate at 37°C for 30 minutes. Wash with PBST three times, pat dry, develop with TMB for 10 min, terminate with 2M H2SO4, and measure absorbance at 450 nm. Figure 2A and Figure 2BAs shown, based on the test results and the comprehensive titer and specificity data of serum antibodies, mouse No. 3 was selected for subsequent experiments.

[0178] 3. Cell fusion and hybridoma culture

[0179] Immunization was enhanced by intraperitoneal injection of antigens. Animals were killed 3 days later to obtain spleen cells for cell fusion. A 37°C water bath was prepared in a clean bench. SP2 / 0 cells and spleen cells were added to a 50mL centrifuge tube at a ratio of 1:10 and mixed. Centrifuge at 500g for 10min, aspirate the supernatant, flick the bottom of the centrifuge tube to loosen the cell pellet slightly; slowly drip 1mL of 45% PEG1450 solution preheated to 37°C within 90s; and shake the centrifuge tube gently continuously; the centrifuge tube was in a 37°C water bath during the whole process. Then gradually add DMEM culture medium to the cell mixture, drip 1mL dropwise in the first minute, add 2mL in the second minute, add 3mL in the third minute, add 4mL in the fourth minute, add 5mL in the fifth minute, and shake while adding in a 37°C water bath. Then incubate at 37°C for 15min, centrifuge at 500g for 5min, and remove the supernatant. Add 5 mL of DMEM medium containing HAT, gently suspend the precipitated cells, and finally add DMED medium containing HAT to about 100 mL. Dispense into a 96-well cell culture plate with macrophages, 100 μL / well, and then place the culture plate in a 37°C, 5% CO2 incubator for culture.

[0180] 4. Screening of positive hybridomas

[0181] Use ELISA to select positive clones. Observe the growth of hybridoma cells. After seven days, when the cell culture supernatant turns yellow, take an appropriate amount of cell supernatant for ELISA antibody detection; according to the ELISA results, select clones with high OD values ​​that are more than twice higher than the negative control, spread them on 96-well plates, and perform the first subclone screening; after seven to ten days, perform ELISA to detect antibodies again, select clones with high OD values, spread them on 96-well plates, and perform the second subclone screening, so that there is about 1 cell in each well; after seven to ten days of culture, perform ELISA to detect antibodies again, select clones with high OD values, spread them on 96-well plates, and perform the third subclone screening, so that there is about 1 cell in each well. For the screening results of anti-Nova-1 antigen positive hybridomas, see Figure 3A (5C2), Figure 3B (5D2), Figure 3C (1F10), Figure 3D (2E9) and Figure 3E(5A2). After three rounds of screening, three positive clones with high OD values ​​were selected and named: 1F10, 2E9, and 5C2. The Nova-1 protein and Nova-2 protein prepared by the above method were coated on ELISA plates (Nova-2 protein was used as a control protein) to verify the specificity of the antibody. Since the two hybridomas 2E9 and 5C2 could react with the Nova-2 protein, the positive hybridoma 1F10 with good specificity was selected to continue the subsequent experiments. At the same time, the mouse antibody was purified from the culture supernatant of the positive hybridoma 1F10, and the protein concentration measured by the BCA method was 0.1 mg / mL (Solebo).

[0182] Example 2 Identification of anti-Nova-1 monoclonal antibody sequences

[0183] The positive hybridoma 1F10 used for antibody production was grown to a certain scale (cell number>3×10 6 After that, the cells were lysed with Trizol, and the total RNA of the hybridoma cell lysate was extracted using the Quick-RNA MicroPrep Kit. The RNA was separated by agarose gel electrophoresis, and then 5'RACE was performed using the Clontech SMARTer RACE 5' / 3'kit to obtain cDNA, which was used as a template. Max DNA Polymerase was used for PCR amplification to extract the antibody V region cDNA, which was then sequenced by a sequencing company, and the CDR region of the antibody amino acid sequence was marked using the Kabat method. SEQ ID NO.13 is the nucleotide sequence of the light chain variable region, and SEQ ID NO.15 is the nucleotide sequence of the heavy chain variable region.

[0184] Example 3 Construction of anti-Nova-1 monoclonal antibody sequence recombinant vector

[0185] 1. Construction of recombinant vector

[0186] According to the nucleotide sequences of the light chain variable region and the heavy chain variable region of the 1F10 antibody obtained in Example 2, the nucleotide sequence of the light chain variable region of the 1F10 antibody (shown in SEQ ID NO.13) was amplified by molecular biology methods using Fast Pfu DNA Polymerase (Quanshijin), and connected with the human IgG1 light chain constant region sequence SEQ ID NO.17 to the vector pcDNA3.1, marked as pcDNA3.1-Nova-1-VL; the nucleotide sequence SEQ ID NO.15 of the heavy chain variable region of the monoclonal antibody encoding the Nova-1 protein was amplified, and connected with the human IgG1 heavy chain constant region sequence SEQ ID NO.18 to the vector pcDNA3.1, marked as pcDNA3.1-Nova-1-VH, and the connected recombinant plasmid was sent to Sangon Biotechnology for sequencing, and the sequenced recombinant plasmid was collected for use.

[0187] 2. Construction of mutants

[0188] The antibody sequence was amplified using Fast Pfu DNA Polymerase (Full Gold) by conventional molecular biology methods to construct mutants of the antibody sequence. The amino acid S at position 115 of the amino acid sequence of the light chain variable region shown in SEQ ID NO.1 was mutated to G, and the amino acid T at position 116 was mutated to S. The new amino acid sequence after mutation was SEQ ID NO.5, and the corresponding nucleotide sequence was SEQ ID NO.14. The nucleotide sequence shown in SEQ ID NO.14 and the human IgG1 light chain constant region sequence SEQ ID NO.17 were connected to the vector pcDNA3.1, and the sequence was marked as pcDNA3.1-Nova-1-VL1. The amino acid W at position 120 of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO.7 was mutated to D, and the amino acid Y at position 125 was mutated to S. The new amino acid sequence after mutation was SEQ ID NO.5, and the corresponding nucleotide sequence was SEQ ID NO.16. The nucleotide sequence shown in SEQ ID NO.16 and the human IgG1 heavy chain constant region sequence SEQ ID NO.18 were connected to the vector pcDNA3.1, and the sequence was marked as pcDNA3.1-Nova-1-VH1. The connected recombinant plasmid was sent to Biotech for sequencing, and the sequenced recombinant plasmid was collected for use.

[0189] Example 4 Preparation of anti-Nova-1 human-mouse chimeric antibody

[0190] The following four groups of recombinant vectors were co-transfected into Expi293F cells (Thermo Company) using PEI according to the Thermo instructions for transient expression: Group 1 was co-transfected with recombinant plasmid pcDNA3.1-Nova-1-VL and recombinant plasmid pcDNA3.1-Nova-1-VH; Group 2 was co-transfected with recombinant plasmid pcDNA3.1-Nova-1-VL1 and recombinant plasmid pcDNA3.1-Nova-1-VH; Group 3 was co-transfected with recombinant plasmid pcDNA3.1-Nova-1-VL and recombinant plasmid pcDNA3.1-Nova-1-VH1; Group 4 was co-transfected with recombinant plasmid pcDNA3.1-Nova-1-VL1 and recombinant plasmid pcDNA3.1-Nova-1-VH1. The supernatant was collected 3 days after transfection and purified using protein A filler. The protein concentration measured by BCA method was 0.1 mg / mL (Solebo).

[0191] Example 5 Application of anti-Nova-1 human-mouse chimeric antibody

[0192] 1. ELISA to verify the function of anti-Nova-1 human-mouse chimeric antibody

[0193] The purified Nova-1 protein (100 ng / well) was coated with carbonate buffer solution with a pH value of 9.6 at 4°C overnight; the next day, the cells were washed with PBST for 3 times, each time for 3 min, and patted dry each time; the wells were blocked with 2% BSA and incubated at 37°C for 1 h; the cells were washed with PBST for 3 times, each time for 3 min, and patted dry each time; the 1F10 mouse monoclonal antibody (0.1 mg / mL) purified in Example 1, the human-mouse chimeric antibody purified in Example 4, and the commercial Nova-1 antibody (thermo, PA5-95571) were incubated at 37°C for 1 h, washed with PBST for 3 times, each time for 3 min, and patted dry; the corresponding goat anti-mouse or goat anti-human IgG-HRP (Jackson) diluted 1:5000 was incubated at 37°C for 30 min, washed with PBST for 3 times, patted dry, developed with TMB for 10 min, terminated with 2M H2SO4, and the absorbance was measured at 450 nm. As shown in Table 1, the activity of the human-mouse chimeric antibody after transformation is basically the same as that of the mouse monoclonal antibody before transformation.

[0194] Table 1 ELISA validation of the function of anti-Nova-1 antibodies

[0195]

[0196] 2. Dot blot method to verify the function of anti-Nova-1 human-mouse chimeric antibody

[0197] Use a pipette to immobilize the anti-human Fab antibody (Sigma, concentration of 100 ng / μL, located in the upper left corner of the trapezoidal membrane) and Nova-1 antigen (concentration of 50 ng / μL, second point in the first row) on the right-angle trapezoidal NC membrane with a circular imprint, bake in a 37°C oven for 10 min, place in a 24-well plate at room temperature, and incubate the positive hybridoma 1F10 mouse monoclonal antibody (0.1 mg / mL) purified in Example 1, the anti-Nova-1 human-mouse chimeric antibody purified in Example 4, and the commercial Nova-1 antibody (thermo, PA5-95571) at 37°C for 1 h, wash with PBST, 4 min / time, for a total of 2 washes; dilute AP-labeled sheep anti-human IgG antibody (Jackson) 2000 times, add to each well, incubate at room temperature for 30 min, use 1% After removing the washing solution in the well, add 300 μL / well of substrate diluent BCIP / NBT (substrate: reaction buffer = 1:49), stand at room temperature and away from light for 4 minutes to obtain the color development result, discard the substrate diluent in the well, rinse the test strip with distilled water 1-2 times, take it out to dry or dry it at 37℃-45℃. Figure 4 It can be seen that the activity of the modified human-mouse chimeric antibodies is basically the same as that of the mouse monoclonal antibodies before the modification. Compared with commercial antibodies, the mouse monoclonal antibodies prepared in the present invention do not react with anti-human Fab antibodies and have better specificity. The four modified human-mouse chimeric antibodies can bind to anti-human Fab antibodies and can be used as substitutes for human serum antibodies to meet the needs of diagnostic kits.

[0198] Table 2 below records the sequences related to the above content of the present invention.

[0199] Table 2 Sequences related to the present invention

[0200]

[0201]

[0202] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A group of monoclonal antibodies to Nova-1 protein, characterized in that: Including monoclonal antibody 1F10 and its mutants; wherein: The monoclonal antibody 1F10 comprises a light chain and a heavy chain, wherein the variable region of the light chain of the monoclonal antibody 1F10 comprises three light chain complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.2, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO.4; the variable region of the heavy chain of the monoclonal antibody 1F10 comprises three heavy chain complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the heavy chain complementary determining region CDR1 is shown in SEQ ID NO.8, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.9, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.10; The monoclonal antibody 1F10 mutant includes a light chain and a heavy chain, and the variable region of the light chain of the monoclonal antibody 1F10 mutant includes three light chain complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.2, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO.6; the variable region of the heavy chain of the monoclonal antibody 1F10 mutant includes three heavy chain complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the heavy chain complementary determining region CDR1 is shown in SEQ ID NO.8, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.9, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.

12.

2. The monoclonal antibody against Nova-1 protein according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.7; The amino acid sequence of the light chain variable region of the monoclonal antibody 1F10 mutant is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F10 mutant is shown in SEQ ID NO.

11.

3. A nucleotide encoding a monoclonal antibody against the Nova-1 protein according to claim 2, characterized in that: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.13, and the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.15; The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1F10 mutant is shown in SEQ ID NO.14; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F10 mutant is shown in SEQ ID NO.

16.

4. A carrier, characterized in that The vector comprises the nucleotide sequence of the monoclonal antibody against the Nova-1 protein according to claim 1.

5. A host cell, characterized in that The host cell comprises the vector of claim 4.

6. Use of the monoclonal antibody against Nova-1 protein according to claim 1 or 2, the nucleotide encoding the monoclonal antibody against Nova-1 protein according to claim 3, the vector according to claim 4 or the host cell according to claim 5 in the preparation of a reagent / kit for detecting Nova-1 protein.

7. Use of the monoclonal antibody against Nova-1 protein according to claim 1 or 2, the nucleotide encoding the monoclonal antibody against Nova-1 protein according to claim 3, the vector according to claim 4 or the host cell according to claim 5 in the preparation of a reagent / kit for diagnosis and / or prognosis of human paraneoplastic movement disorders.

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