Antibody for resisting 25-OH-VD and antibody complex thereof, and reagent and kit for detecting 25-OH-VD

Abstract

The invention discloses an antibody for resisting 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 anti-25-OH-VD and the antibody compound thereof disclosed by the invention comprise a heavy chain complementarity determining region and a light chain complementarity determining region, and 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] The present disclosure claims priority to Chinese patent application number 202310759993.X filed with the Chinese Patent Office on June 25, 2023, entitled “Antibodies against 25-OH-VD and its antibody complexes, reagents and kits for detecting 25-OH-VD”, the entire contents of which are incorporated by reference into the present disclosure. Technical Field

[0003] The present invention relates to the technical field of antibodies, in particular to an antibody against 25-OH-VD and an antibody complex thereof, and a reagent and a kit for detecting 25-OH-VD. Background Art

[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 human skin after exposure to ultraviolet light, and a small amount is ingested from food or supplements. Vitamin D not only affects calcium and phosphorus metabolism, but also has a wide range of physiological effects. It is an essential substance for maintaining human health, cell growth and development, and is closely related to a variety of diseases. Vitamin D is converted into 25-hydroxyvitamin D (25-OH-VD) through hydroxylation in the liver. The level of serum 25-OH-VD can reflect the body's vitamin D storage level and is related to the clinical symptoms of vitamin D deficiency. If the index is too high, it may be vitamin D poisoning, and if the index is too low, it may be vitamin D deficiency.

[0005] 25-Hydroxyvitamin D testing is usually done through serum testing, which can determine whether the body's vitamin D levels are abnormal. If the test reveals that the 25-Hydroxyvitamin D level is too low, it is common in calcium and phosphorus absorption disorders, which may manifest as abnormal bone and tooth development, osteoporosis, delayed teething, and other symptoms. In severe cases, it may also lead to rickets and osteomalacia, which manifest as osteoporosis and muscle aches, and increase the risk of leg cramps, falls, and fractures. If the 25-Hydroxyvitamin D level is too high, it may cause vitamin D poisoning and hypercalcemia, which manifests as loss of appetite, vomiting, and diarrhea.

[0006] Since 25-(OH)-VD is a small molecule, most small molecules have only one antigenic determinant. Therefore, the detection methods of 25-hydroxyvitamin D commonly used at present are all competitive methods based on chemiluminescence, enzyme-linked immunosorbent assay, electrochemiluminescence, immunochromatography, etc. Limited by the defects of the methodology of the competitive method itself, such as insufficient accuracy, its clinical application has many limitations and controversies. With the development of detection technology, the quantitative detection of 25-hydroxyvitamin D can be better achieved using the sandwich method. The small molecule sandwich method is a non-competitive immunoassay method based on anti-immune complex antibodies, but it requires an antibody raw material for 25-OH-VD and its antibody complex. The present invention is based on this demand and develops an antibody raw material for anti-25-OH-VD and its antibody complex. Summary of the Invention

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

[0008] In order to achieve the above-mentioned purpose, according to one aspect of the present invention, an antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises three complementarity determining regions having a heavy chain variable region of any one of the amino acid sequences of SEQ ID NOs: 22, 23, 24, and 25 and three complementarity determining regions having a light chain variable region of any one of the amino acid sequences of SEQ ID NOs: 30, 31, and 32.

[0009] In order to achieve the above object, according to a second aspect of the present invention, an antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises the following complementarity determining regions:

[0010] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0011] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0012] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3 or 17;

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

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

[0015] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0016] In order to achieve the above-mentioned object, according to the 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 in any one of SEQ ID NOs: 22, 23, 24, and 25; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 30, 31, and 32.

[0017] In order to achieve the above-mentioned purpose, according to the 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 shown in any one of SEQ ID NOs: 26, 27, 28, and 29; and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 33, 34, and 35.

[0018] In order to achieve the above object, according to the fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or a functional fragment thereof.

[0019] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or kit is provided, wherein the reagent or kit comprises the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.

[0020] To achieve the above-mentioned object, according to a seventh aspect of the present invention, a method for detecting 25-OH-VD is provided, comprising: a) allowing a test sample to bind to a 25-OH-VD antibody under conditions sufficient for an antibody / antigen binding reaction to occur to form a complex 1; and b) allowing the above-mentioned antibody or functional fragment thereof, antibody conjugate, reagent or kit to contact the complex 1 in a) to form a complex 2; and c) detecting the presence of the complex 2, wherein the presence of the complex 2 indicates the presence of 25-OH-VD in the test sample.

[0021] In order to achieve the above object, according to the eighth aspect of the present invention, a nucleic acid is provided, which encodes the above antibody or a functional fragment thereof.

[0022] In order to achieve the above object, according to the ninth aspect of the present invention, a vector is provided, wherein the vector comprises the above nucleic acid.

[0023] To achieve the above object, according to the tenth aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid, vector or expresses the above antibody or a functional fragment thereof.

[0024] In order to achieve the above object, according to the eleventh aspect of the present invention, a method for preparing the above antibody or a functional fragment thereof is provided, which comprises culturing the above cell.

[0025] In order to achieve the above-mentioned object, according to the twelfth aspect of the present invention, there is provided a use of the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit in detecting or preparing a product for detecting 25-OH-VD. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 These are the results of reducing SDS-PAGE of antibodies 15H7 Rmb1 to 15H7 Rmb6. DETAILED DESCRIPTION

[0028] In the first aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, which comprises three complementarity determining regions having a heavy chain variable region of any one of the amino acid sequences SEQ ID NOs: 22, 23, 24, and 25 and three complementarity determining regions having a light chain variable region of any one of the amino acid sequences SEQ ID NOs: 30, 31, and 32.

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

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

[0031] 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, so long as they exhibit the desired biological activity.

[0032] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

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

[0034] Methods for defining CDRs are well known in the art, including the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200200201154, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by 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, and the definitions in different literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present 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 numbering of all CDR definitions in Table 1 is based on 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." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0038] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0039] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0040] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0041] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if 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 of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

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

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

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

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

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

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

[0049] According to an embodiment 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] In a second aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises the following complementarity determining regions:

[0053] HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0054] HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0055] HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or 17.

[0056] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 18.

[0057] LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5.

[0058] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0059] 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 region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

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

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

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

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

[0064] The HFR3 comprises / is SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0065] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0066] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0067] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0068] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

[0069] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto;

[0070] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or functional fragments thereof provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0071] In an optional embodiment, the HFR1 includes / is represented by the amino acid sequence of SEQ ID NO: 7, 19 or 20.

[0072] In an optional embodiment, the LFR1 includes / is represented by the amino acid sequence of SEQ ID NO: 11 or 21.

[0073] In a third aspect, an embodiment of the present invention provides 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 NOs: 22, 23, 24, and 25, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 30, 31, and 32.

[0074] In an optional embodiment, the heavy chain variable region and light chain variable region of the first aspect and the third aspect are selected from any one of the following combinations:

[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 alternative embodiment, the antibody or functional fragment thereof further comprises a constant region.

[0077] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0078] In an optional embodiment, 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 alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

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

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

[0082] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

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

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

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

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

[0087] The functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they are derived. Based on the disclosure herein, those skilled in the art will readily appreciate that the functional fragments of the above-mentioned antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain the above-mentioned functional fragments.

[0088] Functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0089] In a fourth aspect, 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 shown in any one of SEQ ID NOs: 26, 27, 28, and 29, and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 33, 34, and 35.

[0090] In an optional embodiment, the antibody described in the first, second, third, and fourth aspects above comprises any combination of the following heavy chains 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] In a fifth aspect, the present invention provides an antibody conjugate comprising the above-mentioned antibody or a functional fragment thereof.

[0093] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative coupled to the antibody or a functional fragment thereof.

[0094] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or a functional fragment thereof.

[0095] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0096] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0097] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0098] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0099] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0100] In an optional embodiment, 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 an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

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

[0103] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0104] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0105] In an optional embodiment, the above-mentioned antibody conjugate further comprises a solid phase carrier coupled to the antibody or its functional fragment.

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

[0107] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0108] In a sixth aspect, the present invention provides a reagent or a kit, which comprises the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.

[0109] As mentioned above, 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 antibodies or functional fragments only bind to 25-OH-VD and its antibody complex, and do not bind to 25-OH-VD or 25-OH-VD antibodies. Therefore, the reagent or kit containing the 25-OH-VD and its antibody complex antibody or its functional fragment can effectively perform qualitative or quantitative detection of 25-OH-VD. The reagent or kit provided by the present invention can be used, for example, for detection involving the use of the specific binding properties of 25-OH-VD, such as immunoblotting and immunoprecipitation. As mentioned above, the antibodies or functional fragments thereof in some embodiments or examples of the present invention have higher binding activity with 25-OH-VD, and therefore the reagent or kit containing the antibodies or their functional fragments has higher detection sensitivity or specificity.

[0110] In a seventh aspect, the present invention provides a method for detecting 25-OH-VD, comprising: providing a method for detecting 25-OH-VD, comprising: a) first allowing a sample to be tested to bind to a 25-OH-VD antibody under conditions sufficient for an antibody / antigen binding reaction to occur to form a complex 1; and b) allowing the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit to contact the complex 1 in a) to form a complex 2; and c) detecting the presence of the complex 2, wherein the presence of the complex 2 indicates 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 the antibody or functional fragment thereof described in any one of the above items, and the heavy chain complementary 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 complementary 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] In an eighth aspect, 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 vector containing the above-mentioned nucleic acid molecule.

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

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

[0117] In a twelfth aspect, the present invention provides use of the above-mentioned antibodies or functional fragments thereof, antibody conjugates, or the above-mentioned reagents, kits, or antibody pairs in detecting 25-OH-VD or preparing products for detecting 25-OH-VD.

[0118] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (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 that can recombinantly express the antibody or its functional fragment as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it falls within the scope of protection of the present invention.

[0119] To further clarify the objectives, technical solutions, and advantages of the embodiments of the present invention, the technical solutions described in the embodiments are described below in a clear and complete manner. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available. Exemplary heavy and light chain sequences of the 25-OH-VD antibody described in the examples below are 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 those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0121] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

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

[0123] Example 1 Antibody Discovery of Antibodies Against 25-Hydroxyvitamin D and 25-Hydroxyvitamin D Antibody Complex

[0124] (1) Immunization of mice

[0125] An emulsified preparation 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 sera were collected on days 0, 14, 28, 42, and 69, respectively. The spleens of the mice were then surgically removed and spleen cell suspensions were prepared.

[0126] (2) Cell fusion

[0127] The spleen cell suspension and mouse myeloma cells were mixed, and cell fusion was performed by electrofusion to prepare mouse hybridoma cells, which were added into HAT hybridoma screening medium and then placed in an incubator for culture.

[0128] (3) Hybridoma screening

[0129] After the hybridoma cells were cultured for 7 days, the hybridoma cell supernatant was identified by ELISA screening method to screen out hybridoma cell lines that could specifically recognize and bind to the complex of 25-hydroxyvitamin D and 25-hydroxyvitamin D antibody.

[0130] The hybridoma cell line against the complex of 25-hydroxyvitamin D and 25-hydroxyvitamin D antibody was subcloned by the limiting dilution method. After 7 days, the supernatant of the monoclonal hybridoma cell was screened and identified by the ELISA screening method to obtain a positive monoclonal hybridoma cell line against the complex of 25-hydroxyvitamin D and 25-hydroxyvitamin D antibody. The antibody secreted by this cell line only binds to the complex formed by 25-OH-VD and 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 does not bind to 25-OH-VD or 25-OH-VD antibody.

[0131] (4) Hybridoma antibody gene sequencing

[0132] RNA from the monoclonal hybridoma cell line of the anti-25-hydroxyvitamin D and 25-hydroxyvitamin D antibody complex was extracted and reverse transcribed into cDNA. The antibody gene fragment was then amplified by PCR. Next, 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 25-hydroxyvitamin D antibody complex antibody

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

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

[0136] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0137] (2) Recombinant antibody production

[0138] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed with 100 μg of culture medium and re-dissolved. This was also used as a cell diluent. Plasmid DNA and transfection reagent diluents were prepared separately using culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed, and allowed to stand at room temperature for 15 minutes. The mixture was slowly added to the cell diluent over 1 minute, mixed, and sampled. The viability of the cells after transfection was recorded and observed. The cells were cultured in a 35°C constant temperature incubator at 120 rpm and 8% CO2. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. The electrophoresis pattern is shown in the figure. Two bands were 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).

[0139] The resulting antibody was named complex antibody 15H7 Rmb1. Mutations were performed on the complex antibody 15H7 Rmb1 to generate a mutant antibody that only binds to the complex formed by 25-OH-VD and its antibody, and does not bind to 25-OH-VD or the 25-OH-VD antibody. The sequences of the heavy (H) and light (L) chains of the above-mentioned antibody are shown in the following table:

[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 Performance testing of antibodies

[0143] 1. Performance evaluation

[0144] Antibodies 15H7 Rmb1 to 15H7 Rmb6, along with a control antibody, were used as labeled antibodies with acridinium esters. Another antibody (from Feipeng Bio) was used as a coating antibody to label magnetic beads. The performance of these antibodies was tested using a double-antibody sandwich assay on a chemiluminescence platform (Yingkai Shine I2910).

[0145] Reaction mode: 1) Add 50 μl of 25-hydroxyvitamin D antigen control and 50 μl of magnetic bead-labeled antibody (0.05%), react at 37°C for 10 minutes, and wash three times with PBST; 2) Add 50 μl of acridinium ester-labeled antibody, react at 37°C for 10 minutes, and wash three times with PBST; 3) Add 200 μl of acridinium ester substrate and read the instrument. Specific performance evaluation results are as follows:

[0146] Note: C0~C7: represent different concentrations of standard products, RLU: represents the luminescence signal intensity

[0147] Table 3 Performance evaluation data

[0148]

[0149] 2. Stability assessment

[0150] The above-mentioned antibody was placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for status observation, and the 21-day sample was tested for activity (luminescence platform). The results showed that the complex antibody 15H7 Rmb4 showed no significant changes in protein status after 21 days under all three test conditions, and the activity did not decrease with increasing test temperature, indicating that the expressed antibody was stable. Table 4 below shows the luminescence activity test results after 21 days of testing.

[0151] Table 4 Stability data

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

[0153] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0154] Some of the amino acid sequences involved in this application are shown in Table 5:

[0155]

[0156]

[0157]

Claims

1. An antibody or a functional fragment thereof, comprising three complementary determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NOs: 22, 23, 24, and 25 and three complementary determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NOs: 30, 31, and 32.

2. The antibody or functional fragment thereof according to claim 1, characterized in that: The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment comprises the following complementarity determining region: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3 or 17; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 18; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6; Optionally, the antibody or its functional fragment further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; The HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

4. An antibody or a functional fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 22, 23, 24, and 25; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 30, 31, and 32; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations:

5. The antibody or functional fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or its functional fragment further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is human; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto; Optionally, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

6. An antibody or a functional fragment thereof, comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 26, 27, 28, and 29; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 33, 34, and 35.

7. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or a functional fragment thereof according to any one of claims 1 to 6; Optionally, the antibody conjugate further comprises biotin or a biotin derivative coupled to the antibody or a functional fragment thereof; Optionally, the antibody conjugate further comprises a label coupled to the antibody or a functional fragment thereof; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody or its functional fragment; Optionally, the solid support is selected from microspheres, plates and membranes.

8. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1 to 6 or the antibody conjugate according to claim 7.

9. A method for detecting 25-OH-VD, characterized in that: include: a) allowing the sample to be tested to bind to the 25-OH-VD antibody to form a complex 1 under conditions sufficient for an antibody / antigen binding reaction to occur; and b) contacting the antibody or functional fragment thereof according to any one of claims 1 to 6, the antibody conjugate according to claim 7, or the reagent or kit according to claim 8 with the complex 1 in a) to form a complex 2; and c) detecting the presence of the complex 2, wherein the presence of the complex 2 indicates the presence of 25-OH-VD in the test sample.

10. A nucleic acid, characterized in that It encodes the antibody or its functional fragment according to any one of claims 1 to 6.

11. A carrier, characterized in that It contains the nucleic acid according to claim 10.

12. A cell, characterized in that It contains the nucleic acid according to claim 10 or the vector according to claim 11.

13. A method for preparing the antibody or functional fragment thereof according to any one of claims 1 to 6, characterized in that: It includes: Cultivate the cell of claim 12.

14. Use of the antibody or functional fragment thereof according to any one of claims 1 to 6, the antibody conjugate according to claim 7, or the reagent or kit according to claim 8 in detecting or preparing a product for detecting 25-OH-VD.

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