Antibodies against 25-OH-VD and antibody complexes thereof, reagents and kits for detecting 25-OH-VD

Abstract

The application discloses an antibody against 25-OH-VD and an antibody complex thereof or a reagent and a kit for detecting 25-OH-VD, and relates to the field of antibodies. The antibody against 25-OH-VD and the antibody complex thereof disclosed by the application comprise 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 activity.

Description

[0001] Cross-reference to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202310759993.X, filed on June 25, 2023, entitled "Antibody against 25-OH-VD and its antibody complex, reagent and kit for detecting 25-OH-VD", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically, to an antibody against 25-OH-VD and its antibody complex, as well as reagents and kits for detecting 25-OH-VD. Background Technology

[0004] 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 body through skin irradiation by ultraviolet radiation, with a small portion ingested from food or supplements. Vitamin D not only affects calcium and phosphorus metabolism but also has a wide range of physiological functions. It is an essential substance for maintaining human health, cell growth and development, and is closely related to many diseases. In the liver, vitamin D is converted into 25-hydroxyvitamin D (25-OH-VD) through hydroxylation. The level of serum 25-OH-VD reflects the body's vitamin D storage level and is related to the clinical symptoms of vitamin D deficiency. Excessively high levels may indicate vitamin D poisoning, while excessively low levels may indicate vitamin D deficiency.

[0005] 25-hydroxyvitamin D testing is usually done via serum analysis to determine if the body's vitamin D levels are abnormal. Low 25-hydroxyvitamin D levels are commonly seen in cases of calcium and phosphorus malabsorption, which may manifest as abnormal bone and teeth development, leading to osteoporosis, delayed teething, and in severe cases, rickets and osteomalacia, causing symptoms such as osteoporosis, muscle aches, and increased risk of leg cramps, falls, and fractures. High 25-hydroxyvitamin D levels can cause vitamin D poisoning, leading to hypercalcemia, with symptoms such as loss of appetite, vomiting, and diarrhea.

[0006] Because 25-(OH)-VD is a small molecule, and most small molecules have only one antigenic determinant, currently commonly used methods for detecting 25-hydroxyvitamin D are all competitive methods based on chemiluminescence, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence, and immunochromatography. Due to inherent methodological limitations of competitive methods, such as insufficient accuracy, their clinical application faces considerable limitations and controversy. With the development of detection technology, sandwich assays can better achieve the quantitative detection of 25-hydroxyvitamin D. The small-molecule sandwich assay is a non-competitive immunoassay method based on anti-immune complex antibodies, but it requires antibody raw materials targeting 25-OH-VD and its antibody complexes. This invention addresses this need by developing an antibody raw material targeting 25-OH-VD and its antibody complexes. Summary of the Invention

[0007] This application provides an antibody or its functional fragment thereof, which provides an important source of raw materials for the detection of 25-OH-VD and has good activity.

[0008] To achieve the above objectives, according to one aspect of the present invention, an antibody or functional fragment thereof is provided, the antibody or functional fragment thereof comprising three complementary determining regions having a heavy chain variable region having any one of the amino acid sequences SEQ ID NO:22, 23, 24, 25 and three complementary determining regions having a light chain variable region having any one of the amino acid sequences SEQ ID NO:30, 31, 32.

[0009] To achieve the above objectives, according to a second aspect of the present invention, an antibody or a functional fragment thereof is provided, said antibody or functional fragment thereof comprising the following complementarity-determining region:

[0010] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1;

[0011] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2;

[0012] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3 or 17;

[0013] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4 or 18;

[0014] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5;

[0015] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

[0016] To achieve the above objectives, according to a third aspect of the present invention, an antibody or a functional fragment thereof is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID NO: 22, 23, 24, 25; and the amino acid sequence of the light chain variable region is shown as any one of SEQ ID NO: 30, 31, 32.

[0017] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody or a functional fragment thereof is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 26, 27, 28, 29; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 33, 34, 35.

[0018] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibody described above or a functional fragment thereof.

[0019] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the antibody described above or a functional fragment thereof or the antibody conjugate described above.

[0020] To achieve the above objectives, according to a seventh aspect of the present invention, a method for detecting 25-OH-VD is provided, comprising: a) binding a test sample to a 25-OH-VD antibody to form a complex 1 under conditions sufficient to allow an antibody / antigen binding reaction to occur; and b) contacting the antibody or its functional fragment, antibody-conjugate, reagent, or kit with the complex 1 in a) to form a complex 2; and 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.

[0021] To achieve the above objectives, according to an eighth aspect of the present invention, a nucleic acid is provided that encodes the aforementioned antibody or a functional fragment thereof.

[0022] To achieve the above objectives, according to a ninth aspect of the present invention, a vector is provided, the vector comprising the above-described nucleic acid.

[0023] To achieve the above objectives, according to a tenth aspect of the present invention, a cell is provided, said cell comprising the above-described nucleic acid, vector, or expressing the above-described antibody or a functional fragment thereof.

[0024] To achieve the above objectives, according to an eleventh aspect of the present invention, a method for preparing the above-described antibody or a functional fragment thereof is provided, the method comprising culturing the above-described cells.

[0025] To achieve the above objectives, according to the twelfth aspect of the present invention, there is provided the use of the above-described antibody or its functional fragment, antibody conjugate, reagent or kit in the detection or preparation of a product for detecting 25-OH-VD. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The results are from the reducing SDS-PAGE of antibodies 15H7 Rmb1 to 15H7 Rmb6. Detailed Implementation

[0028] In a first aspect, embodiments of the present invention provide an antibody or a functional fragment thereof, the antibody or the functional fragment thereof comprising three complementary determining regions having a heavy chain variable region having any one of the amino acid sequences SEQ ID NO:22, 23, 24, 25 and three complementary determining regions having a light chain variable region having any one of the amino acid sequences SEQ ID NO:30, 31, 32.

[0029] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to those of HCDR1, HCDR2, and HCDR3 in the same heavy chain variable region defined in the antibody or its functional fragment described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to those of LCDR1, LCDR2, and LCDR3 in the same light chain variable region defined in the antibody or its functional fragment described in the first aspect.

[0030] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:22; and LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:30.

[0031] In this 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.

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

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

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

[0035] Table 1: CDR Definition 1

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0051] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0052] Secondly, embodiments of the present invention provide an antibody or a functional fragment thereof, the antibody or the functional fragment thereof comprising the following complementarity-determining regions:

[0053] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1.

[0054] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2.

[0055] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3 or 17.

[0056] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4 or 18.

[0057] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5.

[0058] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

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

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

[0061] In an optional embodiment, the antibody or its functional fragment further comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

[0062] The HFR1 includes / is such as SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it;

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

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

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

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

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

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

[0069] The LFR4 includes / is, for example, SEQ ID NO:14 or an amino acid sequence having at least 80% identity with it;

[0070] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the antibody 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 frame regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) mentioned above.

[0071] In an optional embodiment, the HFR1 includes / such as the amino acid sequence shown in SEQ ID NO:7, 19 or 20.

[0072] In an optional embodiment, the LFR1 includes / such as the amino acid sequence shown in SEQ ID NO:11 or 21.

[0073] Thirdly, embodiments of the present invention provide an antibody or a functional fragment thereof, 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 any one of SEQ ID NO: 22, 23, 24, 25, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 30, 31, 32.

[0074] In an optional implementation, the heavy chain variable region and the light chain variable region described in the first and third aspects above are selected from any combination of the following:

[0075] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:22 SEQ ID NO:30 2 SEQ ID NO:24 SEQ ID NO:30 3 SEQ ID NO:25 SEQ ID NO:30 4 SEQ ID NO:23 SEQ ID NO:30 5 SEQ ID NO:22 SEQ ID NO:32 6 SEQ ID NO:22 SEQ ID NO:31 .

[0076] In an optional implementation, the antibody or its functional fragment may further include a constant region.

[0077] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

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

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

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

[0081] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0082] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0083] In an optional implementation, the species source of the constant region is human.

[0084] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region sequence (CL) is as shown in SEQ ID NO:16.

[0085] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region (SEQ ID NO: 15 or 16).

[0086] In an optional implementation, the functional fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

[0087] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned functional fragments.

[0088] The functional fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by, for example, automated peptide synthesizers sold by Applied BioSystems.

[0089] Fourthly, the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:26, 27, 28, 29, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:33, 34, 35.

[0090] In optional embodiments, the antibodies described in the first, second, third, and fourth aspects above include any combination of the heavy and light chains:

[0091] combination Heavy chain Light chain 1 SEQ ID NO:26 SEQ ID NO:33 2 SEQ ID NO:28 SEQ ID NO:33 3 SEQ ID NO:29 SEQ ID NO:33 4 SEQ ID NO:27 SEQ ID NO:33 5 SEQ ID NO:26 SEQ ID NO:35 6 SEQ ID NO:26 SEQ ID NO:34 .

[0092] Fifthly, the present invention provides an antibody conjugate comprising the antibody or a functional fragment thereof described above.

[0093] In an optional embodiment, the antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or a functional fragment thereof.

[0094] In an optional embodiment, the antibody conjugate further includes a marker conjugated to the antibody or a functional fragment thereof.

[0095] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0096] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0097] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0098] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0099] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0100] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0101] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0102] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0103] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0104] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0105] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody or a functional fragment thereof.

[0106] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0107] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0108] In a sixth aspect, the present invention provides a reagent or kit comprising the antibody or its functional fragment or the antibody conjugate described above.

[0109] As previously stated, the antibodies or functional fragments thereof in some embodiments or examples of the present invention can effectively bind to 25-OH-VD and its antibody complex, and the antibody or functional fragment binds only to 25-OH-VD and its antibody complex, without binding to 25-OH-VD or the 25-OH-VD antibody. Therefore, reagents or kits containing the antibody or functional fragment of the 25-OH-VD and its antibody complex can effectively perform qualitative or quantitative detection of 25-OH-VD. The reagents or kits provided by the present invention can be used, for example, for detections involving the specific binding properties of 25-OH-VD, such as immunoblotting and immunoprecipitation. As previously stated, the antibodies or functional fragments thereof in some embodiments or examples of the present invention have higher binding activity to 25-OH-VD; therefore, reagents or kits containing the antibody or functional fragment have higher detection sensitivity or specificity.

[0110] In a seventh aspect, the present invention provides a method for detecting 25-OH-VD, comprising: a) under conditions sufficient to induce an antibody / antigen binding reaction, first binding a test sample to a 25-OH-VD antibody to form a complex 1; and b) contacting the antibody or its functional fragment, antibody conjugate, reagent, or kit with the complex 1 in a) to form a complex 2; and 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.

[0111] On the other hand, the present invention provides an antibody pair, comprising antibody 1 and antibody 2, wherein antibody 1 is any of the antibodies described above or a functional fragment thereof, and the heavy chain complementarity-determining region sequence of antibody 2 is shown in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and the light chain complementarity-determining region sequence is shown in SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43.

[0112] In an optional embodiment, the heavy chain sequence of antibody 2 is shown in SEQ ID NO:36, and the light chain sequence is shown in SEQ ID NO:37.

[0113] Eighthly, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.

[0114] In a ninth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0115] In a tenth aspect, the present invention provides cells containing the above-described carrier.

[0116] In one aspect, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing cells as described above.

[0117] In a twelfth aspect, the present invention provides the use of the above-described antibody or its functional fragment, antibody conjugate, or the above-described reagent or kit, and antibody pair in the detection of 25-OH-VD or in the preparation of products for the detection of 25-OH-VD.

[0118] Based on the amino acid sequence of the antibody or its functional fragment disclosed in this invention, those skilled in the art will readily conceive of preparing the antibody or its functional fragment using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the antibody or its functional fragment can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this invention, it falls within the protection scope of this invention.

[0119] 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. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without a specified manufacturer are all commercially available conventional products. The heavy and light chain sequences of the 25-OH-VD antibody described in the following embodiments are exemplarily shown in SEQ ID NO:36 and SEQ ID NO:37, respectively.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0121] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0122] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0123] Example 1: Antibody discovery of anti-25-hydroxyvitamin D and anti-25-hydroxyvitamin D antibody complex.

[0124] (1) Mice's immunity

[0125] An emulsion of 25-hydroxyvitamin D and 25-hydroxyvitamin D antibody complex prepared with incomplete Freund's adjuvant was subcutaneously injected into B / C mice, and pre- and post-immunization serum was collected on days 0, 14, 28, 42, and 69, respectively; the spleen of the mice was then surgically removed to prepare spleen cell suspensions.

[0126] (2) Cell fusion

[0127] Spleen cell suspension and mouse myeloma cells were mixed and fused by electrofusion to prepare mouse hybridoma cells. The cells were then added to HAT hybridoma selection medium and cultured in an incubator.

[0128] (3) Hybridoma screening

[0129] Seven days after hybridoma cell culture, the hybridoma cell supernatant was identified by ELISA to screen hybridoma cell lines that could specifically recognize and bind to the complex of 25-hydroxyvitamin D and 25-hydroxyvitamin D antibody.

[0130] Hybridoma cell lines containing anti-25-hydroxyvitamin D and anti-25-hydroxyvitamin D antibody complexes were subcloned using a limiting dilution method. Seven days later, the supernatant of monoclonal hybridoma cells was screened and identified using ELISA to obtain positive monoclonal hybridoma cell lines against anti-25-hydroxyvitamin D and anti-25-hydroxyvitamin D antibody complexes. The antibodies secreted by these cell lines only bind to 25-OH-VD and the complex formed by its antibody (the light and heavy chain sequences of the 25-hydroxyvitamin D antibody are not limited to SEQ ID NO:36 and SEQ ID NO:37), and do not bind to 25-OH-VD or 25-OH-VD antibodies.

[0131] (4) Hybridoma antibody gene sequencing

[0132] RNA was extracted from monoclonal hybridoma cell lines containing anti-25-hydroxyvitamin D and anti-25-hydroxyvitamin D antibody complexes and reverse transcribed into cDNA. The antibody gene fragment was then amplified by PCR. Subsequently, the antibody gene fragment was ligated and inserted into a sequencing T vector (purchased from Takara). Finally, the antibody gene was sequenced to obtain the gene sequence of the antibody variable region. The obtained mouse antibody sequence was then humanized using humanization technology.

[0133] Example 2: Preparation of recombinant anti-25-hydroxyvitamin D and anti-25-hydroxyvitamin D antibody complex antibody

[0134] (1) Construction of recombinant antibody expression plasmid

[0135] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The 0.73kb Light Chain gene fragment and the 1.47kb Heavy Chain gene fragment were amplified by PCR.

[0136] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0137] (2) Production of recombinant antibodies

[0138] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE, as shown in the figure. The reducing SDS-PAGE showed two bands: one with a Mr of 50 kDa (heavy chain) and the other with a Mr of 28 kDa (light chain).

[0139] The resulting antibody was named complex antibody 15H7 Rmb1. Mutation of complex antibody 15H7 Rmb1 yielded a mutant antibody. This mutant antibody binds only to the complex formed by 25-OH-VD and its antibody, and does not bind to 25-OH-VD or its antibody. The heavy chain (H) and light chain (L) sequences of the above antibody are shown in the table below:

[0140] Table 2 Antibody Sequences

[0141] Antibody name Heavy chain Light chain Antibody 15H7 Rmb1 SEQ ID NO:26 SEQ ID NO:33 Antibody 15H7 Rmb2 SEQ ID NO:28 SEQ ID NO:33 Antibody 15H7 Rmb3 SEQ ID NO:29 SEQ ID NO:33 Antibody 15H7 Rmb4 SEQ ID NO:27 SEQ ID NO:33 Antibody 15H7 Rmb5 SEQ ID NO:26 SEQ ID NO:35 Antibody 15H7 Rmb6 SEQ ID NO:26 SEQ ID NO:34

[0142] Example 3: Antibody Performance Detection

[0143] 1. Performance Evaluation

[0144] The antibodies 15H7 Rmb1 to 15H7 Rmb6 and the control antibody were used as labeled antibodies to label acridinium ester; another antibody (from Phytobio) was used as the coating antibody to label magnetic beads. The performance of the antibodies was tested using a double-antibody sandwich method on a chemiluminescence platform (Shine I2910).

[0145] Reaction pattern: 1) 50 μl of 25-hydroxyvitamin D antigen quality control + 50 μl of magnetic bead-labeled antibody (concentration 0.05%), reacted at 37℃ for 10 min, washed 3 times with PBST; 2) 50 μl of acridine ester-labeled antibody was added, reacted at 37℃ for 10 min, washed 3 times with PBST; 3) 200 μl of acridine ester substrate was added, and the instrument reading was recorded. Specific performance evaluation results are as follows:

[0146] Note: C0~C7: represent standards of different concentrations; RLU: represents the intensity of the luminescence signal.

[0147] Table 3 Performance Evaluation Data

[0148]

[0149] 2. Stability assessment

[0150] The above-mentioned antibody was placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7 days, 14 days, and 21 days for observation of its state. The activity of the 21-day sample was detected (luminescent platform). The results showed that no significant changes in the protein state of the complex antibody 15H7 Rmb4 were observed after 21 days under the three testing conditions, and the activity did not decrease with increasing testing temperature, indicating that the expressed antibody was stable. Table 4 below shows the luminescent activity detection results after 21 days of testing.

[0151] Table 4 Stability Data

[0152] Sample concentration (ng / ml) 320 20 0 4℃, 21-day sample 2119000 378900 1698 -80℃, 21-day sample 2059000 397500 1723 37℃, 21-day sample 2150000 356300 1698

[0153] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0154] The partial amino acid sequences involved in this application are shown in Table 5:

[0155]

[0156]

[0157]

Claims

1. An antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex, characterized in that, The antibody against 25-OH-VD and its antibody complex, or its antigen-binding fragment, has three complementary determinant regions of the heavy chain variable region as shown in any of SEQ ID NO: 22, 23, 24, 25, and three complementary determinant regions of the light chain variable region as shown in any of SEQ ID NO: 30, 32; or The antibody against 25-OH-VD and its antibody complex or its antigen-binding fragment has three complementary determinant regions of the heavy chain variable region as shown in any of SEQ ID NO: 22, 24, 25 and three complementary determinant regions of the light chain variable region as shown in SEQ ID NO:

31. The complementary determination region of the variable region is defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex, characterized in that, The antibody or its antigen-binding fragment against 25-OH-VD and its antibody complex includes the following complementarity-determining regions: HCDR1, its amino acid sequence is shown in SEQ ID NO:

1. HCDR2, its amino acid sequence is shown in SEQ ID NO:

2. HCDR3, its amino acid sequence is shown in SEQ ID NO:

3. LCDR1, whose amino acid sequence is shown in SEQ ID NO:4 or 18. LCDR2, whose amino acid sequence is shown in SEQ ID NO:5, and LCDR3, whose amino acid sequence is shown in SEQ ID NO:6; or HCDR1, its amino acid sequence is shown in SEQ ID NO:

1. HCDR2, its amino acid sequence is shown in SEQ ID NO:

2. HCDR3, its amino acid sequence is shown in SEQ ID NO:

17. LCDR1, whose amino acid sequence is shown in SEQ ID NO:

4. LCDR2, whose amino acid sequence is shown in SEQ ID NO:5, and LCDR3, whose amino acid sequence is shown in SEQ ID NO:

6.

3. The antibody against 25-OH-VD and its antibody complex according to claim 1 or 2, or its antigen-binding fragment, is characterized in that... The antibody against 25-OH-VD and its antibody complex, or its antigen-binding fragment, also has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

4. The antibody against 25-OH-VD and its antibody complex according to claim 3, characterized in that, The HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it; The HFR2 comprises SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it; The HFR4 comprises SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it; The LFR1 includes SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it; The LFR2 comprises SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it; The LFR3 includes SEQ ID NO:13 or an amino acid sequence having at least 80% identity with it; The LFR4 includes SEQ ID NO:14 or an amino acid sequence that is at least 80% identical to it.

5. An antibody against 25-OH-VD and its antibody complex, or an antigen-binding 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 shown in SEQ ID NO:22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:32; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

31.

6. The antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex according to any one of claims 1, 2, 4, and 5, characterized in that, The antibody against 25-OH-VD and its antibody complex, or its antigen-binding fragment, further includes a constant region.

7. The antibody against 25-OH-VD and its antibody complex according to claim 6, characterized in that, The constant region includes the heavy chain constant region and / or the light chain constant region.

8. The antibody against 25-OH-VD and its antibody complex according to claim 7, characterized in that, The heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

9. The antibody against 25-OH-VD and its antibody complex according to claim 7, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

10. The antibody against 25-OH-VD and its antibody complex according to claim 6, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

11. The antibody against 25-OH-VD and its antibody complex according to claim 6, characterized in that, The species origin of the constant region is human.

12. The antibody against 25-OH-VD and its antibody complex according to claim 7, characterized in that, The heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% identity with it; the light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity with it.

13. The antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex according to any one of claims 1, 2, 4, and 5, characterized in that, The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv.

14. An antibody against 25-OH-VD and its antibody complex, or an antigen-binding fragment thereof, wherein the antibody against 25-OH-VD and its antibody complex comprises a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:26, and the amino acid sequence of the light chain is shown in SEQ ID NO:33; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:28, and the amino acid sequence of the light chain is shown in SEQ ID NO:33; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:29, and the amino acid sequence of the light chain is shown in SEQ ID NO:33; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:27, and the amino acid sequence of the light chain is shown in SEQ ID NO:33; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:26, and the amino acid sequence of the light chain is shown in SEQ ID NO:35; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:26, and the amino acid sequence of the light chain is shown in SEQ ID NO:

34.

15. An antibody conjugate, characterized in that, The antibody conjugate comprises an antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex as described in any one of claims 1 to 14, and biotin conjugated to the antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex.

16. An antibody conjugate, characterized in that, The antibody conjugate comprises an antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex as described in any one of claims 1 to 14, and a label or solid-phase carrier conjugated to the antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex.

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

18. The antibody conjugate according to claim 16, characterized in that, The solid support is selected from microspheres, plates, and membranes.

19. A reagent or kit, characterized in that, The reagent or kit comprises an antibody against 25-OH-VD and its antibody complex as described in any one of claims 1 to 14, or an antigen-binding fragment thereof, or an antibody conjugate as described in any one of claims 15 to 18.

20. The use of the antibody against 25-OH-VD and its antibody complex as described in any one of claims 1 to 14, or the antigen-binding fragment thereof, or the antibody conjugate as described in any one of claims 15 to 18, in the preparation of a product for detecting 25-OH-VD.

21. The use according to claim 20, characterized in that, include: a) Under conditions sufficient to allow antibody / antigen binding reaction to occur, the test sample is bound to the 25-OH-VD antibody to form complex 1; and b) Contacting the antibody against 25-OH-VD and its antibody complex as described in any one of claims 1 to 14, or the antibody conjugate as described in any one of claims 15 to 18, 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.

22. A nucleic acid, characterized in that, It encodes the antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex as described in any one of claims 1 to 14.

23. A carrier, characterized in that, It contains the nucleic acid as described in claim 22.

24. A cell characterized by, It contains the nucleic acid as described in claim 22 or the vector as described in claim 23.

25. A method for preparing an antibody or antigen-binding fragment thereof against 25-OH-VD and its antibody complex as described in any one of claims 1-14, characterized in that, It includes: Culture the cells as described in claim 24.

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