Antibodies against s100 proteins and uses thereof

Abstract

The application discloses an antibody against S100 protein and application thereof, and relates to the field of antibodies. The antibody against S100 protein provided by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of S100 protein, and has good activity and sensitivity.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202311433224.7, filed on October 31, 2023, entitled "An antibody against S100 protein and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically, to an antibody against S100 protein and its application. Background Technology

[0004] S100 proteins were discovered in bovine brain by Moore in 1965 and named for their solubility in 100% ammonium sulfate solution. S100 proteins are acidic calcium-binding proteins composed of 21 members with a relative molecular mass of 10,000-21,000. Their genes are located on chromosome 1q21 and belong to the EF-chiral motif superfamily. Each S100 protein has two calcium-binding regions with an EF-chiral structure (α-helix-loop-α-helix). The calcium-binding region near the C-terminus has a high affinity for calcium ions, while the calcium-binding region near the N-terminus is specific to the S100 family. The intermediate hinge region and the C-terminal extension region, which vary among different members, are responsible for their specific biological characteristics.

[0005] S100 protein is widely expressed in normal tissue cells derived from the neuroectoderm, mesoderm, and ectoderm, such as glial cells, Schwann cells, melanocytes, mammary epithelial cells, or myoepithelial cells, or in tumors originating from these cells. The vast majority of S100 proteins are found intracellularly, participating in the regulation of intracellular functions such as proliferation, differentiation, apoptosis, calcium homeostasis, energy metabolism, and migration. When S100 protein is overexpressed in cells, it is released into the bloodstream. Under normal circumstances, S100 protein is mainly metabolized in the kidneys, with a biological half-life of approximately 2 hours. The concentration in normal adult serum is less than 0.2 μg / L, making it difficult to detect; concentrations greater than 0.5 μg / L are considered pathological.

[0006] S100 proteins can specifically bind to calcium ions, regulate protein interactions by activating various enzymes, and play an important role in cell cycle growth and differentiation. Studies have found that 15 members of the S100 family are located in the 1q21 region of chromosome 1, which is prone to gene recombination and can cause S100 gene expression to be out of control. The expression of S100 protein is related to many malignant tumors, so the S100 family is closely related to the occurrence and development of tumors. Studies have confirmed that S100 protein and P53 tumor suppressor protein interact at the nucleic acid level, inhibit protein kinase phosphorylation, and thus affect the regulation of cell growth. S100 protein can also change the adhesion of cells, making cancer cells more likely to adhere to tissues and cause clonal proliferation. The value of S100 protein serological detection in the diagnosis of central nervous system injury has been recognized by the clinic, and its value as a tumor marker has gradually been recognized and tested. According to relevant research reports, S100 protein expression is significantly up-regulated in invasive hepatocellular carcinoma, and S100 protein may become a valuable molecular marker for liver cancer classification and invasion prediction. S100 protein is closely related to the occurrence of non-small cell lung cancer and other diseases. The expression of S100 protein in esophageal cancer, colon cancer and malignant melanoma is also significantly up-regulated. With the in-depth study of S100 protein, S100 protein is also closely related to human heart disease, oral cancer, breast cancer and prostate cancer, etc. The main performance is that their protein expression levels are significantly abnormal when the disease occurs, which is of great significance for clinical diagnosis. Therefore, in the current clinical and scientific research field, it is particularly important to accurately, conveniently and quickly detect S100 protein.

[0007] The traditional detection methods of S100 protein mainly include electrochemiluminescence method, radioimmunoassay, immunoradiometric assay and fluorescence immunoassay. The electrochemiluminescence method has high accuracy in quantitative detection and has the advantages of quantification and stability, but it is not suitable for modern automatic immunization instruments and needs special supporting instruments, which has high investment cost and is difficult to popularize and apply. Radioimmunoassay and immunoradiometric assay have the disadvantages of radioactive pollution and harm, short half-life and short stable period, and complex operation, which limit their application. Fluorescence immunoassay requires special fluorescence equipment and is not easy to popularize in clinics.

[0008] The current market detection methods of S100 protein mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence method, flow fluorescence luminescence method, time-resolved fluorescence immunoassay, photochemical chemiluminescence immunoassay and colloidal gold, etc. These detection methods have the characteristics of simple operation, rapid and accurate detection, and do not require complex equipment. These different detection methods have their own advantages and disadvantages, but they all need antibodies for S100 protein.

[0009] Accordingly, there is a strong need in the art for anti-S100 protein antibodies with good performance. SUMMARY

[0010] The present application provides an anti-S100 protein antibody, which provides an important raw material source for the detection of S100 protein, and has good activity and sensitivity.

[0011] To achieve the above object, according to one aspect of the present application, there is provided an anti-S100 protein antibody, which comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 27, 28, 29, 30, 31, 32, and 33, and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 41, 42, 43, 44, and 45.

[0012] To achieve the above object, according to a second aspect of the present application, there is provided an anti-S100 protein antibody, which comprises the following complementarity determining regions:

[0013] HCDR1 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 1 (NYWIH);

[0014] HCDR2 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2 (GEINPSNGRTKFNEKFKT);

[0015] HCDR3 comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 3 (RFFIASIGGAMDY), 17 (RFFLASIGGAMDY), 18 (RFFIASLGGAMDY), or 19 (RFFLASLGGAMDY);

[0016] LCDR1 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 4 (SASSSVSSSYLY);

[0017] LCDR2 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 5 (GTSNLAS); and

[0018] LCDR3 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 6 (HQWSSYPPT).

[0019] To achieve the above objectives, according to a third aspect of the present invention, an antibody against S100 protein is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 41, 42, 43, 44, 45.

[0020] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody against S100 protein is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 46, 47, 48, 49, 50.

[0021] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibodies described above.

[0022] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the antibody or antibody conjugate described above.

[0023] To achieve the above objectives, according to an eighth aspect of the present invention, the use of the above-described antibody, antibody-drug conjugate, reagent, or kit in the preparation of a product for detecting S100 protein is provided.

[0024] To achieve the above objectives, the present invention also provides a nucleic acid, a vector, a cell, and a method for preparing the above-mentioned antibody. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The results of reductive SDS-PAGE for Anti-S100 4G8 Rmb1 to Anti-S100 4G8 Rmb11. Detailed Implementation

[0027] In a first aspect, embodiments of the present invention provide an antibody against S100 protein, the antibody comprising three complementary determinant regions having a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:27, 28, 29, 30, 31, 32, 33, and three complementary determinant regions having a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:41, 42, 43, 44, 45.

[0028] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to those of HCDR1, HCDR2, and HCDR3 in the same heavy chain variable region defined in the antibody described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to those of LCDR1, LCDR2, and LCDR3 in the same light chain variable region defined in the antibody described in the first aspect.

[0029] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:27; LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:41.

[0030] In this invention, the term "antibody" is used in the broadest sense, and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. Antigen-binding fragments of antibodies typically have the same binding specificity as the antibody from which they originate, and can be selected from any of F(ab')2, Fab', Fab, Fv, and scFv, as readily understood by those skilled in the art based on the description herein, and can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the antigen-binding fragments of the aforementioned antibodies.

[0031] Antigen-binding fragments of antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by automated peptide synthesizers, such as those sold by Applied BioSystems.

[0032] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0033] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0034] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below; definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0035] Table 1: CDR Definition 1

[0036] CDR Kabat AbM2 IMGT Chothia HCDR1 H31-H35 3 ]]> [H26-H35 3 ]] [H26~H33..5 5 ]] H26-H32..34 4 ]] HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0037] 1The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0038] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0039] 3 If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0040] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0041] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0042] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0043] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0044] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0045] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0046] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0047] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0048] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0049] According to embodiments of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 defined by the Kabat, Chothia, AbM, or IMGT systems are as follows:

[0050] CDR Kabat AbM IMGT Chothia HCDR1 H31~H35 H26~H35 H26~H33 H26~H32 HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0051] Secondly, embodiments of the present invention provide an antibody against the S100 protein, the antibody comprising the following complementarity-determining region:

[0052] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1 (NYWIH);

[0053] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2 (GEINPSNGRTKFNEKFKT);

[0054] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3 (RFFIASIGGAMDY), 17 (RFFLASIGGAMDY), 18 (RFFIASLGGAMDY), or 19 (RFFLASLGGAMDY);

[0055] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4 (SASSSVSSSYLY);

[0056] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5 (GTSNLAS); and

[0057] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6 (HQWSSYPPT).

[0058] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0059] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0060] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0061] In an optional embodiment, the antibody described in the first or second aspect further comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0062] In an optional embodiment, the HFR1 includes / such as SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it;

[0063] The HFR2 includes / is such as SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it;

[0064] The HFR3 includes / is, for example, SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it;

[0065] The HFR4 includes / is, for example, SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it;

[0066] The LFR1 includes / such as SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it;

[0067] The LFR2 includes / is, for example, SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it;

[0068] The LFR3 includes / is, for example, SEQ ID NO:13 or an amino acid sequence having at least 80% identity with it; and

[0069] The LFR4 includes / such as SEQ ID NO:14 or an amino acid sequence having at least 80% identity with it.

[0070] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the antibody against S100 protein provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) mentioned above.

[0071] In an optional embodiment, the HFR1 includes / as shown in SEQ ID NO:20, an amino acid sequence.

[0072] In an optional embodiment, the HFR2 includes / as shown in SEQ ID NO:21, an amino acid sequence.

[0073] In an optional embodiment, the HFR3 includes / as shown in SEQ ID NO:22, an amino acid sequence.

[0074] In an optional embodiment, the LFR1 includes / such as the amino acid sequence shown in SEQ ID NO:23, 24 or 25.

[0075] In an optional embodiment, the LFR2 includes / as shown in SEQ ID NO:26, an amino acid sequence.

[0076] Thirdly, embodiments of the present invention provide an antibody against S100 protein, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 41, 42, 43, 44, 45.

[0077] In an optional implementation, the heavy chain variable region and the light chain variable region described in the first or third aspect above are selected from any combination of the following:

[0078]

[0079]

[0080] In optional embodiments, the antibodies described in the first, second, and third aspects above further include a constant region.

[0081] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0082] In an optional implementation, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0083] In an optional embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0084] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0085] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0086] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0087] In an optional implementation, the species source of the constant region is mice.

[0088] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region sequence (CL) is as shown in SEQ ID NO:16.

[0089] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region (SEQ ID NO: 15 or 16).

[0090] In an optional embodiment, the antibody includes any one of F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0091] Fourthly, the present invention provides an antibody against S100 protein, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 46, 47, 48, 49, 50.

[0092] In an optional embodiment, the antibody described in the first, second, third, or fourth aspect above includes any combination of the heavy and light chains:

[0093]

[0094]

[0095] Fifthly, the present invention provides an antibody conjugate comprising the antibodies described above.

[0096] In an optional embodiment, the antibody conjugate further includes biotin or a biotin derivative conjugated with the antibody.

[0097] In an optional embodiment, the antibody conjugate further includes a marker conjugated to the antibody.

[0098] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0099] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0100] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0101] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0102] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0103] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0104] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0105] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0106] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0107] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0108] In an optional embodiment, the colloidal metal is colloidal gold.

[0109] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0110] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0111] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0112] In a sixth aspect, the present invention provides a reagent or kit comprising the antibody or antibody conjugate described above.

[0113] As previously mentioned, the antibodies in some embodiments or examples of this invention can effectively bind to the S100 protein. Therefore, reagents or kits containing the S100 protein antibody can effectively perform qualitative or quantitative detection of the S100 protein. The reagents or kits provided by this invention can be used, for example, for detections involving the specific binding properties of the S100 protein and its antibody, such as immunoblotting and immunoprecipitation. As previously mentioned, the antibodies in some embodiments or examples of this invention have higher binding activity or affinity to the S100 protein; therefore, reagents or kits containing the antibody have higher detection sensitivity or specificity.

[0114] In a seventh aspect, the present invention provides a method for detecting S100 protein, comprising: a) contacting the antibody, antibody conjugate, reagent or kit described above with S100 protein in a sample to be tested under conditions sufficient to cause an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample;

[0115] In an optional embodiment, the immune complex further includes a second antibody that binds to the antibody.

[0116] In an optional embodiment, the immune complex further includes a second antibody that binds to the S100 protein.

[0117] Eighthly, the present invention provides the use of the above-described anti-S100 protein antibody, antibody conjugate, or the above-described reagent or kit in the preparation of products for detecting S100 protein.

[0118] Ninthly, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0119] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0120] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0121] In a twelfth aspect, the present invention provides a method for preparing an anti-S100 protein antibody, comprising: culturing cells as described above.

[0122] Based on the amino acid sequence of the anti-S100 protein antibody disclosed in this invention, those skilled in the art will readily conceive of preparing the anti-S100 protein antibody using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the anti-S100 protein antibody of this invention, it falls within the protection scope of this invention.

[0123] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0125] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0126] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0127] Example 1: Preparation of Anti-S100 4G8 Monoclonal Antibody

[0128] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit and pMD-18T vector were purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting the Anti-S100 4G8 monoclonal antibody was a hybridoma cell line prepared in our laboratory and was revived for later use.

[0129] (1) Antibody gene preparation

[0130] mRNA was extracted from hybridoma cell lines secreting Anti-S100 4G8 monoclonal antibody, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after an A-addition reaction with rTaq DNA polymerase, and transformed into DH5α competent cells. After bacterial growth, four clones of the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0131] (2) Sequence analysis of the variable region gene of Anti-S100 4G8 antibody

[0132] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was 324 bp, with a 57 bp leader peptide sequence preceding it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 366 bp, belonging to the VH1 gene family, with a 57 bp leader peptide sequence preceding it.

[0133] (3) Construction of recombinant antibody expression plasmid

[0134] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The 0.70kb Light Chain gene fragment and the 1.40kb Heavy Chain gene fragment were amplified by PCR.

[0135] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0136] 2. Recombinant antibody production

[0137] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE, as shown in the figure. The reducing SDS-PAGE showed two bands: one with a Mr of 50 kDa (heavy chain) and the other with a Mr of 28 kDa (light chain).

[0138] The resulting antibody was named Anti-S100 4G8Rmb1. Mutations were performed on Anti-S100 4G8Rmb1 to obtain a mutant antibody. The heavy chain (H) and light chain (L) sequences of the above antibody are shown in the table below:

[0139] Table 2: Antibody Sequences

[0140] Antibody name Heavy chain Light chain Anti-S100 4G8Rmb1 SEQ ID NO: 34 SEQ ID NO: 46 Anti-S100 4G8Rmb2 SEQ ID NO: 36 SEQ ID NO: 46 Anti-S100 4G8Rmb3 SEQ ID NO: 37 SEQ ID NO: 46 Anti-S100 4G8Rmb4 SEQ ID NO: 40 SEQ ID NO: 46 Anti-S100 4G8Rmb5 SEQ ID NO: 39 SEQ ID NO: 46 Anti-S100 4G8Rmb6 SEQ ID NO: 35 SEQ ID NO: 46 Anti-S100 4G8Rmb7 SEQ ID NO: 38 SEQ ID NO: 46 Anti-S100 4G8Rmb8 SEQ ID NO: 34 SEQ ID NO: 48 Anti-S100 4G8Rmb9 SEQ ID NO: 34 SEQ ID NO: 50 Anti-S100 4G8Rmb10 SEQ ID NO: 34 SEQ ID NO: 49 Anti-S100 4G8Rmb11 SEQ ID NO: 34 SEQ ID NO: 47

[0141] Example 2: Antibody Performance Detection

[0142] 1. Activity identification

[0143] Dilute S100 antigen (purchased from Hytest, catalog number Cat.#8S9h) to 3ug / ml with coating buffer (mainly NaHCO3), 100uL per well, and incubate overnight at 4°C; the next day, wash twice with washing buffer (mainly Na2HPO4 + NaCl), and blot dry; add blocking buffer (20% BSA + 80% PBS), 120uL per well, incubate at 37°C for 1 hour, and blot dry; add diluted purified antibody and anti-antibody. Add antibody, 100 μL / well, incubate at 37°C for 30 min; wash 5 times with washing buffer and pat dry; add goat anti-mouse IgG-HRP, 100 μL / well, incubate at 37°C for 30 min; wash 5 times with washing buffer and pat dry; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), incubate for 10 min; add stop solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0144] Notes: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0145] Table 3: Activity Data

[0146] Concentration (ng / ml) 500.00 250.00 125.00 62.50 31.25 0.00 Control 0.922 0.529 0.312 0.187 0.023 0.005 Anti-S100 4G8Rmb1 1.946 1.755 1.199 0.703 0.434 0.011 Anti-S100 4G8Rmb2 1.090 0.583 0.388 0.288 0.105 0.014 Anti-S100 4G8Rmb3 2.095 1.907 1.670 1.083 0.624 0.006 Anti-S100 4G8Rmb4 1.272 0.644 0.269 0.137 0.102 0.008 Anti-S100 4G8Rmb5 1.953 1.727 1.037 0.526 0.295 0.003 Anti-S100 4G8Rmb6 Anti-S100 4G8Rmb7 1.709 1.232 0.699 0.338 0.253 0.004 Anti-S100 4G8Rmb7 1.864 1.250 0.780 0.487 0.223 0.012 Anti-S100 4G8Rmb8 1.861 1.629 0.804 0.480 0.270 0.006 Anti-S100 4G8Rmb9 2.017 1.891 1.077 0.654 0.420 0.000 Anti-S100 4G8Rmb10 2.070 1.438 0.657 0.314 0.241 0.000 Anti-S100 4G8Rmb11 1.686 0.961 0.541 0.266 0.195 0.000

[0147] 2. Stability assessment

[0148] The above-mentioned antibodies were placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7, 14, and 21 days for observation of their state, and the activity of the 21-day sample was tested. The results showed that no significant changes in protein state were observed under the three testing conditions after 21 days, and the activity did not decrease with increasing testing temperature, indicating that the above-mentioned antibodies are stable. Table 4 below shows the OD results of enzyme immunoassay for antibody Anti-S100 4G8Rmb3 after 21 days of testing.

[0149] Table 4: Stability Data

[0150] Sample concentration (ng / ml) 125 62.5 0 4°C, 21 days sample 1.627 1.031 0.017 -80°C, 21 days sample 1.633 1.047 0.006 37°C, 21 days sample 1.636 1.039 0.035

[0151] 3. Performance testing of the chemiluminescence platform

[0152] 3.1 Antibody Coating Process

[0153] 10 mg / mL carboxyl magnetic beads were washed three times with MES buffer. The beads were resuspended in MES buffer, and EDC was added to a final concentration of 1 mg / mL. The mixture was shaken at 25°C and incubated for 30 min. The beads were then resuspended in MES buffer again, and Anti-S100-A antibody solution (from Feifeipeng Biotechnology) was added to a final concentration of 0.2 mg / mL. This solution was used as the working solution for the magnetic microparticles. The mixture was shaken at 25°C and incubated for 120 min. The mixture was stored in Tris buffer at 2–8°C.

[0154] 3.2 Antibody labeling process

[0155] Anti-S100 4G8Rmb1 to Anti-S100 4G8Rmb11 (3 mg / ml) and control antibody Anti-S100-B (from Phytobio) were replaced with PBS (100 mM PB, 50 mM sodium chloride, pH 8.0) using a Zeba desalting column (10K MWCO). Acridinium ester was prepared into a 4 mM solution using DMSO. 10 eq of acridinium ester was added to the Anti-NTProBNP-A antibody solution, and the reaction was carried out at 25°C for 2 hours. Excess reagents were removed by desalting, and the acridinium ester-modified antibody was used as the acridinium ester working solution and stored at 4°C for later use.

[0156] 3.3 Testing Process

[0157] Detection was performed on the Shine i2910 fully automated chemiluminescence immunoassay analyzer:

[0158] 1) Add 50 μl of S100 antigen quality control, 50 μl of sample processing reagent, and 50 μl of magnetic bead-labeled antibody (concentration 0.05%), and react at 37°C for 20 min; 2) Add 50 μl of acridine ester-labeled antibody, react at 37°C for 20 min, and wash 3 times with PBST; 3) Add 100 μl of pre-excitation solution and 100 μl of excitation solution, and detect the relative luminescence intensity (RLU), and read the instrument value.

[0159] Remark:

[0160] Sample: refers to clinical samples containing different concentrations of S100 calibrators and Roche values.

[0161] 3.4 Test Results

[0162] 3.4.1 Test results of calibrators

[0163] The detection results of Anti-S100 4G8Rmb1 to Anti-S100 4G8Rmb11 against S100 calibrators are shown in Table 5. The results show that Anti-S100 4G8Rmb1 to Anti-S100 4G8Rmb11 can effectively detect S100 calibrators within a concentration range of 2 to 20 ng / ml.

[0164] Table 5: Detection Results of Chemiluminescence Platform Calibrators

[0165] Calibrator concentration (ng / ml) 0 0.2 2 20 Anti-S100 4G8Rmb1 923 81827 615086 6585527 Anti-S100 4G8Rmb2 684 55517 440239 4182502 Anti-S100 4G8Rmb3 678 82668 629387 5982944 Anti-S100 4G8Rmb4 1133 71811 517713 5320443 Anti-S100 4G8Rmb5 967 72437 544070 5502263 Anti-S100 4G8Rmb6 642 74656 600588 5486459 Anti-S100 4G8Rmb7 1176 90545 690169 6781368 Anti-S100 4G8Rmb8 967 89921 708367 6544050 Anti-S100 4G8Rmb9 639 74408 592781 5297158 Anti-S100 4G8Rmb10 905 75831 603801 5701452 Anti-S100 4G8Rmb11 380 47299 389368 3428602

[0166] 3.4.2 Clinical Sample Test Results

[0167] The detection results of clinical samples using antibodies Anti-S100 4G8Rmb1 to Anti-S100 4G8Rmb11 are shown in Table 6. The results show that the chemiluminescence reagent composed of antibodies Anti-S100 4G8Rmb1 to Anti-S100 4G8Rmb11 has a clinical correlation R2 > 0.98 with Roche S100 reagent, which is significantly better than the control antibody.

[0168] Table 6: Clinical Sample Test Results

[0169]

[0170] The amino acid sequences involved in this application are shown in Table 7:

[0171] Table 7: Amino Acid Sequences

[0172]

[0173]

[0174]

[0175] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against S100 protein, said antibody comprising three complementary determinant regions of a heavy chain variable region as shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33 and three complementary determinant regions of a light chain variable region as shown in any one of SEQ ID NO: 41, 42, 43, 44, 45; The complementary determination region of the variable region is defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody against S100 protein, characterized in that, The antibody includes the following complementarity-determining regions: HCDR1, whose amino acid sequence is shown in SEQ ID NO:1; HCDR2, the amino acid sequence of which is shown in SEQ ID NO:2; HCDR3, whose amino acid sequence is shown in SEQ ID NO:3, 17, 18 or 19; LCDR1, whose amino acid sequence is shown in SEQ ID NO:4; LCDR2, whose amino acid sequence is shown in SEQ ID NO:5; and LCDR3, whose amino acid sequence is shown in SEQ ID NO:

6.

3. The antibody according to claim 1 or 2, characterized in that, The antibody also contains HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

4. The antibody according to claim 3, characterized in that, The HFR1 includes SEQ ID NO:7 or an amino acid sequence that has at least 80% identity with it; The HFR2 comprises SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it; The HFR3 includes SEQ ID NO:9 or an amino acid sequence that has at least 80% identity with it; The HFR4 includes SEQ ID NO:10 or an amino acid sequence that is at least 80% identical to it; The LFR1 includes SEQ ID NO:11 or an amino acid sequence that has at least 80% identity with it; The LFR2 includes SEQ ID NO:12 or an amino acid sequence that has at least 80% identity with it; The LFR3 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:13; and The LFR4 includes SEQ ID NO:14 or an amino acid sequence that is at least 80% identical to it.

5. An antibody against S100 protein, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 41; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:27; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:42, 43, 44, and 45.

6. The antibody according to any one of claims 1, 2, 4, and 5, characterized in that, The antibody also contains a constant region.

7. The antibody according to claim 6, characterized in that, The constant region includes the heavy chain constant region and / or the light chain constant region.

8. The antibody according to claim 7, characterized in that, The heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

9. The antibody according to claim 7, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

10. The antibody according to claim 6, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

11. The antibody according to claim 6, characterized in that, The species source of the constant region is mice.

12. The antibody according to claim 7, characterized in that, The heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% identity with it.

13. The antibody according to claim 7, characterized in that, The light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity with it.

14. An antibody against S100 protein, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40; the amino acid sequence of the light chain is as shown in SEQ ID NO: 46; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:34; the amino acid sequences of the light chain are shown in SEQ ID NO:47, 48, 49, and 50.

15. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the antibody as described in any one of claims 1 to 14 and biotin conjugated to the antibody.

16. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody as described in any one of claims 1 to 14 and a label or solid-phase carrier conjugated to the antibody.

17. The antibody conjugate according to claim 16, characterized in that, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

18. A reagent or kit, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 14 or the antibody conjugate as described in any one of claims 15 to 17.

19. Use of the antibody according to any one of claims 1 to 14, the antibody conjugate according to any one of claims 15 to 17, or the reagent or kit according to claim 18 in the preparation of a product for detecting S100 protein.

20. The use according to claim 19, characterized in that, The uses include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, the antibody of any one of claims 1 to 14, the antibody conjugate of any one of claims 15 to 17, or the reagent or kit of claim 18 is contacted with the S100 protein in the sample to be tested to form an immune complex; and b) Detect the presence of the immune complex, the presence of which indicates the presence of S100 protein in the sample to be tested.

21. The use according to claim 20, characterized in that, The immune complex further includes a second antibody, which binds to the antibody.

22. The use according to claim 20, characterized in that, The immune complex also includes a second antibody that binds to the S100 protein.

23. A nucleic acid, characterized in that, It encodes the antibody as described in any one of claims 1 to 14.

24. A carrier, characterized in that, It contains the nucleic acid as described in claim 23.

25. A cell characterized in that, It contains the nucleic acid as described in claim 23 or the vector as described in claim 24.

26. A method for preparing the antibody according to any one of claims 1 to 14, characterized in that, It includes: Culture the cells as described in claim 25.

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