25-oh-VD complex antibody and use thereof

Abstract

Disclosed in the present disclosure is a 25-OH-VD complex antibody, relating to the field of antibodies. The 25-OH-VD complex antibody disclosed in the present disclosure comprises a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for the detection of 25-OH-VD, and has good specificity and activity.

Description

25-OH-VD complex antibody and application thereof

[0001] Cross-reference to related applications

[0002] The present disclosure claims priority to the Chinese patent application No. 202410841411.7, filed on June 26, 2024, entitled "25-OH-VD complex antibody and application thereof", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to the field of antibody technology, in particular, to a 25-OH-VD complex antibody and application thereof. BACKGROUND

[0004] 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 polycyclic hydrophenanthrene compound. Vitamin D is mainly synthesized by human skin after ultraviolet irradiation, and a small part is obtained from food or supplements. Vitamin D not only affects calcium and phosphorus metabolism, but also has a wide range of physiological effects, and is an essential substance for maintaining human health, cell growth and development, and is closely related to many diseases. Vitamin D is converted to 25-hydroxyvitamin D in the liver by hydroxylation, and then converted to active 1, 25-dihydroxyvitamin D in the kidney. The level of serum 25-OH-VD can reflect the storage level of vitamin D in the body, and is related to the clinical symptoms of vitamin D deficiency. If the index is too high, it may be vitamin D poisoning, and if the index is too low, it may be vitamin D deficiency.

[0005] 25-hydroxyvitamin D detection is usually determined by serum detection, which can determine whether the vitamin D content in the body is abnormal. If the 25-hydroxyvitamin D content is found to be too low in the examination, it is common in calcium and phosphorus absorption disorders, which may manifest as abnormal development of bones and teeth, osteoporosis, delayed tooth eruption, and other symptoms, and may even cause rickets, chondropathy, and other symptoms, such as osteoporosis, muscle aches, which can increase the risk of leg cramps, falls, and fractures. If the 25-hydroxyvitamin D is too high, it may cause vitamin D poisoning, leading to hypercalcemia, which manifests as loss of appetite, vomiting, diarrhea, and other symptoms.

[0006] The current standard for judging the nutritional status of adult vitamin D is: vitamin D deficiency is serum 25-hydroxyvitamin D level < 50 nmol / L (1 nmol / L = 0.4 ng / ml), insufficient is 50-75 nmol / L, sufficient is > 75 nmol / L. More and more epidemiological and laboratory evidence shows that serum 25-hydroxyvitamin D level is related to the occurrence of various diseases such as childhood rickets, diabetes, chronic kidney disease, hepatitis, immune dysfunction (Sjogren's syndrome, multiple sclerosis, rheumatoid arthritis), asthma, osteoporosis, Parkinson's disease, cardiovascular disease, hypertension, type 2 diabetes, tumors (prostate cancer, colon cancer, breast cancer, etc.). Therefore, the detection of 25-hydroxyvitamin D has very important significance for the diagnosis and prevention of diseases in clinical practice.

[0007] 25-(OH)-VD can be used to determine the concentration of vitamin D, which is an analyte difficult to accurately measure because of its high lipophilicity and high affinity binding to vitamin D binding protein DBP.

[0008] Since 25-hydroxyvitamin D is a small molecule, the current commonly used 25-hydroxyvitamin D detection method is a competition method based on chemiluminescence, enzyme-linked immunoassay, electrochemiluminescence, and immunochromatography. With the development of detection technology, sandwich method can also be used for quantitative detection of 25-hydroxyvitamin D, but it requires 25-hydroxyvitamin D antibody complex antibody raw materials. Therefore, there is a strong demand for 25-hydroxyvitamin D complex antibody with good performance among those skilled in the art. SUMMARY

[0009] The present application provides a 25-OH-VD complex antibody, which provides an important raw material source for the detection of 25-OH-VD, and the antibody has good specificity and activity.

[0010] In order to achieve the above purpose, according to one aspect of the present disclosure, a 25-OH-VD complex antibody is provided, and the aforementioned antibody comprises the following complementarity determining regions:

[0011] HCDR1, comprising or consisting of the amino acid sequence represented by SEQ ID NO: 1 (HYGVG);

[0012] HCDR2, comprising or consisting of the amino acid sequence represented by SEQ ID NO: 2 (GIDNGGDTGTNRALKS);

[0013] HCDR3, comprising or consisting of the amino acid sequence represented by SEQ ID NO: 3 (SNDGNWGTPHDIDY);

[0014] LCDR1 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4 (SGSSSNVGAYGVG);

[0015] LCDR2 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 (STTTRPP);

[0016] LCDR3 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 (AAEDNSGYGV).

[0017] To achieve the above object, according to a second aspect of the present disclosure, a 25-OH-VD complex antibody is provided, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 15; and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 17.

[0018] To achieve the above object, according to a third aspect of the present disclosure, a 25-OH-VD complex antibody is provided, wherein the antibody comprises three heavy chain complementarity determining regions of any of the heavy chain variable regions of the second aspect and three light chain complementarity determining regions of the light chain variable region.

[0019] To achieve the above object, according to a fourth aspect of the present disclosure, a 25-OH-VD complex antibody is provided, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 16; and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 18.

[0020] To achieve the above object, according to a fifth aspect of the present disclosure, an antibody conjugate is provided, wherein the antibody conjugate comprises the antibody as described above.

[0021] To achieve the above object, according to a sixth aspect of the present disclosure, a reagent or kit is provided, wherein the reagent or kit comprises the antibody as described above or the antibody conjugate as described above.

[0022] To achieve the above object, according to a seventh aspect of the present disclosure, a use of the antibody, the antibody conjugate, the reagent or the kit as described above in detecting 25-OH-VD, diagnosing a disease related to vitamin D metabolism, preparing a product for detecting 25-OH-VD or preparing a product for diagnosing a disease related to vitamin D metabolism is provided.

[0023] To achieve the above object, according to an eighth aspect of the present disclosure, a method for diagnosing a disease related to vitamin D metabolism is provided, wherein the method comprises:

[0024] a) allowing a test sample to bind to a 25-OH-VD antibody to form a complex 1 under conditions sufficient for an antibody / antigen binding reaction to occur; and

[0025] b) contacting the antibody of any one of claims 1-6, the antibody conjugate of claim 7, or the reagent or kit of claim 8 with the complex 1 in a) to form a complex 2; and

[0026] c) detecting the presence of the complex 2, the presence of the complex 2 indicating the presence of 25-OH-VD in the test sample.

[0027] To achieve the above object, according to a ninth aspect of the present disclosure, there is provided a method for detecting 25-OH-VD, the method comprising:

[0028] a) allowing a test sample to bind to a 25-OH-VD antibody to form a complex 1 under conditions sufficient for an antibody / antigen binding reaction to occur; and

[0029] b) contacting the antibody of any one of claims 1-6, the antibody conjugate of claim 7, or the reagent or kit of claim 8 with the complex 1 in a) to form a complex 2; and

[0030] c) detecting the presence of the complex 2, the presence of the complex 2 indicating the presence of 25-OH-VD in the test sample.

[0031] To achieve the above object, the present disclosure further provides a nucleic acid molecule, a vector, a cell and a method for preparing the above antibody. BRIEF DESCRIPTION OF DRAWINGS

[0032] To more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings needed in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some embodiments of the present disclosure, and therefore should not be considered as a limitation on the scope, and other related drawings can also be obtained by those skilled in the art without creative labor.

[0033] Figure 1: Standard curve graph of clinical sample correlation. DETAILED DESCRIPTION

[0034] In a first aspect, the embodiments of the present disclosure provide a 25-OH-VD complex antibody, the aforementioned antibody comprising the following complementarity determining regions:

[0035] HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 (HYGVG);

[0036] HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2 (GIDNGGDTGTNRALKS);

[0037] HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3 (SNDGNWGTPHDIDY);

[0038] LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 (SGSSSNVGAYGVG);

[0039] LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5 (STTTRPP);

[0040] LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6 (AAEDNSGYGV).

[0041] In the present disclosure, 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, as long as they exhibit the desired biological activity.

[0042] In the present disclosure, the term "25-OH-VD complex" refers to a complex formed by free 25-OH-VD and free anti-25-OH-VD antibody (can specifically bind 25-OH-VD) binding together, i.e. antigen-antibody complex, for example, the complex formed by 25-OH-VD and anti-25-OH-VD antibody (Feng Biotech, Catalog No: VD-REAB-E1-009) in the embodiments of the present disclosure.

[0043] The term "25-OH-VD complex antibody" refers to an antibody that binds to the aforementioned antigen-antibody complex, which has properties including: (1) does not bind free 25-OH-VD antigen; nor does it bind free anti-25-OH-VD antibody; and / or (2) strongly binds to 25-OH-VD complex, weakly or not at all to free anti-25-OH-VD antibody or free 25-OH-VD antigen; the strength of binding can be 1-fold, 1.5-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 80-fold, or 100-fold difference in binding activity.

[0044] In the present disclosure, the term "antigen-binding fragment" is a substance comprising CDRs of an antibody, which lacks some of the amino acids present in the full-length chain, but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabody, single chain antibody molecules (scFv), scFv dimers (bivalent diabody), and minimal recognition units of an antibody. The above antigen-binding fragments are capable of binding to the same antigen as the parent antibody.

[0045] Antigen-binding fragments generally have the same binding specificity as the antibody from which they are derived. It is readily understood by one skilled in the art in light of the teachings of the present disclosure that the above antigen-binding fragments can be obtained by methods such as enzymatic digestion, including pepsin or papain, and / or by chemical reduction to break disulfide bonds. The above antigen-binding fragments are readily obtained by one skilled in the art based on the structural basis of the intact antibody disclosed in the present disclosure.

[0046] Antigen-binding fragments can also be obtained by recombinant genetic technology, which is also known to one skilled in the art, or by, for example, an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems and the like.

[0047] In the present disclosure, the term "complementarity determining region," "CDR," or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to the regions comprising one or more, or even all, of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the detailed description of the present disclosure, CDRs refer to the highly variable regions of the heavy and light chains of antibodies.

[0048] In the present disclosure, the heavy chain complementarity determining region is denoted as HCDR, which includes HCDR1, HCDR2, and HCDR3; and the light chain complementarity determining region is denoted as LCDR, which includes LCDR1, LCDR2, and LCDR3.

[0049] Methods for defining CDRs are well known in the art and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system set forth in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" is described in Chothia et al., J Mol Biol 196:901-917 (1987). Still other CDR definition methods can not strictly follow one of the above schemes but will still overlap at least a portion of the CDR regions defined by Kabat, although they can be shortened or lengthened according to prediction or experimental results for particular residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, with slight variations in the definitions in different literature. One of skill in the art can routinely determine which residues comprise particular CDRs given the variable region amino acid sequence of a given antibody. It is noted that CDRs defined by other methods not listed in Table 1 are also within the scope of the present disclosure.

[0050] Table 1: CDR Definitions 1 1 The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with amino acid positions on the heavy chain indicated by "H + number" and amino acid positions on the light chain indicated by "L + number". One of ordinary skill in the art can unambiguously correlate this Kabat numbering system to any variable region sequence without depending on any experimental data other than the sequence itself. As used herein, the "Kabat numbering" refers to the numbering system set forth in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). 2 "AbM" as used in Table 1 with a lower case "b" refers to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular. 3 If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B. 4If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34. 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35. According to embodiments of the present disclosure, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 is numbered by the Kabat system.

[0051] According to embodiments of the present disclosure, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 is defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM, or Contact.

[0052] In some alternative embodiments of the present disclosure, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is defined by the Kabat system.

[0053] In some alternative embodiments of the present disclosure, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is defined by the Chothia system.

[0054] In some alternative embodiments of the present disclosure, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is defined by the IMGT system.

[0055] In some alternative embodiments of the present disclosure, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is defined by the AbM system.

[0056] In some alternative embodiments of the present disclosure, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is defined by the Contact system.

[0057] In some alternative embodiments of the present disclosure, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact system.

[0058] Embodiments of the present disclosure provide a 25-OH-VD complex antibody, the aforementioned antibody comprising the following complementarity determining regions:

[0059] HCDR1 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1 (HYGVG);

[0060] HCDR2 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2 (GIDNGGDTGTNRALKS);

[0061] HCDR3 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3 (SNDGNWGTPHDIDY);

[0062] LCDR1 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 4 (SGSSSNVGAYGVG);

[0063] LCDR2 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 5 (STTTRPP);

[0064] LCDR3 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 6 (AAEDNSGYGV).

[0065] According to embodiments of the present disclosure, the above-mentioned HCDRs and LCDRs are defined by the Kabat system.

[0066] In the present disclosure, the “framework region” or “FR” region, which includes heavy chain framework regions and light chain framework regions, refers to the regions of the antibody heavy chain variable region and light chain variable region other than the CDRs; wherein the heavy chain framework regions can be further subdivided into regions adjacent to CDRs separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions can be further subdivided into regions adjacent to CDRs separated by CDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0067] In the present disclosure, 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.

[0068] In an alternative embodiment, the antibody of the first aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0069] In an alternative embodiment, the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 of the antibody described above have the amino acid sequences of HFR1, HFR2, HFR3, HFR4 in the order of SEQ ID NO: 7 to SEQ ID NO: 10, and LFR1, LFR2, LFR3, LFR4 in the order of SEQ ID NO: 11 to SEQ ID NO: 14; or amino acid sequences having at least 80% identity to the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 sequences.

[0070] HFR1, HFR2, HFR3, HFR4 in the order of SEQ ID NO: 7 to SEQ ID NO: 10, and LFR1, LFR2, LFR3, LFR4 in the order of SEQ ID NO: 11 to SEQ ID NO: 14; or amino acid sequences having at least 80% identity to the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 sequences.

[0071] In the present disclosure, the term "identity" percentage refers to the extent to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. The alignment of the percentage of amino acid sequence identity can be performed in various ways in the art, such as the software well known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA, etc.

[0072] In other embodiments, the amino acid sequences of each framework region of the 25-OH-VD complex antibody provided by the present disclosure can 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 to the corresponding framework region described above.

[0073] In a second aspect, the embodiments of the present disclosure provide a 25-OH-VD complex antibody, 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 shown in SEQ ID NO: 15; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17.

[0074] In a third aspect, the embodiments of the present disclosure provide a 25-OH-VD complex antibody, wherein the antibody comprises three heavy chain complementarity determining regions of any of the heavy chain variable regions in the second aspect and three light chain complementarity determining regions of the light chain variable region.

[0075] In an alternative embodiment, the complementarity determining regions described in the third aspect above are defined by any one of Kabat, Chothia, IMGT, AbM or Contact system or a combination of multiple systems.

[0076] In an alternative embodiment, the antibody described in the first aspect, the second aspect or the third aspect further comprises a constant region.

[0077] In an alternative embodiment, the constant region comprises a heavy chain constant region and a light chain constant region.

[0078] In an alternative embodiment, the heavy chain constant region is selected from any one of IgG, IgA, IgM, IgE, IgD heavy chain constant region or a combination of multiple constant region segments.

[0079] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0080] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0081] In an alternative embodiment, the light chain constant region is selected from kappa or lambda light chain constant region.

[0082] In an alternative embodiment, the species origin of the constant region is bovine, equine, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0083] In an alternative embodiment, the species origin of the constant region is ovine.

[0084] In this context, the division of variable and constant region sequences refers to the IMGT division method, see Lefranc, M.-P., Giudicelli, V., Ginovart, C., Jabado- the international ImMunoGeneTics database. Nucl. Acids Res., 29(1): 207-209 (2001). DOI: 10.1093 / nar / 29.1.207. PMID: 11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGH C, IMGT Repertoire. the international ImMunoGenetics 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. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. The variable region divided by different methods and the variable region divided by IMGT will have some differences in the C-terminal or N-terminal of the constant region. Other methods known in the art for dividing the variable region or the constant region are also within the protection scope of the present disclosure.

[0085] In an optional embodiment, the constant region of the above-mentioned antibody is:

[0086] a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 19; and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 20; or an amino acid sequence with at least 80% identity to the heavy chain constant region and the light chain constant region.

[0087] In other embodiments, the above-mentioned constant region sequence can 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 to the constant region (SEQ ID NO: 19, 20).

[0088] In an optional embodiment, the above-mentioned antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv, and scFv.

[0089] In a fourth aspect, the present disclosure provides a 25-OH-VD complex antibody, wherein the amino acid sequence of the heavy chain of the above-mentioned antibody is as shown in SEQ ID NO: 16; and the amino acid sequence of the light chain is as shown in SEQ ID NO: 18.

[0090] In a fifth aspect, the present disclosure provides an antibody conjugate, wherein the above-mentioned antibody conjugate includes the above-mentioned antibody.

[0091] In an optional embodiment, the above-mentioned antibody conjugate includes biotin or a biotin derivative.

[0092] In an optional embodiment, the above-mentioned antibody conjugate further includes a label or a purification tag.

[0093] In optional embodiments, the above-mentioned markers refer to a class of substances having properties such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or probed by instruments, through which qualitative or quantitative detection of the corresponding target objects can be achieved.

[0094] In optional embodiments, the above-mentioned markers include but are not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0095] In actual use, the skilled person in the art can select appropriate markers according to the detection conditions or actual needs, and no matter what kind of marker is used, it falls within the protection scope of the present disclosure.

[0096] In optional embodiments, the above-mentioned fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (such as but not limited to fluorescein isothiocyanate (FITC), hydroxyl fluorescein (FAM), tetra-chloro fluorescein (TET), etc. or their analogues), rhodamine dyes and their derivatives (such as but not limited to red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogues), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogues), Alexa series dyes and their derivatives (such as but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogues), and protein dyes and their derivatives (such as but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0097] In optional embodiments, the above-mentioned enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0098] In optional embodiments, the above-mentioned radioisotopes include but are not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99mTc、 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.

[0099] In optional embodiments, the chemiluminescent reagent described above includes but is not limited to luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bispyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lucigenin and its derivatives, and peroxymonosulfate and its derivatives.

[0100] In optional embodiments, the nanoparticle-based label described above includes but is not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0101] In optional embodiments, the colloids described above include but are not limited to colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0102] In optional embodiments, the colloidal metals described above include but are not limited to colloidal gold, colloidal silver, and colloidal selenium.

[0103] In optional embodiments, the antibody conjugate described above further includes a solid-phase carrier conjugated to the antibody described above.

[0104] In optional embodiments, the solid-phase carrier described above is selected from microspheres, plates, and membranes.

[0105] In optional embodiments, the solid-phase carrier described above includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0106] In a sixth aspect, the embodiments of the present disclosure provide a reagent or kit, wherein the aforementioned reagent or kit includes the aforementioned antibody or the aforementioned antibody conjugate.

[0107] As described above, the antibody in some embodiments of the present disclosure can effectively bind to the complex formed by 25-OH-VD and the anti-25-OH-VD antibody, and the antibody only binds to the complex formed by 25-OH-VD and the anti-25-OH-VD antibody, does not bind to 25-OH-VD and the anti-25-OH-VD antibody, or weakly binds to the anti-25-OH-VD antibody and does not bind to 25-OH-VD. Therefore, the reagent or kit comprising the above-mentioned 25-OH-VD complex antibody can effectively detect 25-OH-VD qualitatively or quantitatively. The reagent or kit provided by the present disclosure can be used in, for example, immunoblotting, immunoprecipitation and other detections involving the use of 25-OH-VD specific binding performance. As described above, the antibody in some embodiments of the present disclosure has higher binding activity to the complex formed by 25-OH-VD and the anti-25-OH-VD antibody, and therefore the reagent or kit comprising the above-mentioned antibody has higher detection sensitivity or specificity.

[0108] In a seventh aspect, the embodiments of the present disclosure provide a method for detecting 25-OH-VD, comprising: a) allowing a sample to be tested to bind to an anti-25-OH-VD antibody to form a complex 1 under conditions sufficient for an antibody / antigen binding reaction to occur; and b) allowing the above-mentioned antibody, antibody conjugate, reagent or kit to contact the complex 1 in a) to form a complex 2; and c) detecting the presence of the complex 2, and the presence of the complex 2 indicates the presence of 25-OH-VD in the test sample.

[0109] In an eighth aspect, the embodiments of the present disclosure provide a method for diagnosing a disease related to vitamin D metabolism, comprising: a) allowing a sample to be tested to bind to an anti-25-OH-VD antibody to form a complex 1 under conditions sufficient for an antibody / antigen binding reaction to occur; and b) allowing the above-mentioned antibody, antibody conjugate, reagent or kit to contact the complex 1 in a) to form a complex 2; and c) detecting the presence of the complex 2, and the presence of the complex 2 indicates the presence of 25-OH-VD in the test sample.

[0110] In optional embodiments, the above-mentioned diseases include, but are not limited to, childhood rickets, diabetes, chronic kidney disease, hepatitis, Sjogren's syndrome, multiple sclerosis, rheumatoid arthritis, asthma, osteoporosis, Parkinson's disease, cardiovascular disease, hypertension, type 2 diabetes, and tumors.

[0111] In a ninth aspect, the embodiments of the present disclosure provide the use of the above-mentioned 25-OH-VD complex antibody, antibody conjugate in detecting 25-OH-VD, diagnosing a disease related to vitamin D metabolism, preparing a product for detecting 25-OH-VD, or preparing a product for diagnosing a disease related to vitamin D metabolism.

[0112] It is to be understood that the products of the present disclosure include, but are not limited to, reagents, kits, test strips, or reagent panels.

[0113] In a tenth aspect, the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody.

[0114] In an eleventh aspect, the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.

[0115] In a twelfth aspect, the present disclosure provides a cell containing the above-mentioned vector.

[0116] In a thirteenth aspect, the present disclosure provides a method of producing a 25-OH-VD complex antibody, comprising: culturing the above-mentioned cell.

[0117] In the present disclosure, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, and includes both RNA and / or DNA that can be single-stranded or double-stranded, or multiple copies. Examples of nucleic acid molecules include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, a DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. When the nucleic acid molecule encodes a protein or polypeptide, the coding is optionally the coding sense or antisense strand. The nucleic acid molecule can be naturally occurring, synthetic, or recombinant, or any combination thereof. "Nucleic acid molecule", "nucleic acid", and "polynucleotide" are used interchangeably.

[0118] In the present disclosure, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and expressed, e.g., to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. The vector can be used to transform, transduce, or transfect a host cell, so that the genetic element carried by the vector is expressed within the host cell. For example, the vector includes: a plasmid, a phagemid, a cosmid, an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC), a bacteriophage such as a lambda phage or a M13 phage, and an animal virus, etc. The vector can contain various elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector can contain a replication initiation site. The vector can also include components that assist its entry into a cell, including but not limited to, a viral particle, a liposome, or a protein coat. The vector can be an expression vector or a cloning vector.

[0119] In the present disclosure, the term "recombinant cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced. The exogenous polynucleotide can or can not be integrated into the genome of the "recombinant cell". When the vector is contained in the recombinant cell, the vector can be introduced into a mammalian cell, a recombinant cell is constructed, and then the recombinant cell is used to express the antibody or antigen binding fragment provided by the present disclosure. The corresponding antibody can be obtained by culturing the recombinant cell. The available mammalian cell can be a CHO cell or the like.

[0120] Based on the amino acid sequences of the 25-OH-VD complex antibodies disclosed in the present disclosure, it is easy for those skilled in the art to conceive that the 25-OH-VD complex antibodies can be prepared by genetic engineering technology or other technologies (chemical synthesis, recombinant expression), for example, the antibodies can be isolated and purified from the culture products of recombinant cells capable of recombinantly expressing the antibodies described in any of the above, which is easy for those skilled in the art to achieve. Therefore, no matter what technology is used to prepare the 25-OH-VD complex antibodies of the present disclosure, it all falls within the protection scope of the present disclosure.

[0121] In order to make the purposes, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. In the embodiments, no specific conditions are specified, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0122] 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. The techniques employed or contemplated herein are standard methodologies unless otherwise indicated. Materials, methods, and examples are illustrative only and not limiting.

[0123] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety.

[0124] The features and advantages of the present disclosure are further described in the following detailed description in connection with the appended drawings.

[0125] Example 1 Antibody discovery of monoclonal antibodies

[0126] 1. Immunizing an animal

[0127] The 25-hydroxy vitamin D and 25-hydroxy vitamin D antibody complex is used as an immunogen, emulsified with incomplete Freund's adjuvant (Sigma), and the immune response of the sheep is obtained by subcutaneous injection. The pre-immune and post-immune sera are collected on days 0, 14, 28, 42 and 69, respectively. The serum titers are detected, and three sheep with titers meeting the requirements are selected. The whole blood of the sheep is collected, and PBMC cells are prepared using a lymphocyte separation kit.

[0128] 2. Construction of phage library

[0129] The RNA in the PBMC of the sheep is extracted and reverse transcribed into cDNA. The specific sheep antibody gene amplification primers are used to amplify the heavy chain variable region and light chain variable region gene fragments from the cDNA template. Then, the heavy chain variable region and light chain variable region gene fragments are sequentially inserted into the phage vector V02 (constructed in the laboratory) by enzyme digestion and ligation. Finally, the ligated phage plasmid is electroporated into TG1 competent cells. The next day, single clone colonies are selected for PCR identification, and the antibody gene sequencing is performed to evaluate the quality of the phage library. After passing the evaluation, the phage library is screened.

[0130] 3. Screening of phage library

[0131] The phage library TG1 bacterial liquid is inoculated into a flask, and when the bacterial liquid concentration reaches an appropriate concentration (OD600 is 0.8-1.0), the helper phage is added for 1 hour of infection. Then, the culture is continued overnight. The next day, the bacterial liquid is centrifuged, and the supernatant is collected. The displayed phage library is purified by salt precipitation.

[0132] The phage library is subjected to 3-4 rounds of magnetic bead panning. Then, the single clone phage infection colonies are selected for antibody supernatant expression. Next, the single clone phage antibody expression supernatant is screened and identified by ELISA screening method. Finally, one single clone phage with the best 25-hydroxy vitamin D and 25-hydroxy vitamin D antibody complex is selected and named phage 6D10.

[0133] 4. Sequencing of phage antibody gene

[0134] The phage 6D10 is subjected to antibody gene sequencing. Through sequence analysis, the unique sheep single clone antibody sequence is obtained by removing duplicate and invalid sequences. The obtained sheep single clone antibody sequence is subjected to eukaryotic recombinant expression verification.

[0135] Example 2 Recombinant cell preparation

[0136] 1. Construction of recombinant antibody expression plasmid

[0137] pcDNATM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed by introducing a polyclonal enzyme cutting site, hereinafter referred to as 3.4A expression vector; the variable region gene obtained according to step 4 in Example 1 is used to design light chain variable region and heavy chain variable region gene specific primers, respectively, with restriction endonuclease enzyme cutting sites and protective bases at both ends, and light chain gene fragments and heavy chain gene fragments are amplified by PCR amplification method.

[0138] The heavy chain and light chain gene fragments are double enzyme cut using restriction endonuclease, the 3.4A vector is double enzyme cut using restriction endonuclease, and the recovered heavy chain gene and light chain gene are connected to the 3.4A expression vector, respectively, to obtain heavy chain and light chain recombinant expression plasmids.

[0139] 2. Recombinant cell preparation

[0140] The HEK293 cells were recovered in advance and subcultured to 200 ml system, so that the cell density reached 3-5 x 10 6 cells / ml cell density reached the selected antibody concentration and cells, cell viability > 95%; centrifugal washing of cells, resuspension with culture medium, and at the same time, the cell density was adjusted to 3,0 x 10 6 cells / ml, and the cells were washed and resuspended with culture medium, and at the same time, as a cell diluent. The plasmid DNA and transfection reagent diluent in step 1 were prepared with culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed well, and placed at room temperature for 15 min; the mixture was slowly added to the cell diluent within 1 min, mixed well, sampled and counted, and the cell viability after transfection was recorded and observed. The antibody expressed in the supernatant of the above recombinant cells was named antibody 6D10Rmb, and the antibody was a 25-OH-VD complex antibody. The above recombinant cells were placed in a constant temperature incubator at 35°C for culture, with a rotation speed of 120 rpm and a CO2 content of 8%.

[0141] The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0142] Table 2: Antibody sequence

[0143] 3. Specific detection of supernatant antibody

[0144] 3.1 Binding experiment of supernatant antibody with 25-OH-VD and anti-25-OH-VD antibody complex (detection mode 1)

[0145] (1) ELISA plate coating

[0146] ELISA plate coated with PK1-SA8 (avidin, concentration: 2.2 mg / ml) 3 ug / mL, 100ul / well, 4℃ overnight; 1x PBST wash 2 times; 1% casein, 120ul / well, 37℃ blocking for 1h, tap dry, ready for use.

[0147] (2) Preparation of 25-OH-VD and Anti 25-OH-VD antibody complex

[0148] Incubate antibody Anti 25-OH-VD-BIO (antibody Anti 25-OH-VD-BIO is antibody Anti 25-OH-VD conjugated with biotin) at a working concentration of 1 ug / mL and 0.1 ug / mL free 25-OH-VD (from Fierbio) in a centrifuge tube at 37℃ shaking incubator for 1.5h, forming 25-OH-VD and Anti 25-OH-VD-BIO complex. Add the incubated 25-OH-VD and Anti 25-OH-VD-BIO complex to the ELISA detection plate in step (1), 100ul / well, 37℃ incubate for 1h; discard the supernatant, do not tap the plate and do not wash, ready for use.

[0149] (3) Preparation of supernatant antibody and control

[0150] Take the supernatant of the recombinant cells of antibody 6D10Rmb (cultured for 12 days) and dilute it with 1% casein at an appropriate dilution.

[0151] Control 1: 1% casein

[0152] Control 2: Antibody A, diluted with 1% casein to 0.1 ug / ml respectively

[0153] Note: Antibody A strongly binds to 25-OH-VD and Anti 25-OH-VD complex, and weakly binds to Anti 25-OH-VD and 25-OH-VD.

[0154] (4) Detection reading

[0155] Add the antibody supernatant and controls prepared in step (3) to the ELISA in step (2), 100ul / well. After gently mixing, incubate at 37℃ for 45min; 1x PBST wash 3 times; add rabbit anti-goat FC-HRP (1% casein diluted 15000 times), 100ul / well, 37℃ incubate for 45min; 1x PBST wash 3 times; add A and B solutions, 50ul / well each, show for 10min, add stop solution, 50ul / well, read. The results are shown in the following table.

[0156] 3.2 Same experimental procedure as detection mode 1, replace the antibody complex in step (2) with 1 ug / mL Anti 25-OH-VD-BIO (same as Anti 25-OH-VD-BIO in step (2) of detection mode 1), and conduct the supernatant antibody binding experiment with Anti 25-OH-VD antibody (detection mode 2). The experimental results are shown in Table 3.

[0157] 3.3 Same experimental procedure as detection mode 1, replace the antibody complex in step (2) with 0.1 ug / mL free 25-OH-VD-BIO (free 25-OH-VD conjugated with biotin in step (2) of detection mode 1), and conduct the supernatant antibody binding experiment with 25-OH-VD (detection mode 3). The experimental results are shown in Table 3.

[0158] Table 3: Supernatant antibody binding experiment data

[0159] The results show that the antibody 6D10Rmb only binds to 25-OH-VD and 25-OH-VD antibody complex, and does not bind to 25-OH-VD and 25-OH-VD antibody, with good specificity.

[0160] Example 3: Production of recombinant antibody

[0161] After 13 days, centrifuge the supernatant of the above recombinant cells to collect the sample, and use affinity chromatography column to purify the antibody.

[0162] Example 4: Performance detection of antibody

[0163] 1. Fluorescence platform performance detection

[0164] 1.1 Antibody labeling

[0165] T-line labeling: Take 100 ul of 1% content of fluorescent microspheres, add 900 ul of activation buffer after mixing, centrifuge to remove the supernatant, add 1 mL of activation buffer and ultrasonic mixing, then add activator, avoid light and shake mixing for 20 min, then centrifuge to remove the supernatant, add the same volume of coupling buffer (MES buffer, pH = 6.0) as the microspheres, ultrasonic mixing, then add 0.1-0.2 mg of 25-OH-VD antibody (from Feifeng Biological, item number: VD-REAB-E1-010), avoid light and shake mixing for 3 h, finally add blocking buffer for blocking, avoid light and shake mixing for 45 min, then terminate the labeling, centrifuge to remove the supernatant, dissolve the microspheres with microsphere preservation solution, ultrasonic mixing, as 25-OH-VD antibody label, store at 4°C for use.

[0166] C marker (goat anti-chicken IgY): Take 100ul 1% solid content fluorescent microspheres, add 900ul activation buffer and mix well, centrifuge to remove supernatant, add 1mL activation buffer and mix well by ultrasonic, then add activator, avoid light, mix well by shaking for 20min, centrifuge to remove supernatant, add the same volume of coupling buffer (MES buffer, pH = 6.5) as the microspheres, mix well by ultrasonic, then add 0.4-0.8mg goat anti-chicken IgY antibody, avoid light, mix well by shaking for 3h, finally add blocking buffer for blocking, avoid light, mix well by shaking for 45min, then terminate the labeling, centrifuge to remove supernatant, reconstitute the microspheres with microsphere preservation solution, mix well by ultrasonic and store at 4℃ for use.

[0167] 1.2 Preparation of microsphere working solution

[0168] Dilute the 25-OH-VD antibody marker to 10-20% and the goat anti-chicken IgY antibody marker to 1-2% with the microsphere dilution solution, then mix them together and use a spray pad instrument to spray the markers on glass fiber.

[0169] 1.3 Preparation of dried microsphere pad

[0170] Place the sprayed fluorescent pad in a 50℃ oven and dry for more than 2h.

[0171] 1.4 Sample pad treatment

[0172] Dilute the sample pad dilution solution to 0.4mg / ml and spread it on glass fiber, then place it in a 50℃ oven and dry overnight.

[0173] 1.5 NC membrane coating

[0174] T line: dilute the antibody 6D10Rmb and the control antibody (from Feifeng Biological, item number: VD-REAB-E1-008) to 1.0mg / ml with the coating dilution solution and coat them;

[0175] C line: dilute the chicken IgY antibody to 1.0mg / ml with the coating dilution solution and coat it;

[0176] After completing the T and C line coating, place them in a 50℃ oven and dry overnight.

[0177] 1.6 Preparation of fluorescent chromatography strip

[0178] Use a strip cutter to cut the fluorescent chromatography strip to the desired width, assemble it, and then add samples for detection.

[0179] 1.7 Detection of different concentrations of 25-OH-VD samples

[0180] The sample containing different concentrations of 25-OH-VD sample was added to the sample diluent, mixed well and then loaded: 75 uL of 3.5 mm wide strip was loaded, the test card was reacted for 15 min, the test card was immediately inserted into the instrument for reading, and the T / C was calculated, and the specific results are shown in Table 4. The results show that the signal-to-noise ratio of the antibody 6D10Rmb is better than that of the control antibody, and the sensitivity is higher.

[0181] Table 4: Fluorescence platform performance data

[0182] 1.8 Clinical sample detection

[0183] The clinical sample was added to the sample diluent, mixed well and then loaded: 75 uL of 3.5 mm wide strip was loaded, the test card was reacted for 15 min, the test card was immediately inserted into the instrument for reading, and the T / C was calculated, and the detection results are shown in Table 5, and the linear correlation standard curve is shown in Figure 1. The results show that the detection reagent composed of the antibody 6D10Rmb has a correlation R 2 >0.92 with the assigned clinical sample, has good clinical correlation, high detection sensitivity, and good linear range.

[0184] Table 5: Clinical sample detection results

[0185] 1.9 25-OH-VD analog cross-reaction detection

[0186] The anhydrous ethanol, calcitriol, vitamin D2 and vitamin D3 (from Feipeng Biology) were added to the sample diluent, mixed well and then loaded: 75 uL of 3.5 mm wide strip was loaded, the test card was reacted for 15 min, the test card was immediately inserted into the instrument for reading, and the T / C was calculated, and the detection results are shown in Table 6. The results show that the detection composed of the antibody 6D10Rmb has no obvious cross-reaction with calcitriol, vitamin D2 and vitamin D3, and has good specificity.

[0187] Table 6: Cross-reaction detection results

[0188] Some of the amino acid sequences involved in the present application are shown in Table 7:

[0189] Table 7: Amino acid sequences

[0190] The above merely describes preferred embodiments of the present disclosure and is not intended to limit the present disclosure. The present disclosure can have various modifications and changes for those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure. Industrial applicability

[0191] The anti-25-OH-VD complex antibody provided by the present disclosure can specifically bind to 25-OH-VD complex, has high detection sensitivity and specificity, and can be used for the detection of 25-OH-VD. Therefore, the anti-25-OH-VD complex antibody provided by the present disclosure has excellent practical performance and broad market application prospect.

Claims

1. A 25-OH-VD complex antibody, characterized in that, The antibody contains the following complementarity-determining regions: HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1 (HYGVG); HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2 (GIDNGGDTGTNRALKS); HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3 (SNDGNWGTPHDIDY); LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4 (SGSSSNVGAYGVG); LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5 (STTTRPP); LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6 (AAEDNSGYGV).

2. The antibody according to claim 1, characterized in that, The antibodies include HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4, and the amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are as follows: The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:7 to SEQ ID NO:10, and LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:11 to SEQ ID NO:14; or amino acid sequences that have at least 80% identity with the sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

3. A 25-OH-VD complex antibody, 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 shown in SEQ ID NO:15; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

17.

4. A 25-OH-VD complex antibody, characterized in that, The antibody comprises the three heavy chain complementarity-determining regions of the heavy chain variable region and the three light chain complementarity-determining regions of the light chain variable region as described in claim 3. Optionally, the complementary determination region of the variable region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

5. The antibody according to any one of claims 1 to 4, characterized in that, The antibody also includes a constant region; Optionally, the constant region includes a heavy chain constant region and a light chain constant region; Optionally, 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; Optionally, 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; Optionally, 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; Optionally, the species source of the constant region is sheep; Optionally, the antibody constant region is: The amino acid sequence of the heavy chain constant region as shown in SEQ ID NO:19; and the amino acid sequence of the light chain constant region as shown in SEQ ID NO:20; or an amino acid sequence having at least 80% identity with the heavy chain constant region and the light chain constant region; Optionally, the antibody comprises any one of F(ab')2, Fab', Fab, Fv, and scFv.

6. A 25-OH-VD complex antibody, comprising a heavy chain and a light chain, characterized in that, The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:16; the light chain amino acid sequence is shown in SEQ ID NO:

18.

7. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody according to any one of claims 1 to 6; Optionally, the antibody conjugate further includes biotin or a biotin derivative conjugated with the antibody; Optionally, the antibody conjugate further includes a marker conjugated to the antibody; Optionally, the marker is selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers; Optionally, the antibody conjugate further includes a solid-phase support conjugated to the antibody; Optionally, the solid support is selected from microspheres, plates, and membranes.

8. A reagent or kit, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 6 or the antibody conjugate as described in claim 7.

9. Use of the antibody according to any one of claims 1-6, the antibody conjugate according to claim 7, or the reagent or kit according to claim 8 in the detection of 25-OH-VD, the diagnosis of diseases related to vitamin D metabolism, the preparation of products for the detection of 25-OH-VD, or the preparation of products for the diagnosis of diseases related to vitamin D metabolism; Optionally, the diseases include rickets in children, diabetes, chronic kidney disease, hepatitis, Sjögren's syndrome, multiple sclerosis, rheumatoid arthritis, asthma, osteoporosis, Parkinson's disease, cardiovascular disease, hypertension, type 2 diabetes, and tumors.

10. A method for diagnosing diseases related to vitamin D metabolism, characterized in that, The method includes: a) Under conditions sufficient to induce antibody / antigen binding, the test sample is bound to the 25-OH-VD antibody to form complex 1; and b) Contacting the antibody of any one of claims 1-6, the antibody conjugate of claim 7, or the reagent or kit of claim 8 with complex 1 in a) to form complex 2; and c) Detect the presence of the complex 2, the presence of the complex 2 indicating the presence of 25-OH-VD in the test sample.

11. A method for detecting 25-OH-VD, characterized in that, The method includes: a) Under conditions sufficient to induce antibody / antigen binding, the test sample is bound to the 25-OH-VD antibody to form complex 1; and b) Contacting the antibody of any one of claims 1-6, the antibody conjugate of claim 7, or the reagent or kit of claim 8 with complex 1 in a) to form complex 2; and c) Detect the presence of the complex 2, the presence of the complex 2 indicating the presence of 25-OH-VD in the test sample.

12. A nucleic acid, characterized in that, The nucleic acid encodes the antibody according to any one of claims 1 to 6.

13. A carrier, characterized in that, The vector comprises the nucleic acid as described in claim 12.

14. A cell characterized in that, The cell comprises the nucleic acid of claim 12 or the vector of claim 13.

15. A method for preparing the antibody according to any one of claims 1 to 6, characterized in that, The method includes culturing the cells of claim 14.

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