Anti-25-hydroxyvitamin d antibodies and uses thereof

Abstract

This invention discloses an antibody against 25-hydroxyvitamin D and its applications, relating to the field of antibodies. The anti-25-hydroxyvitamin D antibody disclosed in this invention includes a heavy chain complementarity-determining region and a light chain complementarity-determining region. This antibody provides an important source of raw materials for the detection of 25-hydroxyvitamin D and exhibits excellent affinity or activity.

Description

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202311713295.2, filed on December 13, 2023, entitled "Anti-25-hydroxyvitamin D antibody and its use thereof," the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of antibody technology. Specifically, this invention relates to a 25-hydroxyvitamin D antibody and its uses. More specifically, this invention relates to an antibody for 25-hydroxyvitamin D, a nucleic acid molecule, a vector, a cell or host, a method for preparing the antibody, a conjugate, a reagent or kit and its uses, a method for detecting 25-hydroxyvitamin D, a method for screening 25-hydroxyvitamin D antibodies, and a mutant library. Background Technology

[0004] In 1650, Francis Glisson discovered rickets, a disease prevalent among the wealthy. During the Industrial Revolution in the 18th century, urban overcrowding and pollution increased the incidence of rickets by 40-60%. In 1822, Sniadecki discovered the correlation between rickets and lack of sunlight. With further scientific advancements, research showed that insufficient sunlight exposure leads to lower levels of vitamin D in the body, resulting in diseases such as rickets. Consequently, vitamin D, as an essential nutrient, gained widespread public attention.

[0005] 25-hydroxyvitamin D (25-OH-VD) is the main form of vitamin D in the body. Vitamin D is a steroid derivative, a fat-soluble vitamin, and a cyclopentane-polyhydrophenanthrene compound. Vitamin D is mainly synthesized by the human body through skin irradiation by ultraviolet radiation, with a small portion ingested from food or supplements. Vitamin D not only affects calcium and phosphorus metabolism but also has a wide range of physiological functions, being essential for maintaining human health, cell growth, and development. There are two forms of vitamin D in the human body: vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D is converted to 25-hydroxyvitamin D (25-OH-VD) in the liver through hydroxylation, and then converted to the active form, 1,25-dihydroxyvitamin D, in the kidneys.

[0006] 25-hydroxyvitamin D testing is typically performed via serum analysis to determine if vitamin D levels in the body are abnormal. Low 25-hydroxyvitamin D levels are commonly associated with calcium and phosphorus malabsorption, potentially manifesting as abnormal bone and teeth development, leading to osteoporosis, delayed teething, and other symptoms. Increasing epidemiological and laboratory evidence suggests that serum 25-hydroxyvitamin D levels are associated with various diseases, including rickets in children, diabetes, chronic kidney disease, hepatitis, immune dysfunctions (Sjögren's syndrome, multiple sclerosis, rheumatoid arthritis), asthma, osteoporosis, Parkinson's disease, cardiovascular disease, hypertension, type 2 diabetes, and various cancers (prostate cancer, colon cancer, breast cancer, etc.). Therefore, 25-hydroxyvitamin D testing is of significant clinical importance for disease diagnosis and prevention.

[0007] Currently, commonly used methods for detecting 25-hydroxyvitamin D include chemiluminescence, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence, and immunochromatography. All of these methods require specific antibodies to be effective. Therefore, there is a strong demand in this field for antibodies that can effectively bind to and detect 25-hydroxyvitamin D. Summary of the Invention

[0008] The present invention aims to provide an antibody against 25-hydroxyvitamin D, a reagent or kit for detecting 25-hydroxyvitamin D.

[0009] In one aspect of the invention, an anti-25-hydroxyvitamin D antibody is provided, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 have the following amino acid sequences:

[0010] HCDR 1:X1NYGVG, where X1 is D or R;

[0011] HCDR 2:GIDKHGX2RGYNRALKX3, where X2 is V or R, and X3 is S or R;

[0012] HCDR 3:VWYSGNGFDFGYFDY;

[0013] LCDR 1: SGX4X5SNIX6X7GNYVS, where X4 is S or R, X5 is S or W, X6 is G or R, and X7 is Y, V, W or F;

[0014] LCDR 2: DSITRAS;

[0015] LCDR 3:ASFDX8SDGGI, where X8 is S, W, or F.

[0016] In a second aspect, the present invention provides an anti-25-hydroxyvitamin D antibody comprising a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:17 or a variant thereof, wherein, compared to the amino acid sequence shown in SEQ ID NO:17, the variant of SEQ ID NO:17 comprises at least one of the mutations D30R, V56R, and S65R at the following sites; the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:18 or a variant thereof, wherein, compared to the amino acid sequence shown in SEQ ID NO:18, the variant of SEQ ID NO:18 comprises at least one of the mutations S25R, S26W, G30R, Y31V / W / F, and S95W / F at the following sites.

[0017] In a third aspect of the invention, the present invention provides an anti-25-hydroxyvitamin D antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein HCDR1, HCDR2, HCDR3 are the heavy chain variable regions defined by the antibody described in the second aspect; and LCDR1, LCDR2, LCDR3 are the light chain variable regions defined by the antibody described in the second aspect.

[0018] In a fourth aspect of the invention, the invention provides a nucleic acid molecule that encodes the antibody described in the first, second, or third aspect.

[0019] In a fifth aspect, the present invention provides a carrier comprising the nucleic acid molecule described in the fourth aspect.

[0020] In a sixth aspect of the invention, the invention provides a cell or host comprising: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expressing the antibody described in the first, second or third aspect.

[0021] In a seventh aspect of the invention, the invention provides a method for preparing the antibody described in the first, second, or third aspect, the method comprising culturing the cells or host described in the sixth aspect.

[0022] In an eighth aspect of the invention, the invention provides a conjugate comprising: the antibody described in the first, second, or third aspect and a conjugate portion thereto.

[0023] In a ninth aspect of the invention, the invention provides a reagent or kit comprising: an antibody as described in the first, second, or third aspect, or a conjugate as described in the seventh aspect.

[0024] In a tenth aspect of the invention, the invention provides for the use of an antibody described in the first, second, or third aspect, a conjugate described in the eighth aspect, or a reagent or kit described in the ninth aspect in the detection of 25-hydroxyvitamin D, the preparation of a product for the detection of 25-hydroxyvitamin D, or the preparation of a product for indicating 25-hydroxyvitamin D-related diseases.

[0025] In an eleventh aspect of the present invention, a method for detecting 25-hydroxyvitamin D is provided, the method comprising: contacting a sample to be tested with an antibody described in the first, second or third aspect, a conjugate described in the eighth aspect or a reagent or kit described in the ninth aspect to form an immune complex.

[0026] In a twelfth aspect of the present invention, a method for screening 25-hydroxyvitamin D antibodies is provided, the method comprising: a) designing primers for replacing amino acids at sites X1, X2, X3, X4, X5, X6, X7, and X8 defined in the antibody of the first aspect, or at mutation sites defined in the antibody of the second aspect; b) constructing a mutant library using the nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the cell of the sixth aspect as templates, with the primers of a); and c) screening 25-hydroxyvitamin D antibodies from the mutant library.

[0027] In a thirteenth aspect of the invention, the invention provides a mutant library comprising the antibodies described in the first, second, or third aspects.

[0028] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0029] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0033] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0034] In this document, the terms “optional,” “optional,” “alternatively,” “optional,” or “optional” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0035] In this document, the term "antibody" is used in the broadest sense, which may include full-length monoclonal antibodies, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, with no restrictions on their specific structure, as long as they exhibit the desired antigen-binding ability.

[0036] In this article, the terms “full-length antibody”, “full-length monoclonal antibody” or “full-length monoclonal antibody” refer to antibodies composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0037] In this document, the terms "polyclonal antibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes. For example, antibodies that recognize two antigenic epitopes (bispecific antibodies, or simply biantibodies), three antigenic epitopes, or four antigenic epitopes are used in a broad sense, and their specific structures are not limited, as long as they can recognize multiple antigenic epitopes. In this invention, at least one of the multiple antigenic epitopes is derived from 25-hydroxyvitamin D.

[0038] In this document, the term "antigen-binding fragment of an antibody" refers to a fragment containing part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but still possesses the performance activity of specifically binding to an antigen. For example, the fragment may contain part or all of the antibody's CDR. Such fragments bind to antigens and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include, but are not limited to, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, scFv-Fc fusion proteins, scFv-Fv fusion proteins, single-domain antibodies, or minimal recognition units. Such fragments can be generated using recombinant nucleic acid technology or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0039] In this article, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only Fab molecules, which consists of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0040] In this paper, the term “F(ab')2 antibody” or “F(ab')2 fragment” has two antigen-binding F(ab') parts linked together by disulfide bonds.

[0041] In this article, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

[0042] In this paper, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by linking the variable regions of the antibody heavy chain and light chain through short peptides.

[0043] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, with a very small molecular weight, accounting for only about 1% of a complete antibody.

[0044] In this paper, 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 all of the major amino acid residues that contribute to the binding affinity of antibodies to the antigens or epitopes they recognize.

[0045] In this paper, heavy chain complementarity-determining regions (heavy chain variable regions CDRs) are referred to as "HCDRs" or "HCDRs", which include HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2), and HCDR3 (also known as CDR-H3); light chain complementarity-determining regions (light chain variable regions CDRs) are referred to as "LCDRs" or "LCDRs", which include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2), and LCDR3 (also known as CDR-L3). Commonly used CDR definition systems in this field include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition", see Chothia et al., J Mol Biol 196:901-917 (1987). Exemplary CDR definitions 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 the CDRs defined in this invention include, but are not limited to, the CDRs defined by the methods in Table 1. CDRs defined using other methods disclosed in the art based on the heavy chain variable region and light chain variable region disclosed in this application are also within the scope of this disclosure.

[0046] Table 1: CDR Definition 1

[0047]

[0048]

[0049] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below). The amino acid number on the heavy chain is represented by "H + number", and the amino acid number on the light chain is represented by "L + number".

[0050] 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.

[0051] 3If 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.

[0052] 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.

[0053] 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.

[0054] Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. 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 stated herein, "Kabat numbering" refers to numbering using the numbering system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983), for which the HCDRs and LCDRs of the antibodies in this application were numbered using the aforementioned system; the specific numbering results are shown in Table 1. It should be noted that the polypeptide sequences of this invention are not numbered according to the Kabat numbering system. However, those skilled in the art can readily convert the sequence numbers in the sequence listing to Kabat numbers.

[0055] In this paper, the term "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 (which may be denoted as VH) and light chain variable region (which may be denoted as VL) excluding the CDR; wherein, the heavy chain frame region is denoted as "HFR" and can be further subdivided into adjacent regions separated by CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region is denoted as "LFR" and can be further subdivided into adjacent regions separated by CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0056] In this paper, the variable region of the heavy chain is obtained by connecting the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the variable region of the light chain is obtained by connecting the following CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0058] Without substantially affecting antibody activity (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain variants of the antibody sequence. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences of this invention can have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0059] It should be noted that the location or number of the mutation sites defined in the specification and claims of this invention also needs to be adjusted according to the number and location of the added and / or deleted amino acids.

[0060] In this paper, the term "at least 80% homology" refers to at least 80% homology with each reference sequence, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. The term "at least 90% homology" refers to at least 90% homology with each reference sequence, which can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0061] In this paper, the term “variant” or “mutant” can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid mutations.

[0062] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into and / or between cells or hosts. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0063] In this document, the term "cell" generally refers to a cell whose genetic material has been modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0064] This invention proposes an anti-25-hydroxyvitamin D antibody, a nucleic acid molecule, a vector, a cell or host, a method for preparing the antibody, a conjugate, a reagent or kit and its uses, a method for detecting 25-hydroxyvitamin D, a method for screening 25-hydroxyvitamin D antibodies, and a mutant library, which will be described in detail below.

[0065] Antibody

[0066] In a first aspect, the present invention provides an anti-25-hydroxyvitamin D antibody. According to embodiments of the present invention, the antibody comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 have the following amino acid sequences:

[0067] HCDR 1:X1NYGVG, where X1 is D or R;

[0068] HCDR 2:GIDKHGX2RGYNRALKX3, where X2 is V or R, and X3 is S or R;

[0069] HCDR 3:VWYSGNGFDFGYFDY;

[0070] LCDR 1: SGX4X5SNIX6X7GNYVS, where X4 is S or R, X5 is S or W, X6 is G or R, and X7 is Y, V, W or F;

[0071] LCDR 2: DSITRAS;

[0072] LCDR 3:ASFDX8SDGGI, where X8 is S, W, or F.

[0073] According to an embodiment of the present invention, HCDR1 is defined as the amino acid sequence at Kabat positions H30-H35, HCDR2 is defined as the amino acid sequence at Kabat positions H50-H65, HCDR3 is defined as the amino acid sequence at Kabat positions H95-H102, LCDR1 is defined as the amino acid sequence at Kabat positions L24-L34, LCDR2 is defined as the amino acid sequence at Kabat positions L50-L56, and LCDR3 is defined as the amino acid sequence at Kabat positions L89-L97.

[0074] As can be seen from the aforementioned principles of CDR definition, different systems will yield different CDRs, even for the same heavy chain variable region or the same light chain variable region. In this application, "HCDR 1:X1NYGVG" refers to HCDR 1 whose amino acid sequence includes or is X1NYGVG. This can also be adjusted according to different systems to obtain HCDR 1 with sequences shorter or longer than X1NYGVG.

[0075] In some optional embodiments of the present invention, X1 is D;

[0076] In some optional embodiments of the present invention, X1 is R;

[0077] In some optional embodiments of the present invention, X2 is V;

[0078] In some optional embodiments of the present invention, X2 is R;

[0079] In some optional embodiments of the present invention, X3 is S;

[0080] In some optional embodiments of the present invention, X3 is R;

[0081] In some optional embodiments of the present invention, X4 is S;

[0082] In some optional embodiments of the present invention, X4 is R;

[0083] In some optional embodiments of the present invention, X5 is S;

[0084] In some optional embodiments of the present invention, X5 is W;

[0085] In some optional embodiments of the present invention, X6 is G;

[0086] In some optional embodiments of the present invention, X6 is R;

[0087] In some optional embodiments of the present invention, X7 is Y;

[0088] In some optional embodiments of the present invention, X7 is V;

[0089] In some optional embodiments of the present invention, X7 is W;

[0090] In some optional embodiments of the present invention, X7 is F;

[0091] In some optional embodiments of the present invention, X8 is S;

[0092] In some optional embodiments of the present invention, X8 is W;

[0093] In some optional embodiments of the present invention, X8 is F;

[0094] In some optional embodiments of the present invention, X1 being D, X2 being V, X3 being S, X4 being S, X5 being S, X6 being G, X7 being Y, and X8 being S do not simultaneously hold true.

[0095] In some optional embodiments of the present invention, X1, X2, X3, X4, X5, X6, X7 and X8 are selected from any combination of the following:

[0096]

[0097]

[0098] The antibody described in the first aspect of this invention has excellent activity, affinity, stability, or specificity.

[0099] The antibody described in the first aspect of the present invention has improved activity, affinity, stability or specificity.

[0100] In a second aspect, the present invention provides an anti-25-hydroxyvitamin D antibody. According to embodiments of the invention, the antibody or functional fragment comprises: a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:17 or a variant thereof, wherein, compared to the amino acid sequence shown in SEQ ID NO:17, the variant of SEQ ID NO:17 comprises a mutation at at least one of the following sites: D30R, V56R, and S65R; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:18 or a variant thereof, wherein, compared to the amino acid sequence shown in SEQ ID NO:18, the variant of SEQ ID NO:18 comprises a mutation at at least one of the following sites: S25R, S26W, G30R, Y31V / W / F, and S95W / F.

[0101] It should be noted that the numbering of the variable sites in the heavy chain is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO:17 from the N-terminus to the C-terminus. For example, position 30 refers to the 30th position of the amino acid sequence shown in SEQ ID NO:17 starting from the N-terminus; "D30R" means that the aspartic acid at position 30 of the amino acid sequence shown in SEQ ID NO:17 is replaced by arginine.

[0102] The numbering of the variable sites in the light chain was obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO:18 from the N-terminus to the C-terminus. For example, position 25 refers to the 25th position of the amino acid sequence shown in SEQ ID NO:18 starting from the N-terminus; "S25R" means that the serine at position 25 of the amino acid sequence shown in SEQ ID NO:18 is replaced by arginine; and "S95W / F" means that the serine at position 95 of the amino acid sequence shown in SEQ ID NO:18 can be replaced by tryptophan or phenylalanine.

[0103] According to embodiments of the present invention, the antibody may further include at least one of the following additional technical features:

[0104] In some alternative embodiments of the present invention, the mutation at the 30th position is D30R;

[0105] In some alternative embodiments of the present invention, the mutation at position 56 is V56R;

[0106] In some alternative embodiments of the present invention, the mutation at the 65th position is S65R;

[0107] In some alternative embodiments of the present invention, the mutation at the 25th position is S25R;

[0108] In some optional embodiments of the present invention, the mutation at the 26th position is S26W;

[0109] In some alternative embodiments of the present invention, the mutation at the 30th position is G30R;

[0110] In some optional embodiments of the present invention, the mutation at the 31st position is Y31V;

[0111] In some alternative embodiments of the present invention, the mutation at the 31st position is Y31W;

[0112] In some optional embodiments of the present invention, the mutation at the 31st position is Y31F;

[0113] In some alternative embodiments of the present invention, the mutation at the 95th position is S95W;

[0114] In some alternative embodiments of the present invention, the mutation at the 95th position is S95F;

[0115] In some alternative embodiments of the present invention, the heavy chain variable region and the light chain variable region are selected from any combination of the following:

[0116]

[0117]

[0118] It should be noted that the comma in the table above represents "and", that is, a combination mutation. For example, "D30R,V56R,S65R" means a combination mutation of three mutations: D30R, V56R, and S65R.

[0119] In a third aspect, the present invention provides an anti-25-hydroxyvitamin D antibody. According to embodiments of the present invention, the antibody comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein HCDR1, HCDR2, HCDR3 are the heavy chain variable regions defined by the antibody described in the second aspect; and LCDR1, LCDR2, LCDR3 are the light chain variable regions defined by the antibody described in the second aspect.

[0120] It should be noted that HCDR1, HCDR2, and HCDR3 in the antibody described in the third aspect are HCDR1, HCDR2, and HCDR3 of the same heavy chain variable region defined in the antibody described in the second aspect, and LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 of the same light chain variable region defined in the antibody described in the second aspect.

[0121] For example, when the heavy chain variable region defined in the antibody described in the second aspect has no mutation compared to SEQ ID NO:17, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are HCDR1:DNYGVG; HCDR2:GIDKHGVRGYNRALKS; and HCDR3:VWYSGNGFDFGYFDY, respectively; then the HCDR1, HCDR2, and HCDR3 contained in the antibody described in the third aspect are also HCDR1:DNYGVG; HCDR2:GIDKHGVRGYNRALKS; and HCDR3:VWYSGNGFDFGYFDY, respectively.

[0122] For example, when the light chain variable region defined in the antibody described in the second aspect has a mutation at the S25R site compared to SEQ ID NO:18, the LCDR1, LCDR2, and LCDR3 of the light chain variable region are LCDR1:SGRSSNIGYGNYVS; LCDR2:DSITRAS; and LCDR3:ASFDSSDGGI, respectively; then the LCDR1, LCDR2, and LCDR3 contained in the antibody described in the third aspect are also LCDR1:SGRSSNIGYGNYVS; LCDR2:DSITRAS; and LCDR3:ASFDSSDGGI, respectively.

[0123] According to an embodiment of the present invention, HCDR1 is defined as the amino acid sequence at Kabat positions H30-H35, HCDR2 is defined as the amino acid sequence at Kabat positions H50-H65, HCDR3 is defined as the amino acid sequence at Kabat positions H95-H102, LCDR1 is defined as the amino acid sequence at Kabat positions L24-L34, LCDR2 is defined as the amino acid sequence at Kabat positions L50-L56, and LCDR3 is defined as the amino acid sequence at Kabat positions L89-L97.

[0124] In some optional 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.

[0125] According to embodiments of the present invention, the antibody described in the first or third aspect further includes at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0126] In an optional embodiment of the present invention, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0127] According to embodiments of the present invention, HFR1 comprises an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology therewith; HFR2 comprises an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology therewith; HFR3 comprises an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology therewith; HFR4 comprises an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology therewith; LFR1 comprises an amino acid sequence as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% homology therewith; LFR2 comprises an amino acid sequence as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% homology therewith; LFR3 comprises an amino acid sequence as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% homology therewith; and LFR4 comprises an amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% homology therewith.

[0128] According to embodiments of the present invention, the antibody described in the first, second, or third aspect above may further include at least one of the following technical features:

[0129] According to an embodiment of the present invention, the affinity KD of the antibody is <10. -9 M.

[0130] According to an embodiment of the present invention, the affinity KD of the antibody is <10. -10 M.

[0131] According to an embodiment of the present invention, the affinity KD of the antibody is <10. -11 M.

[0132] According to an embodiment of the present invention, the affinity KD of the antibody is <10. -12 M.

[0133] According to an embodiment of the present invention, the affinity KD of the antibody is <10. -13 M.

[0134] According to an embodiment of the present invention, the antibody further includes a constant region; wherein the constant region includes at least one of a heavy chain constant region and a light chain constant region.

[0135] According to embodiments of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0136] According to an embodiment of the present invention, 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.

[0137] In an optional embodiment of the present invention, 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.

[0138] In an optional embodiment of the present invention, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0139] According to an embodiment of the present invention, the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0140] In an optional embodiment of the present invention, the heavy chain constant region includes or is an amino acid sequence as shown in SEQ ID NO:15 or having at least 80% identity with it; or the light chain constant region includes or is a light chain constant region as shown in SEQ ID NO:16 or having at least 80% identity with it.

[0141] According to an embodiment of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region, and the N end of the light chain constant region is connected to the C end of the light chain variable region.

[0142] In this paper, the partitioning of the variable and constant regions is based on the IMGT partitioning method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:housemouse(Mus musculus)IGHC,IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. theinternational ImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus of the variable region or the N-terminus of the constant region delineated by IMGT. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this invention.

[0143] Those skilled in the art will understand that the features and advantages described above with respect to the first aspect of the antibody also apply to the antibody described in the second or third aspect, and will not be repeated here.

[0144] Nucleic acid molecules, vectors, cells or hosts, and methods for preparing antibodies.

[0145] In the process of preparing or obtaining the antibodies described in the first, second or third aspects, nucleic acid molecules expressing these antibodies can be linked to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0146] In a fourth aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the antibody described in the first, second, or third aspect. The nucleic acid molecule according to embodiments of the present invention can encode the aforementioned antibody.

[0147] According to embodiments of the present invention, the nucleic acid molecule includes DNA or RNA.

[0148] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the molecular sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0149] In a fifth aspect, the present invention provides a vector. According to embodiments of the present invention, the vector comprises the nucleic acid molecule described in the fourth aspect. When the above-mentioned nucleic acid molecule is ligated to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or can be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked. In this document, "operably linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the vector of some specific embodiments of the present invention is introduced into suitable recipient cells, the aforementioned antibody expression can be effectively achieved under the mediation of a regulatory system, thereby achieving large-scale in vitro production of antibodies.

[0150] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0151] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector.

[0152] In a sixth aspect, the present invention provides a cell or host. According to embodiments of the invention, the cell or host comprises: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expressing the antibody described in the first, second, or third aspect. Using this cell, under suitable conditions, the aforementioned antibody can be effectively expressed intracellularly.

[0153] According to an embodiment of the present invention, the cell is obtained by introducing the carrier described in the fifth aspect into the cell.

[0154] It should be noted that the cells used in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0155] In an optional embodiment of the present invention, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, but do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0156] It should be noted that the "suitable conditions" mentioned in this invention refer to conditions suitable for the expression of the antibody described in this invention. Those skilled in the art will readily understand that suitable conditions for antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for antibody expression based on the specific environment of their laboratory.

[0157] In a seventh aspect, the present invention provides a method for preparing the antibodies described in the first, second, or third aspects. According to embodiments of the invention, the method includes culturing the cells or host described in the sixth aspect. The methods according to some specific embodiments of the invention can efficiently obtain large quantities of the antibodies.

[0158] Based on the amino acid sequence of the antibody disclosed herein, those skilled in the art will readily conceive of preparing the antibody using genetic engineering or other techniques (chemical synthesis, recombinant expression), such as isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody as 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 antibody disclosed herein, it falls within the protection scope of this disclosure.

[0159] Those skilled in the art will understand that the features and advantages described above for the antibodies in relation to the first, second, or third aspects also apply to the nucleic acid molecule, vector, cell or host, and the method of preparing the antibody, which will not be repeated here.

[0160] Conjugates, reagents or kits and their uses

[0161] In an eighth aspect, the present invention provides a conjugate. According to embodiments of the invention, the conjugate comprises: an antibody as described in the first, second, or third aspect, and a conjugated portion thereto. The conjugate according to embodiments of the invention can specifically bind to 25-hydroxyvitamin D and can be used for the qualitative or quantitative detection of 25-hydroxyvitamin D, or for indicating 25-hydroxyvitamin D-related diseases.

[0162] According to embodiments of the present invention, the above-mentioned coupling may further include at least one of the following additional technical features:

[0163] According to an embodiment of the present invention, the coupling portion includes at least one of a purification tag, an affinity substance, a marker, and a solid-phase support.

[0164] According to an embodiment of the present invention, the purification label includes at least one of the following: His label, Flag label, GST label, MBP label, SUMO label, and C-Myc label.

[0165] In this document, the affinity substance may be, for example, biotin, biotin derivatives or streptavidin, one of the complementary sense and antisense strands of nucleic acids.

[0166] In some alternative embodiments of the present invention, the affinity substance includes at least one selected from biotin, biotin derivatives, or streptavidin.

[0167] In this article, "marker" refers to a class of substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc., which enable qualitative or quantitative detection of the corresponding target.

[0168] According to embodiments of the present invention, the marker includes at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0169] 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.

[0170] According to embodiments of the present invention, 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).

[0171] According to embodiments of the present invention, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0172] According to embodiments of the present invention, the radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、177 Lu、 172 Lu and 18 F.

[0173] According to embodiments of the present invention, 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.

[0174] According to embodiments of the present invention, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0175] According to embodiments of the present invention, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0176] According to embodiments of the present invention, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0177] In this document, the solid support can be a substance that can be suspended or dispersed in a liquid phase (e.g., solid supports such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as perforated plates, microfluidic paths, glass capillaries, nanopillars, and monolithic columns).

[0178] According to an embodiment of the present invention, the solid support comprises at least one selected from microspheres, plates, and membranes.

[0179] In a ninth aspect of the invention, a reagent or kit is provided. According to embodiments of the invention, the reagent or kit comprises: an antibody as described in the first, second, or third aspect, or a conjugate as described in the eighth aspect. As previously mentioned, the antibodies in some specific embodiments or examples of the invention are capable of binding to 25-hydroxyvitamin D; therefore, reagents or kits containing said antibodies can effectively perform qualitative or quantitative detection of 25-hydroxyvitamin D. The reagents or kits provided by the invention can be used, for example, for detections involving the specific binding properties of 25-hydroxyvitamin D and its antibodies, such as immunoblotting and immunoprecipitation. As previously mentioned, the mutant antibodies of the invention have improved 25-hydroxyvitamin D binding activity, affinity, stability, or specificity; therefore, reagents or kits containing said antibodies have improved detection sensitivity or specificity.

[0180] The above kit may contain any one or more of the following: processing solution, anti-25-hydroxyvitamin D antibody, 25-hydroxyvitamin D quality control, anti-IgG antibody, instructions for use, or literature. Anti-25-hydroxyvitamin D antibody can be used for various types of diagnostic tests, such as detecting the presence of various diseases, drugs, or other proteins in vitro or in vivo. For example, it can be used to test for related diseases by detecting the serum or blood of the subject.

[0181] In a tenth aspect of the invention, the invention provides for the use of an antibody described in the first, second, or third aspect, a conjugate described in the eighth aspect, or a reagent or kit described in the ninth aspect in the detection of 25-hydroxyvitamin D, indicative of 25-hydroxyvitamin D-related diseases, in the preparation of a product for the detection of 25-hydroxyvitamin D, or in the preparation of a product for indicative of 25-hydroxyvitamin D-related diseases.

[0182] According to embodiments of the present invention, the "product" includes, but is not limited to, reagents, test strips, reagent plates, or reagent kits.

[0183] According to embodiments of the present invention, the “instruction” includes, but is not limited to, prediction, diagnosis, prognosis, or medication guidance.

[0184] According to an embodiment of the present invention, the "25-hydroxyvitamin D-related disease" refers to a disease that uses 25-hydroxyvitamin D as a marker.

[0185] In one or more embodiments of this disclosure, 25-hydroxyvitamin D-related diseases include, but are not limited to, rickets, diabetes, chronic kidney disease, hepatitis, Sjögren's syndrome, multiple sclerosis, rheumatoid arthritis, asthma, osteoporosis, Parkinson's disease, cardiovascular disease, hypertension, type 2 diabetes, tumors, osteomalacia, or vitamin D poisoning.

[0186] In an eleventh aspect, the present invention provides a method for detecting 25-hydroxyvitamin D. According to an embodiment of the present invention, the method includes: contacting the sample to be tested with an antibody described in the first, second, or third aspect, a conjugate described in the eighth aspect, or a reagent or kit described in the ninth aspect to form an immune complex.

[0187] According to an embodiment of the present invention, based on the signal of the immune complex, it is determined whether the sample to be tested contains 25-hydroxyvitamin D or the content of 25-hydroxyvitamin D.

[0188] According to an embodiment of the present invention, the immune complex further includes a second antibody, which binds to the antibody.

[0189] According to an embodiment of the present invention, the signal includes a fluorescence signal.

[0190] Methods, Mutant Library

[0191] In a twelfth aspect of the invention, a method for screening 25-hydroxyvitamin D antibodies is provided. According to an embodiment of the invention, the method comprises: a) designing primers for replacing amino acids at one, two, three, four, five, six, seven, or eight sites of X1, X2, X3, X4, X5, X6, X7, and X8 defined in the antibody of the first aspect, or at mutation sites defined in the antibody of the second aspect; b) constructing a mutant library using the primers of a) as a template, employing the nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the cell or host of the sixth aspect;

[0192] c) Screen for 25-hydroxyvitamin D antibodies from the mutant library.

[0193] According to an embodiment of the present invention, the mutant library is a single-point saturation mutant library.

[0194] According to embodiments of the present invention, the 25-hydroxyvitamin D antibody includes or is the antibody described in the first, second, or third aspect.

[0195] In a thirteenth aspect of the invention, the invention provides a mutant library comprising the antibodies described in the first, second, or third aspects.

[0196] According to an embodiment of the present invention, the mutant library is obtained by the method described in the twelfth aspect.

[0197] In a fourteenth aspect of the invention, a method for indicating 25-hydroxyvitamin D-related diseases is provided. According to embodiments of the invention, the method comprises: contacting a sample to be tested from a subject with an antibody described in the first, second, or third aspect, a conjugate described in the eighth aspect, or a reagent or kit described in the ninth aspect to form an immune complex.

[0198] According to an embodiment of the present invention, the state of the subject's 25-hydroxyvitamin D-related disease is indicated based on the signal of the immune complex.

[0199] According to embodiments of the present invention, the method described in the fourteenth aspect above may further include at least one of the following technical features:

[0200] According to an embodiment of the present invention, the immune complex further includes a second antibody, which binds to the antibody.

[0201] According to an embodiment of the present invention, the signal includes a fluorescence signal.

[0202] As used herein, the term "subject" refers to a vertebrate, preferably a mammal, and most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, racing animals, and pets. Tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro are also included.

[0203] The amino acid sequences involved in this article are shown in Table 2:

[0204] Table 2: Amino Acid Sequences

[0205]

[0206]

[0207] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0208] 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.

[0209] Unless otherwise specified, the practice of this disclosure 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., 2011), each of which is explicitly incorporated herein by reference.

[0210] In this embodiment, restriction endonucleases, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, Taq DNA polymerase was purchased from TaKaRa, gel extraction kits and plasmid extraction kits were commercially available, and primer synthesis and gene sequencing were outsourced. The sequence of the 25-hydroxyvitamin D monoclonal antibody (hereinafter referred to as WT antibody) was derived from the sequencing of sheep phage display antibodies.

[0211] Example 1: Construction and screening of mutant libraries

[0212] 1. Construction of template plasmid for wild-type (WT) 25-hydroxyvitamin D antibody (WT antibody for short)

[0213] (1) WT antibody gene synthesis:

[0214] The nucleotide sequences of VH and VL of the WT antibody were optimized using E. coli codons, and then the antibody gene sequence was synthesized. The amino acid sequences of VH and VL of the WT antibody are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively, and the heavy chain and light chain amino acid sequences are shown in SEQ ID NO:19 and SEQ ID NO:20, respectively.

[0215] (2) Amplification of WT antibody gene fragments:

[0216] The nucleotide sequences of antibodies VH and VL synthesized in step (1) were amplified by DNA polymerase PCR, and then the antibody bands were separated by agarose gel electrophoresis. The antibody gene fragments were then purified using a gel recovery kit.

[0217] (3) Enzyme digestion and ligation of WT antibody gene fragments:

[0218] The antibody gene fragment and the VO1 vector plasmid (including the nucleotide sequence of the constant region) obtained in step (2) were simultaneously digested with restriction endonucleases. The resulting antibody gene fragment and VO1 vector with sticky ends were then purified using a gel extraction kit. Next, the antibody gene fragment and VO1 vector were ligated with T4 DNA ligase at 22°C for 4 hours. The ligation product was then recovered and purified, and the DNA concentration was determined. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells to obtain a bacterial culture. The entire bacterial culture was then plated onto ampicillin-resistant plates and incubated overnight at 37°C.

[0219] (4) Extraction and sequencing verification of WT template plasmid

[0220] Ten single-clone colonies were selected from the overnight culture in step (3). Colony PCR and gel electrophoresis were performed using Taq DNA polymerase. The bacteria with the correct inserted antibody gene sequence were selected for culture and amplification. The plasmid was extracted using a plasmid extraction kit and then sent to a sequencing company for gene sequencing verification.

[0221] 2. Construction of single-point mutation libraries

[0222] This part of the experiment involves performing single-point saturation mutations on the VH and VL full CDR regions of the WT antibody obtained in step 1 to construct a single-point mutation library.

[0223] (1) Primer design and synthesis

[0224] Using degenerate base codons, 68 pairs of upstream and downstream primers for single-point saturation mutations of amino acid sites (68 amino acid sites) in the full CDR region of VH and VL were designed and outsourced to a company for primer synthesis.

[0225] (2) PCR amplification of single-point saturation mutant plasmid

[0226] Using the primers obtained in step (1), the single-point saturated mutant plasmids were amplified by PCR. The reaction system was prepared according to Table 3, and then the PCR reaction conditions in Table 4 were used to amplify and prepare the single-point saturated mutant library plasmids. Finally, the WT template plasmids obtained in step 1 were digested with restriction endonucleases at 37°C for 1 hour to obtain plasmids of 68 mutant libraries.

[0227] Table 3: PCR amplification reaction system

[0228] WT template plasmid 50ng DNA polymerase 1μl DNA polymerase buffer 10μl dNTP (2.5mM) 4μl Upstream primer (10 μM) 1μl Downstream primer (10 μM) 1μl ddH2O Adjust the volume to 50 μl

[0229] Table 4: PCR reaction conditions

[0230] Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ time 5min 30s 30s 2min 5min ∞

[0231] Note: Steps 2 through 4 are repeated 22 times.

[0232] (3) Transformation with single-point saturation mutant plasmids:

[0233] Take 10 μl of the plasmids of the mutant library of amino acid sites in the full CDR region obtained in step (2), transform 10 μl of each reaction product into 100 μl of TG1 Escherichia coli competent cells to obtain bacterial culture, and then spread all the bacterial culture on a plate containing ampicillin resistance and incubate overnight at 37°C.

[0234] 3. Screening of single-point mutation libraries

[0235] (1) Antibody expression in mutant library

[0236] 500 μl of culture medium was added to each 96-well plate beforehand. For each single-point mutation library, 92 single-clone colonies were selected, and WT, negative control (i.e., colonies without the insertion of VH / VL genes) and blank control (i.e., only culture medium, no colonies) colonies were set up. After incubation at 37°C for 5-6 hours, the bacterial culture was transferred to a new 96-well plate and incubated at 37°C for 1-2 hours. Finally, induction medium was added and the plate was incubated overnight at 37°C to express antibodies, resulting in antibody expression supernatants from 68 mutant libraries.

[0237] (2) Mutant library screening and sequencing

[0238] Commercially available 25-OH-VD-BSA was added to 68 ELISA plates at a rate of 0.0125 μg / ml and 100 μl / well. The plates were incubated overnight at 4°C. The plates were then blocked with 1-2% skim milk powder the next day. The antibody expression supernatant of the 68 mutant libraries obtained in step (1) was added to the wells of the ELISA plates at a rate of 100 μl / well. WT, negative control (i.e., colonies without VH / VL genes) and blank control (i.e., only culture medium without colonies) were set up and incubated at room temperature for 2 hours. The plates were then washed, developed, and read using the standard ELISA detection method. Finally, the data results were analyzed, and clones with improved mutations were sent for sequencing. The sequencing results were then analyzed, and the mutation sites of 11 unique mutant candidate clones were selected (see Table 5) for the construction of combined mutant libraries. (The Ratio values ​​in Table 5 represent the degree of affinity enhancement. When the Ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of WT.)

[0239] Table 5: Screening results and mutation sites of candidate clones

[0240]

[0241] Note: In this paper, WT represents no mutation relative to the wild-type sequence (same as Table 6 and Table 7); the positions of the mutated amino acids in this paper are obtained by sequentially numbering the amino acid sequences of VH or VL of WT from the N-terminus to the C-terminus.

[0242] 4. Construction of combined mutant libraries

[0243] (1) Library primer design and synthesis:

[0244] Based on the mutation sites on VH and VL of the 25-hydroxyvitamin D antibody obtained in step 3-(2), amplification primers for the combined mutant library were designed and primers were synthesized.

[0245] (2) Fragment amplification and ligation

[0246] According to the PCR system in Table 3 and the PCR reaction conditions in Table 4, the antibody mutant fragments were amplified, and then the antibody mutant fragments were recovered by gel electrophoresis. The antibody mutant fragments were then spliced ​​into complete antibody fragments (heavy chain variable region or light chain variable region) using the overlap PCR method.

[0247] Finally, the antibody fragment was inserted into the V01 vector by enzyme digestion and ligation to form a complete antibody expression plasmid (see step 1-(3) "WT antibody gene fragment digestion and ligation" for specific steps). 100 ng of plasmid was transformed into 100 μl of TG1 Escherichia coli competent cells, and the entire bacterial culture was plated on an ampicillin-resistant plate and incubated overnight at 37°C.

[0248] 5. Screening of combined mutant libraries

[0249] Thirty-eight randomly selected combined mutant antibodies were subjected to supernatant expression, ELISA screening, and positive clone sequencing analysis. The combined mutation site information and ratio values ​​are shown in Table 6. The results showed that the binding activity of mutant antibodies 1-38 was significantly better than that of WT antibodies.

[0250] Table 6: Information on candidate clones with combined mutations

[0251]

[0252]

[0253] Example 2: Expression of mutant 25-hydroxyvitamin D antibody

[0254] This embodiment expresses the mutant 25-hydroxyvitamin D antibody obtained in Example 1. The specific experimental procedures are as follows:

[0255] 1. Construction of eukaryotic recombinant expression plasmids

[0256] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector that has been constructed. 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 variable region gene sequences in the 49 candidate clones screened in Example 1, VL and VH gene-specific amplification primers and constant region overlap primers for the corresponding antibody sequences were designed. The primers at both ends are respectively equipped with HindIII and EcoRI restriction sites and protective bases. The light chain gene fragment and the heavy chain gene fragment were amplified by PCR amplification.

[0257] The heavy and light chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. The digested antibody light and heavy chain gene fragments and the vector were purified and recovered. The gene fragments encoding the antibody light and heavy chains were then ligated into the 3.4A expression vector and transformed into DH5α *E. coli* competent cells. After bacterial growth, single colonies were picked for PCR identification of positive clones. Positive clones were then sequenced to confirm sequence accuracy. Plasmids were extracted from correctly sequenced clones for later use.

[0258] 2. Sample preparation of recombinant antibodies

[0259] HEK293 cells were revived in advance and passaged to a 200ml volume to achieve a cell density of (3-5)×10⁻⁶ cells / mL. 6 Cells / ml, cell viability >95%; centrifuge to wash cells, rehydrate with culture medium, and adjust cell density to 2.9 × 10⁶ cells / ml. 6 Cells / ml were used as cell dilution buffers. 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. Antibodies were obtained through affinity purification using a protein A affinity chromatography column.

[0260] Example 3: Affinity Analysis

[0261] The 49 mutant antibodies obtained above were analyzed for affinity. The specific steps were as follows: the binding and dissociation curves of the antigen and antibody were tested on a Biacore 8K+ instrument, and the instrument automatically fitted and obtained the affinity constant, binding rate, and dissociation rate. In the affinity test results, KD represents the equilibrium dissociation constant, i.e., the affinity constant; the smaller the KD value, the higher the affinity; ka represents the binding rate; and kd represents the dissociation rate. The results showed that the mutant antibody had a better affinity for 25-hydroxyvitamin D than the wild-type antibody.

[0262] Table 7: Affinity Test Data

[0263]

[0264]

[0265] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0266] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-25-hydroxyvitamin D antibody comprising HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, characterized in that, The amino acid sequences of HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 are as shown below: HCDR 1:X1NYGVG, where X1 is D or R; HCDR 2: GIDKHGX2RGYNRALKX3, where X2 is V or R, and X3 is S or R; HCDR 3: VWYSGNGFDFGYFDY; LCDR 1: SGX4X5SNIX6X7GNYVS, where X4 is S or R, X5 is S or W, X6 is G or R, and X7 is Y, V, W or F; LCDR 2: DSITRAS; LCDR 3: ASFDX8SDGGI, where X8 is S, W, or F; The following conditions cannot be met simultaneously: X1 is D, X2 is V, X3 is S, X4 is S, X5 is S, X6 is G, X7 is Y, and X8 is S. X1, X2, X3, X4, X5, X6, X7, and X8 are selected from any of the following combinations: 。 2. An antibody against 25-hydroxyvitamin D, characterized in that include: Heavy chain variable region and light chain variable region; The heavy chain variable region and the light chain variable region are selected from any combination of the following: 。 3. An anti-25-hydroxyvitamin D antibody comprising HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, characterized in that, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are, in order, the heavy chain variable regions defined by the antibody of any combination as described in claim 2, namely HCDR1, HCDR2, HCDR3 and the light chain variable regions, namely LCDR1, LCDR2, and LCDR3. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 is any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

4. The antibody according to any one of claims 1 to 3, characterized in that, The antibodies include HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

5. The antibody according to claim 4, characterized in that, The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, or mutants thereof.

6. The antibody according to claim 4, characterized in that, The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are derived from at least one of human antibodies, dairy bovine antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

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

8. The antibody according to any one of claims 1-3 and 5-7, characterized in that, The antibody further includes a constant region; wherein the constant region includes a heavy chain constant region and a light chain constant region.

9. The antibody according to claim 8, characterized in that, The heavy chain constant region and the light chain constant region are derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, or mutants thereof.

10. The antibody according to claim 8, characterized in that, The heavy chain constant region and the light chain constant region are derived from at least one of human antibodies, dairy bovine antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

11. The antibody according to claim 8, 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.

12. The antibody according to claim 8, 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.

13. The antibody according to claim 8, characterized in that, The light chain constant region includes light chain constant regions selected from κ-type or λ-type.

14. The antibody according to claim 8, characterized in that, The heavy chain constant region includes or is an amino acid sequence as shown in SEQ ID NO:15 or having at least 80% identity with it; the light chain constant region includes or is an amino acid sequence as shown in SEQ ID NO:16 or having at least 80% identity with it.

15. A nucleic acid, characterized in that, The nucleic acid encodes the antibody according to any one of claims 1 to 14.

16. A carrier, characterized in that, The vector contains the nucleic acid as described in claim 15.

17. A cell characterized in that, The cell contains the nucleic acid of claim 15 or the vector of claim 16.

18. A method for preparing the antibody according to any one of claims 1 to 14, characterized in that, The method includes culturing the cells of claim 17.

19. A coupling, characterized in that, include: The antibody according to any one of claims 1 to 14 and the coupling portion thereto, said coupling portion comprising at least one of a purification tag and a solid-phase carrier.

20. The coupling according to claim 19, characterized in that, The purification label includes at least one of the following: His label, Flag label, GST label, MBP label, SUMO label, and C-Myc label.

21. The coupling according to claim 19, characterized in that, The solid support is selected from at least one of microspheres, plates, and membranes.

22. A coupling agent, characterized in that, include: The antibody and its conjugated portion according to any one of claims 1 to 14, wherein the conjugated portion comprises a marker selected from at least one of fluorescent dyes, enzymes, chemiluminescent reagents and nanoparticle markers.

23. A coupling, characterized in that, include: The antibody and its conjugated portion according to any one of claims 1 to 14, wherein the conjugated portion comprises an affinity substance, characterized in that the affinity substance is selected from at least one of biotin, biotin derivatives, or streptavidin.

24. A reagent or kit, characterized in that, include: The antibody according to any one of claims 1 to 14 or the conjugate according to any one of claims 19 to 23.

25. Use of the antibody according to any one of claims 1 to 14, or the conjugate according to any one of claims 19 to 23, or the reagent or kit according to claim 24, in the preparation of a product for the detection of 25-hydroxyvitamin D.

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