Anti-25-hydroxyvitamin D antibody, reagent and kit for detecting 25-hydroxyvitamin D

Abstract

The present invention discloses an antibody against 25-hydroxyvitamin D or a reagent and a kit for detecting 25-hydroxyvitamin D, relating to the field of antibodies. The antibody against 25-hydroxyvitamin D disclosed by the present invention includes a heavy chain complementary determining region and a light chain complementary determining region. This antibody provides an important raw material source for the detection of 25-hydroxyvitamin D and has good affinity and activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular, to an anti-25-hydroxyvitamin D antibody, a reagent and a kit for detecting 25-hydroxyvitamin D. Background Art

[0002] 25-hydroxyvitamin D 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 skin after ultraviolet irradiation, and a small part is ingested from food or supplements. Vitamin D not only affects calcium and phosphorus metabolism, but also has a wide range of physiological functions. It is an essential substance for maintaining human health, cell growth and development, and is closely related to various diseases. Vitamin D is converted into 25-hydroxyvitamin D (25-OH-VD) in the liver through hydroxylation. The level of 25-OH-VD in serum can reflect the storage level of vitamin D in the human 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.

[0003] 25-hydroxyvitamin D can be used to determine the vitamin D concentration. The concentration of 25-hydroxyvitamin D is considered to be the best index of vitamin D, and can judge whether the vitamin D content in the body is abnormal. If it is found that the content of 25-hydroxyvitamin D is too low, it is common in calcium and phosphorus absorption disorders, and may be manifested as abnormal development of bones and teeth, such as osteoporosis and delayed tooth eruption. In severe cases, it may also lead to rickets and osteomalacia, manifested as osteoporosis and muscle soreness, which will increase the risk of leg cramps, falls and fractures. If 25-hydroxyvitamin D is on the high side, it may cause vitamin D poisoning and hypercalcemia, manifested as loss of appetite, vomiting, diarrhea and other symptoms.

[0004] At present, the commonly used detection methods for 25-hydroxyvitamin D include chemiluminescence method, enzyme-linked immunosorbent assay, electrochemiluminescence method, immunochromatography method, etc. All of the above detection methods require relevant antibodies to achieve. Therefore, there is a strong demand in this field for relevant antibodies with good activity and affinity. Summary of the Invention

[0005] The present application provides an antibody or its functional fragment, which provides an important raw material source for the detection of 25-hydroxyvitamin D and has good activity and affinity.

[0006] To achieve the above object, according to one aspect of the present invention, there is provided an antibody or its functional fragment, wherein the antibody or its functional fragment comprises three complementary determining regions of a heavy chain variable region having the amino acid sequence SEQ ID NO: 17 and three complementary determining regions of a light chain variable region having the amino acid sequence SEQ ID NO: 18.

[0007] To achieve the above object, according to the second aspect of the present invention, there is provided an antibody or a functional fragment thereof, the antibody or the functional fragment thereof comprising the following complementarity determining regions:

[0008] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same;

[0009] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same;

[0010] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same;

[0011] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same;

[0012] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same;

[0013] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same.

[0014] To achieve the above object, according to the third aspect of the present invention, there is provided an antibody or a functional fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region being as shown in SEQ ID NO: 18.

[0015] To achieve the above object, according to the fourth aspect of the present invention, there is provided an antibody or a functional fragment thereof, comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as shown in SEQ ID NO: 19; the amino acid sequence of the light chain being as shown in SEQ ID NO: 20.

[0016] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody conjugate, the antibody conjugate comprising the above antibody or a functional fragment thereof.

[0017] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, the reagent or the kit comprising the above antibody or a functional fragment thereof or the above antibody conjugate.

[0018] To achieve the above object, according to the seventh aspect of the present invention, there is provided a method for detecting 25-hydroxyvitamin D, comprising: a) contacting the above-mentioned antibody or its functional fragment, antibody conjugate, or reagent or kit with 25-hydroxyvitamin D in a test sample under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0019] To achieve the above object, according to the eighth aspect of the present invention, there is provided a nucleic acid encoding the above-mentioned antibody or its functional fragment.

[0020] To achieve the above object, according to the ninth aspect of the present invention, there is provided a vector comprising the above-mentioned nucleic acid.

[0021] To achieve the above object, according to the tenth aspect of the present invention, there are provided cells comprising the above-mentioned nucleic acid, vector, or expressing the above-mentioned antibody or its functional fragment.

[0022] To achieve the above object, according to the eleventh aspect of the present invention, there is provided a method for preparing the above-mentioned antibody or its functional fragment, the method comprising culturing the above-mentioned cells.

[0023] To achieve the above object, according to the twelfth aspect of the present invention, there is provided the use of the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit in the detection or preparation of a product for detecting 25-hydroxyvitamin D. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 Results of the reducing SDS-PAGE of Anti-25-OH-VD 9C9 Rmb. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] In a first aspect, an embodiment of the present invention provides an antibody or its functional fragment, the antibody or its functional fragment comprising three complementarity-determining regions of a heavy-chain variable region having the amino acid sequence SEQ ID NO: 17 and three complementarity-determining regions of a light-chain variable region having the amino acid sequence SEQ ID NO: 18.

[0027] It should be noted that under the Kabat definition, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region SEQ ID NO: 17 are shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively. Then, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the antibody or its functional fragment are also shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively.

[0028] Under the Kabat definition, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region SEQ ID NO: 18 are shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively. Then, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the antibody or its functional fragment are also shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively.

[0029] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0030] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0031] In the present invention, the heavy chain complementary determining regions are denoted as HCDR and include HCDR1, HCDR2, and HCDR3; the light chain complementary determining regions are denoted as LCDR and include LCDR1, LCDR2, and LCDR3.

[0032] The methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. 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). There are other CDR definition methods that may not strictly follow one of the above-mentioned schemes, but will still overlap with at least a part of the CDR region defined by Kabat, although they may be shortened or lengthened according to the prediction or experimental results of specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, with slightly different annotations in different literatures. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also fall within the scope of protection of the present disclosure.

[0033] Table 1: CDR Definitions 1

[0034]

[0035]

[0036] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below), and the amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those of ordinary skill in the art can clearly map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein,

[0037] "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

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

[0039] 3 When neither H35A nor H35B is present, CDR-H1 ends at position 35; when only H35A is present, CDR-H1 ends at position 35A; when both H35A and H35B are present, CDR-H1 ends at position 35B.

[0040] 4 When neither H35A nor H35B is present, CDR-H1 ends at position 32; when only H35A is present, CDR-H1 ends at position 33; when both H35A and H35B are present, CDR-H1 ends at position 34.

[0041] 5 When neither H35A nor H35B is present, CDR-H1 ends at position 33; when only H35A is present, CDR-H1 ends at position 34; when both H35A and H35B are present, CDR-H1 ends at position 35.

[0042] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

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

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

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

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

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

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

[0049] According to an embodiment of the present invention, the Kabat, Chothia, AbM, or IMGT system-defined amino acid sequence corresponding Kabat number positions of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 are as follows:

[0050]

[0051]

[0052] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0053] In an alternative embodiment, an embodiment of the present invention provides an antibody or a functional fragment thereof, and the antibody or the functional fragment thereof includes the following complementarity determining regions:

[0054] HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 1, or consists of the same.

[0055] HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 2, or consists of the same.

[0056] HCDR3, which contains the amino acid sequence shown in SEQ ID NO: 3, or consists of the same.

[0057] LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 4, or consists of the same.

[0058] LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 5, or consists of the same.

[0059] LCDR3, which contains the amino acid sequence shown in SEQ ID NO: 6, or consists of the same.

[0060] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the regions other than the CDRs in the variable regions of the heavy chain and the light chain of the antibody; wherein, the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0061] In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: 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 combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0062] In an alternative embodiment, the antibody or its functional fragment further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0063] The HFR1 comprises / is as shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0064] The HFR2 comprises / is as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0065] The HFR3 comprises / is as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0066] The HFR4 comprises / is as shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0067] The LFR1 comprises / is as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0068] The LFR2 comprises / is as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0069] The LFR3 comprises / is as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

[0070] The LFR4 comprises / is as shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto;

[0071] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14).

[0072] In an alternative embodiment, the antibody or its functional fragment binds 25-hydroxyvitamin D with an affinity of KD < 7.42×10 -8 M.

[0073] In an alternative embodiment, the antibody or its functional fragment binds 25-hydroxyvitamin D with an affinity of KD ≤ 10 -8 M, KD ≤ 10 -9 M, KD ≤ 10 -10 M, KD ≤ 10 -11 M or KD ≤ 10 -12 M.

[0074] In an alternative embodiment, the antibody or its functional fragment binds 25-hydroxyvitamin D with an affinity of KD ≤ 2.21×10 -9 M.

[0075] There are many methods for measuring antibody affinity (KD), which can be classified into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods according to the detection principle. Among them, common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biolayer interferometry (BLI); common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA), etc.

[0076] In an alternative embodiment, the KD is measured by a kinetic detection method; optionally, surface plasmon resonance, for example, by using a biosensor system such as the system.

[0077] In a second aspect, an embodiment of the present invention provides an antibody or its functional fragment, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:18.

[0078] In an alternative embodiment, the antibody or its functional fragment further comprises a constant region.

[0079] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0080] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ-type or λ-type light chain constant regions.

[0081] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cow, porcine, ovine, murine, rat, canine, feline, rabbit, equine, deer, mink, chicken, duck, goose, turkey, gamecock or human.

[0082] In an alternative embodiment, the species origin of the constant region is murine.

[0083] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.

[0084] It should be noted that in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above constant region (SEQ ID NO:15 or 16).

[0085] In an alternative embodiment, the functional fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

[0086] The functional fragment of the above antibody generally has the same binding specificity as its source antibody. Those skilled in the art can easily understand from the content described in the present invention that the functional fragment of the above antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the disclosure of the structure of the complete antibody in the present invention, those skilled in the art can easily obtain the above functional fragments.

[0087] The functional fragment of the above antibody can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0088] In a third aspect, the present invention provides an antibody or its functional fragment, including a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:19, and the amino acid sequence of the light chain is as shown in SEQ ID NO:20.

[0089] In a fourth aspect, the present invention provides an antibody conjugate, which includes the above antibody or its functional fragment.

[0090] In an alternative embodiment, the above antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or its functional fragment.

[0091] In an alternative embodiment, the antibody conjugate further comprises a label conjugated to the antibody or a functional fragment thereof.

[0092] In an alternative embodiment, the label refers 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 an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through these properties.

[0093] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0094] In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs. Whatever label is used, it falls within the protection scope of the present invention.

[0095] In an alternative embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (such as, for example, but not limited to fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, for example, but not limited to red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, for example, but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, for example, but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, for example, but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0096] In an alternative embodiment, the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0097] In alternative embodiments, the radioisotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0098] In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rhodamine and its derivatives, and peroxyoxalate and its derivatives.

[0099] In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0100] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0101] In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0102] In alternative embodiments, the colloidal metal is colloidal gold.

[0103] In alternative embodiments, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its functional fragment.

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

[0105] In alternative embodiments, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0106] In a fifth aspect, the present invention provides a reagent or a kit, which includes the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.

[0107] As described above, the antibodies or functional fragments thereof in some embodiments or examples of the present invention can effectively bind to 25-hydroxyvitamin D. Therefore, reagents or kits containing the 25-hydroxyvitamin D antibodies or functional fragments thereof can effectively qualitatively or quantitatively detect 25-hydroxyvitamin D. Using the reagents or kits provided by the present invention, for example, they can be used in immunoblotting, immunoprecipitation and other detections involving the specific binding performance of 25-hydroxyvitamin D and its antibodies. As described above, the antibodies or functional fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity for 25-hydroxyvitamin D. Therefore, the reagents or kits containing the antibodies or functional fragments thereof have higher detection sensitivity or specificity.

[0108] In a sixth aspect, the present invention provides a method for detecting 25-hydroxyvitamin D, comprising: a) contacting the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit with 25-hydroxyvitamin D in a test sample to form an immune complex under conditions sufficient for an antibody / antigen binding reaction; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample;

[0109] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or its functional fragment.

[0110] In an alternative embodiment, the immune complex further comprises a second antibody that binds to 25-hydroxyvitamin D.

[0111] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its functional fragment.

[0112] In an eighth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0113] In a ninth aspect, the present invention provides a cell containing the above-mentioned vector.

[0114] In a tenth aspect, the present invention provides a method for preparing an antibody or its functional fragment, which comprises: culturing the cells as described above.

[0115] In an eleventh aspect, the present invention provides the use of the above-mentioned antibody or its functional fragment, antibody conjugate or the above-mentioned reagent or kit in detecting 25-hydroxyvitamin D or preparing a product for detecting 25-hydroxyvitamin D.

[0116] Based on the disclosure of the amino acid sequence of the antibody or its functional fragment in the present invention, those skilled in the art can easily conceive of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment. For example, the antibody or its functional fragment can be isolated and purified from the culture product of a recombinant cell capable of recombinantly expressing the antibody or its functional fragment described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the antibody or its functional fragment of the present invention, it falls within the protection scope of the present invention.

[0117] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0118] 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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0119] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); and "Current Protocols in Immunology" (ed. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0120] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0121] Example 1 Preparation of Anti-25-OH-VD 9C9 Monoclonal Antibody

[0122] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting Anti-25-OH-VD 9C9 monoclonal antibody was an existing hybridoma cell line, which was revived for standby.

[0123] (1) Preparation of antibody gene

[0124] mRNA was extracted from the hybridoma cell line secreting Anti-25-OH-VD 9C9 monoclonal antibody. DNA products were obtained by RT-PCR method. After the A-tailing reaction of the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew out, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones of each were sent to the gene sequencing company for sequencing.

[0125] (2) Sequence analysis of the variable region genes of Anti-25-OH-VD 9C9 antibody

[0126] The gene sequences obtained by the above sequencing were analyzed in the kabat antibody database, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 336bp, and there was a 57bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 348bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0127] (3) Construction of recombinant antibody expression plasmid

[0128] pcDNA TM 3.4 The pcDNA3.4 vector was the constructed recombinant antibody eukaryotic expression vector. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI had been introduced into this expression vector, and it was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protection bases at both ends, and 0.74kb Light Chain gene fragments and 1.40kb Heavy Chain gene fragments were amplified by PCR amplification method.

[0129] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0130] 2. Recombinant antibody production

[0131] Resuscitate HEK293 cells in advance, passage and culture them to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. Select antibodies with the appropriate concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the medium, and at the same time, use it as the cell diluent. Prepare plasmid DNA and transfection reagent diluents with the medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, centrifuge to collect the samples. Affinity purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. Two bands are shown after reducing SDS-PAGE, one with an Mr of 50 KD (heavy chain) and the other with an Mr of 28 KD (light chain).

[0132] The obtained antibody was named Anti-25-OH-VD 9C9Rmb1, and the Anti-25-OH-VD 9C9Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0133] Table 2 Antibody sequences

[0134] Antibody Name Heavy Chain Light Chain Anti-25-OH-VD 9C9Rmb1 SEQ ID NO:19 SEQ ID NO:20

[0135] Example 2 Performance detection of the antibody

[0136] 1. Affinity analysis

[0137] Dilute the purified antibody in advance and perform gradient dilution on the 25-OH-VD antigen at the same time; use the CM5 chip pre-coupled with goat anti-mouse IgG to test the binding and dissociation curves of the antigen and antibody on the Biacore 8K+ device, and the instrument automatically fits to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0138] Table 3 Affinity data

[0139] Sample Name KD ka kd Control 7.42E-08 5.76E+03 4.27E-04 Anti-25-OH-VD 9C9Rmb 2.21E-09 2.82E+04 6.22E-05

[0140] 2. Activity identification

[0141] Dilute the 25-OH-VD antigen with the coating solution (main component NaHCO3) to 3 μg / ml, 100 μL per well, and incubate overnight at 4°C; the next day, wash 2 times with the washing solution (main components Na2HPO4 + NaCl), and pat dry; add the blocking solution (20% BSA + 80% PBS), 120 μL per well, at 37°C for 1 h, and pat dry; add the diluted purified antibody and control antibody, 100 μL / well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), for 10 min; add the stop solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0142] Note: Solution A (main components citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components citric acid

[0143] + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0144] Table 4 Activity data

[0145] Concentration (ng / ml) 2.441 1.221 0.610 0.305 0.153 0.000 Control 1.103 0.765 0.341 0.164 0.018 0.011 Anti-25-OH-VD 9C9Rmb 1.687 1.072 0.624 0.336 0.185 0.023

[0146] 3. Stability assessment

[0147] Place the above antibody at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Take samples at 7 days, 14 days, and 21 days for status observation, and perform activity detection on the 21-day samples. The results show that there is no obvious change in the protein state after the antibody is placed for 21 days under the three assessment conditions, and the activity does not show a downward trend with the increase of the assessment temperature, indicating that the above antibody is stable. Table 5 below shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-25-OH-VD 9C9Rmb after 21 days of assessment.

[0148] Table 5 Stability Data

[0149] Sample Concentration (ng / ml) 1.221 0.610 0.000 Sample at 4°C for 21 days 1.074 0.637 0.042 Sample at -80°C for 21 days 1.068 0.644 0.031 Sample at 37°C for 21 days 1.084 0.651 0.041

[0150] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0151] Some amino acid sequences involved in this application are shown as follows:

[0152]

[0153]

Claims

1. An anti-25-hydroxyvitamin D antibody or a functional fragment thereof, wherein the antibody or the functional fragment has three complementarity-determining regions of the heavy chain variable region with the amino acid sequence of SEQ ID NO: 17 and three complementarity-determining regions of the light chain variable region with the amino acid sequence of SEQ ID NO: 18; The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of the variable region is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

2. An anti-25-hydroxyvitamin D antibody or a functional fragment thereof, characterized in that, The antibody or the functional fragment thereof comprises the following complementarity-determining regions: The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3; The amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4; The amino acid sequence of LCDR2 is as shown in SEQ ID NO: 5; The amino acid sequence of LCDR3 is as shown in SEQ ID NO:

6.

3. The antibody or its functional fragment according to claim 1 or 2, characterized in that, The antibody or the functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

4. The antibody or the functional fragment thereof according to claim 3, wherein The HFR1 comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; The LFR4 comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

5. The antibody or its functional fragment according to claim 1 or 2, characterized in that, The antibody or its functional fragment binds 25-hydroxyvitamin D with an affinity of KD < 7.42×10 -8 M.

6. An anti-25-hydroxyvitamin D antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

18.

7. The antibody or its functional fragment according to any one of claims 1 to 2, 4, and 6, characterized in that, The antibody or the functional fragment thereof further comprises a constant region.

8. The antibody or functional fragment thereof according to claim 7, wherein The constant region comprises a heavy chain constant region and a light chain constant region.

9. The antibody or functional fragment thereof according to claim 8, wherein, The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant regions.

10. The antibody or functional fragment thereof according to claim 7, wherein The species source of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose or human.

11. The antibody or functional fragment thereof according to claim 7, wherein The species source of the constant region is mouse.

12. The antibody or functional fragment thereof according to claim 8, wherein The heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity therewith, and the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity therewith.

13. The antibody or its functional fragment according to any one of claims 1 to 2, 4 to 6, characterized in that, The functional fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

14. An anti-25-hydroxyvitamin D antibody or a functional fragment thereof, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 19; the amino acid sequence of the light chain is as shown in SEQ ID NO:

20.

15. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody or its functional fragment according to any one of claims 1 to 13, or the antibody according to claim 14, and biotin or a biotin derivative or a label conjugated to the antibody or its functional fragment.

16. The conjugate according to claim 15, characterized in that, The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

17. An antibody conjugate, characterized in that, The antibody conjugate further comprises the antibody or its functional fragment according to any one of claims 1 to 13, or the antibody according to claim 14, and a solid-phase carrier conjugated to the antibody or its functional fragment.

18. The conjugate according to claim 17, wherein, The solid-phase carrier is selected from microspheres, plates and membranes.

19. A reagent or kit, characterized in that, The reagent or kit comprises the antibody or its functional fragment according to any one of claims 1 to 13, or the antibody according to claim 14, or the antibody conjugate according to any one of claims 15 to 18. Use of the antibody or its functional fragment according to any one of claims 1 to 13, the antibody according to claim 14, the antibody conjugate according to any one of claims 15 to 18, or the reagent or kit according to claim 19 for preparing a product for detecting 25-hydroxyvitamin D, characterized in that, Comprising: a) contacting the antibody or its functional fragment according to any one of claims 1 to 13, the antibody according to claim 14, the antibody conjugate according to any one of claims 15 to 18, or the reagent or kit according to claim 19 with 25-hydroxyvitamin D in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

21. The use according to claim 20, wherein The immune complex further comprises a second antibody that binds to the antibody or its functional fragment.

22. The use according to claim 20, wherein, The immune complex further comprises a second antibody that binds to 25-hydroxyvitamin D.

23. A nucleic acid, characterized in that, It encodes the antibody or its functional fragment according to any one of claims 1 to 13, or the antibody according to claim 14.

24. A carrier, characterized in that, It contains the nucleic acid according to claim 23.

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

26. A method for preparing the antibody or its functional fragment according to any one of claims 1 to 13 or the antibody according to claim 14, characterized in that, It includes: culturing the cell according to claim 25.

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