CFGF23 specific antibody and application thereof in preparation of osteoporosis auxiliary diagnostic reagent

Abstract

The invention relates to a cFGF23 specific antibody and application thereof in preparation of an osteoporosis auxiliary diagnosis reagent, and belongs to the technical field of disease auxiliary diagnosis. The three specific antibodies can specifically recognize the C terminal of the FGF23 eukaryotic protein, and the two capture antibodies and the detection antibody recognize the C terminal (180-205), the C terminal (200-229) and the C terminal (222-251) of the FGF23 protein respectively. When the cFGF23 protein is detected, the capture performance of the detection system on the cFGF23 can be greatly improved through mixed capture of the two capture antibodies.

Description

Technical Field

[0001] The invention belongs to the technical field of disease auxiliary diagnosis, and relates to a cFGF23-specific antibody and an application thereof in the preparation of an osteoporosis auxiliary diagnosis reagent. Background Art

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and degeneration of bone tissue microstructure, which leads to increased bone fragility and a significantly increased risk of fractures. The occurrence of this disease is mainly related to the imbalance in which the rate of bone resorption exceeds the rate of bone formation during bone remodeling. Its etiology is complex, involving multiple factors such as genetics, endocrine, nutrition, and lifestyle. Osteoporosis can be divided into two major categories: primary and secondary. Primary osteoporosis is mainly seen in the elderly and postmenopausal women, due to the decrease in the level of hormones related to bone metabolism (such as estrogen deficiency), which leads to accelerated bone loss; secondary osteoporosis is related to specific diseases (such as hyperparathyroidism, diabetes) or long-term use of certain drugs (such as glucocorticoids).

[0003] Clinically, osteoporosis often has no obvious symptoms in the early stages and is easily overlooked. When the disease progresses to the fracture stage, patients may show symptoms such as pain, shortened height, and hunchback. The most common fracture sites include the hip, vertebral body, and distal radius. Diagnosis mainly relies on bone density measurement (such as dual-energy X-ray absorptiometry, DXA), and a bone density T value ≤-2.5 is one of the diagnostic criteria. In addition, combined with medical history, laboratory tests, and imaging evaluations, the cause can be identified and secondary factors can be excluded. The goal of osteoporosis treatment is to reduce the risk of fractures, mainly including drug therapy (such as bisphosphonates, anti-RANKL monoclonal antibodies, bone formation promoters), nutritional support (calcium and vitamin D supplementation), and lifestyle intervention (exercise, smoking cessation, and alcohol restriction). Early screening and comprehensive management are of great significance for improving patient prognosis.

[0004] According to the International Osteoporosis Foundation (IOF), approximately 200 million people worldwide suffer from osteoporosis. As the global aging population intensifies, the number of people suffering from osteoporosis is expected to continue to increase. Strengthening early screening, preventive measures and health education are of great significance to reducing the risk of fractures and reducing the medical burden.

[0005] Fibroblast growth factor 23 is a member of the fibroblast growth factor (FGFs) family and is mainly secreted by osteocytes and osteoblasts. The FGF23 gene is located on human chromosome 12 and contains 3 exons and 2 introns. The relative molecular mass of the mature FGF23 protein (without the signal peptide) is 26kDa. FGF23 is a powerful calcium-phosphorus regulatory factor that can regulate the balance of blood phosphorus and vitamin D in the body. The full-length FGF23 is a 32kDa glycoprotein consisting of two functional regions, the N-terminal and the C-terminal.

[0006] FGF23 is cleaved by proteases at specific sites, mainly at the paired basic amino acid sequence (RXXR) between Arg176-Arg179. After cleavage, two fragments are generated: the N-terminus contains a signal peptide and an active site that promotes phosphate excretion, and the C-terminus participates in receptor-specific effects by binding to Klotho. Circulating C-terminal FGF23 (cFGF23) refers to the C-terminal fragment produced after FGF23 cleavage, and its level changes in a variety of disease states and is closely related to bone metabolism disorders. Therefore, there are three forms of FGF23 in the blood: full-length FGF23 polypeptide (iFGF23), C-terminal FGF23 polypeptide (cFGF23), and N-terminal FGF23P polypeptide (nFGF23).

[0007] In recent years, studies have found that cFGF23 may play an important role in the occurrence and development of osteoporosis. Increased cFGF23 levels are often regarded as an indirect indicator of changes in iFGF23 activity. Studies have shown that abnormal cFGF23 levels in osteoporosis patients may reflect osteocyte dysfunction, enhanced bone matrix degradation, and systemic abnormalities in bone metabolism. In addition, cFGF23 abnormalities may be associated with low bone density and bone microarchitecture degradation. Its mechanism of action may be achieved by affecting osteoblast activity and regulating bone matrix mineralization. Increased cFGF23 levels may aggravate bone loss, especially in osteoporosis patients with chronic kidney disease or abnormal phosphorus metabolism. This effect is particularly significant. Therefore, it is urgent to develop a highly sensitive cFGF23 detection system, and the main problem to be solved in detecting cFGF23 is the acquisition of high-affinity paired antibodies.

[0008] The main problems with the current cFGF23 detection technology are: (1) the C-terminal fragment of FGF23 is relatively short, resulting in a limited number of paired antibodies that can be screened; and (2) also due to the short C-terminal fragment, there is spatial conformational competition between paired antibodies when detecting FGF23, resulting in insufficient sensitivity of the detection system. Summary of the invention

[0009] In order to perform high-sensitivity detection of cFGF23 protein, the present invention provides three monoclonal antibodies that recognize cFGF23 protein and their use in a chemiluminescent immunoassay kit, providing a tool for auxiliary diagnosis of diseases related to cFGF23 protein, and further providing the use of the antibody or kit in auxiliary diagnosis of osteoporosis.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] 1. A: The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the recognition epitope or binding epitope of the antibody or antigen-binding fragment is the 180-205 segment of FGF23 protein (the sequence is shown in SEQ ID NO: 1 [SAEDDSERDPLNVLKPRARMTPAPAS]), and the antibody or antigen-binding fragment comprises:

[0012] The heavy chain variable region CDRs of at least one of the following:

[0013] (1-1) comprising CDR H1 shown in SEQ ID NO.6; or comprising CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.6;

[0014] (2-1) comprising CDR H2 shown in SEQ ID NO.7; or comprising CDR H2 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.7;

[0015] (3-1) comprising CDR H3 shown in SEQ ID NO.8; or comprising CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.8.

[0016] As an embodiment, the antibody or antigen-binding fragment comprises:

[0017] The CDRs of the heavy chain variable region of at least one of the following:

[0018] (4-1) CDR H1 shown in SEQ ID NO.6; or CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.6;

[0019] (5-1) CDR H2 shown in SEQ ID NO.7; or CDR H2 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.7;

[0020] (6-1) CDR H3 shown in SEQ ID NO.8; or CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.8.

[0021] The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the antibody or antigen-binding fragment comprises:

[0022] The CDRs of the light chain variable region of at least one of the following:

[0023] (7-1) comprising CDR L1 shown in SEQ ID NO.10; or comprising CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.10;

[0024] (8-1) comprising a CDR L2 having an amino acid sequence of IAS; or comprising a CDR L2 having an amino acid sequence of IAS that is 90% or more identical to that of IAS;

[0025] (9-1) comprising CDR L3 shown in SEQ ID NO.11; or comprising CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.11.

[0026] As an embodiment, the antibody or antigen-binding fragment comprises:

[0027] The CDRs of the light chain variable region of at least one of the following:

[0028] (10-1) CDR L1 shown in SEQ ID NO.10; or CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.10;

[0029] (11-1) CDR L2 whose sequence is IAS; or CDR L2 having an amino acid sequence with a sequence of IAS that is 90% or more identical;

[0030] (12-1) CDR L3 shown in SEQ ID NO.11; or CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.11.

[0031] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes at least one of the above (1-1) to (6-1), and the light chain variable region includes at least one of the above (7-1) to (12-1).

[0032] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.6, CDR H2 shown in SEQ ID NO.7, and CDR H3 shown in SEQ ID NO.8, and the light chain variable region includes CDR L1 shown in SEQ ID NO.10, CDR L2 with sequence IAS, and CDR L3 shown in SEQ ID NO.11.

[0033] The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0034] It comprises the amino acid sequence shown in SEQ ID NO.5; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.5.

[0035] As an embodiment, the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0036] The amino acid sequence shown in SEQ ID NO.5; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.5.

[0037] The present invention provides an antibody or antigen-binding fragment that specifically binds to the cFGF23 protein, wherein the antibody or antigen-binding fragment comprises the following light chain variable region:

[0038] It comprises the amino acid sequence shown in SEQ ID NO.9; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.9.

[0039] As an embodiment, the antibody or antigen-binding fragment comprises the following light chain variable region:

[0040] The amino acid sequence shown in SEQ ID NO.9; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.9.

[0041] As an embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO.5 and a light chain variable region shown in SEQ ID NO.9.

[0042] Alternatively, B: The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the antibody recognition epitope or binding epitope of the antibody or antigen-binding fragment is the 200-229 segment of FGF23 protein (the sequence is shown in SEQ ID NO: 2 [TPAPASCSQELPSAEDNSPMASDPLGVVRG]), and the antibody or antigen-binding fragment comprises:

[0043] The heavy chain variable region CDRs of at least one of the following:

[0044] (1-2) comprising CDR H1 shown in SEQ ID NO. 13; or comprising CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 13;

[0045] (2-2) comprising CDR H2 shown in SEQ ID NO. 14; or comprising CDR H2 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 14;

[0046] (3-2) comprising CDR H3 shown in SEQ ID NO.15; or comprising CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.15.

[0047] In some embodiments, the antibody or antigen-binding fragment comprises:

[0048] The CDRs of the heavy chain variable region of at least one of the following:

[0049] (4-2) CDR H1 shown in SEQ ID NO. 13; or CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 13;

[0050] (5-2) CDR H2 shown in SEQ ID NO. 14; or CDR H2 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 14;

[0051] (6-2) CDR H3 shown in SEQ ID NO. 15; or CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 15.

[0052] The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the antibody or antigen-binding fragment comprises:

[0053] The CDRs of the light chain variable region of at least one of the following:

[0054] (7-2) comprising CDR L1 shown in SEQ ID NO. 17; or comprising CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 17;

[0055] (8-2) comprising a CDR L2 having an amino acid sequence of MCS; or comprising a CDR L2 having an amino acid sequence that is 90% or more identical to the amino acid sequence of MCS;

[0056] (9-2) comprising CDR L3 shown in SEQ ID NO.18; or comprising CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.18.

[0057] In some embodiments, the antibody or antigen-binding fragment comprises:

[0058] The CDRs of the light chain variable region of at least one of the following:

[0059] (10-2) CDR L1 shown in SEQ ID NO. 17; or CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 17;

[0060] (11-2) CDR L2 having an amino acid sequence of MCS; or CDR L2 having an amino acid sequence of MCS having an identity of 90% or more;

[0061] (12-2) CDR L3 shown in SEQ ID NO. 18; or CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 18.

[0062] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes at least one of the above (1-2) to (6-2), and the light chain variable region includes at least one of the above (7-2) to (12-2).

[0063] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.13, CDR H2 shown in SEQ ID NO.14, and CDR H3 shown in SEQ ID NO.15, and the light chain variable region includes CDR L1 shown in SEQ ID NO.17, CDR L2 with the sequence of MCS, and CDR L3 shown in SEQ ID NO.18.

[0064] The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0065] It comprises the amino acid sequence shown in SEQ ID NO.12; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.12.

[0066] As an embodiment, the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0067] The amino acid sequence shown in SEQ ID NO.12; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.12.

[0068] The present invention provides an antibody or antigen-binding fragment that specifically binds to the cFGF23 protein, wherein the antibody or antigen-binding fragment comprises the following light chain variable region:

[0069] It comprises the amino acid sequence shown in SEQ ID NO.16; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.16.

[0070] As an embodiment, the antibody or antigen-binding fragment comprises the following light chain variable region:

[0071] The amino acid sequence shown in SEQ ID NO.16; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.16.

[0072] As an embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO.12 and a light chain variable region shown in SEQ ID NO.16.

[0073] Alternatively, C: The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the antibody recognition epitope or binding epitope of the antibody or antigen-binding fragment is the 222-251 segment of FGF23 protein (the sequence is shown in SEQ ID NO: 3 [DPLGVVRGGRVNTHAGGTGPEGCRPFAKFI], and the antibody or antigen-binding fragment comprises:

[0074] The heavy chain variable region CDRs of at least one of the following:

[0075] (1-3) comprising CDR H1 shown in SEQ ID NO. 20; or comprising CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 20;

[0076] (2-3) comprising CDR H2 of SEQ ID NO. 21; or comprising CDR H2 having 90% or more identity with the amino acid sequence of SEQ ID NO. 21;

[0077] (3-3) comprising CDR H3 shown in SEQ ID NO. 22; or comprising CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 22.

[0078] In some embodiments, the antibody or antigen-binding fragment comprises:

[0079] The heavy chain variable region CDRs of at least one of the following:

[0080] (4-3) CDR H1 shown in SEQ ID NO. 20; or CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 20;

[0081] (5-3) CDR H2 shown in SEQ ID NO. 21; or CDR H2 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 21;

[0082] (6-3) CDR H3 shown in SEQ ID NO. 22; or CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 22.

[0083] The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the antibody or antigen-binding fragment comprises:

[0084] The CDRs of the light chain variable region of at least one of the following:

[0085] (7-3) comprising CDR L1 shown in SEQ ID NO. 24; or comprising CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 24;

[0086] (8-3) comprising a CDR L2 having a sequence of LYC; or comprising a CDR L2 having an amino acid sequence having a sequence of LYC that is 90% or more identical;

[0087] (9-3) comprising CDR L3 shown in SEQ ID NO. 25; or comprising CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 25.

[0088] In some embodiments, the antibody or antigen-binding fragment comprises:

[0089] The CDRs of the light chain variable region of at least one of the following:

[0090] (10-3) CDR L1 shown in SEQ ID NO. 24; or CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 24;

[0091] (11-3) CDR L2 having a sequence of LYC; or CDR L2 having an amino acid sequence of LYC that is more than 90% identical;

[0092] (12-3) CDR L3 shown in SEQ ID NO.25; or CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.26.

[0093] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes at least one of the above (1-3) to (6-3), and the light chain variable region includes at least one of the above (7-3) to (12-3).

[0094] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.20, CDR H2 shown in SEQ ID NO.21, and CDR H3 shown in SEQ ID NO.22, and the light chain variable region includes CDR L1 shown in SEQ ID NO.24, CDR L2 with sequence LYC, and CDR L3 shown in SEQ ID NO.25.

[0095] The present invention provides an antibody or antigen-binding fragment that specifically binds to cFGF23 protein, wherein the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0096] It comprises the amino acid sequence shown in SEQ ID NO.19; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.19.

[0097] As an embodiment, the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0098] The amino acid sequence shown in SEQ ID NO.19; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.19.

[0099] The present invention provides an antibody or antigen-binding fragment that specifically binds to the cFGF23 protein, wherein the antibody or antigen-binding fragment comprises the following light chain variable region:

[0100] It comprises the amino acid sequence shown in SEQ ID NO.23; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.23.

[0101] As an embodiment, the antibody or antigen-binding fragment comprises the following light chain variable region:

[0102] The amino acid sequence shown in SEQ ID NO.23; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.23.

[0103] As an embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO.19 and a light chain variable region shown in SEQ ID NO.23.

[0104] 2. The present invention also provides a hybridoma cell line that secretes antibodies or antigen-binding fragments that specifically bind to cFGF23 protein. The hybridoma cell lines that secrete the A, B, C antibodies or their antigen-binding fragments are named 3B11, 9D3 and 2D5, respectively.

[0105] Hybridoma cell line 3B11, deposited in China Center for Type Culture Collection, deposit number CCTCC NO: C202513, deposit date December 19, 2024, deposit address: Wuhan University, Wuhan, China, classification name: Hybridoma cell line 3B11;

[0106] Hybridoma cell line 9D3, deposited in China Center for Type Culture Collection, deposit number CCTCC NO: C202502, deposit date December 19, 2024, deposit address: Wuhan University, Wuhan, China, classification name: Hybridoma cell line 9D3;

[0107] Hybridoma cell line 2D5 is deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: C202514, the deposit date December 19, 2024, the deposit address: Wuhan University, Wuhan, China, and the classification name: Hybridoma cell line 2D5.

[0108] 3. The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment.

[0109] In a specific embodiment, the DNA encoding the heavy chain variable region of the 3B11 antibody is shown as SEQ ID NO.26, the DNA encoding the light chain variable region of the 3B11 antibody is shown as SEQ ID NO.27, the DNA encoding the heavy chain variable region of the 9D3 antibody is shown as SEQ ID NO.28, the DNA encoding the light chain variable region of the 9D3 antibody is shown as SEQ ID NO.29, the DNA encoding the heavy chain variable region of the 2D5 antibody is shown as SEQ ID NO.30, and the DNA encoding the light chain variable region of the 2D5 antibody is shown as SEQ ID NO.31.

[0110] 4. The present invention also provides a vector containing the above nucleic acid.

[0111] In a specific embodiment, the vector is a pcDNA3.1 vector.

[0112] 6. The present invention provides a host cell containing the above-mentioned vector.

[0113] In a specific embodiment, the host cell is an Escherichia coli DH5α competent cell, a 293F cell or a CHO-S cell.

[0114] 7. The present invention further provides a capture reagent for cFGF23 protein. The capture reagent comprises at least one selected from the following antibodies or antigen-binding fragments:

[0115] (1) the antibody or antigen-binding fragment as described in A above,

[0116] (2) the antibody or antigen-binding fragment as described in B above,

[0117] (3) The antibody or antigen-binding fragment as described in C above.

[0118] 8. The present invention further provides a detection reagent for cFGF23 protein. The detection reagent comprises at least one selected from the following antibodies or antigen-binding fragments:

[0119] (1) the antibody or antigen-binding fragment as described in A above,

[0120] (2) the antibody or antigen-binding fragment as described in B above,

[0121] (3) The antibody or antigen-binding fragment as described in C above.

[0122] As an embodiment, when used to detect cFGF23 protein, the detection reagent comprises an antibody or antigen-binding fragment in A, an antibody or antigen-binding fragment in B, and an antibody or antigen-binding fragment in C, so as to recognize different segments of the C-terminus of the FGF23 protein and improve the capture performance of the detection system for cFGF23.

[0123] 9. The present invention provides the use of the antibody or antigen-binding fragment, the hybridoma cell line, the nucleic acid, the vector, the host cell, the capture reagent, and the detection reagent in any of the following aspects:

[0124] (1) preparing a cFGF23 protein detection kit;

[0125] (2) Preparation of osteoporosis auxiliary diagnosis kit;

[0126] (3) Assist in the diagnosis of osteoporosis.

[0127] 10. The present invention provides a cFGF23 protein detection kit, the kit comprising: a capture antibody and a detection antibody;

[0128] The capture antibody and the detection antibody are both selected from at least one of A, B, and C, and the capture antibody is different from the detection antibody;

[0129] As an embodiment, (1) the capture antibody is selected from at least one of the antibodies or antigen-binding fragments described in A and B above, and the detection antibody is selected from one of the antibodies or antigen-binding fragments described in C above;

[0130] (2) the capture antibody is selected from at least one of the antibodies or antigen-binding fragments described in B and C above, and the detection antibody is selected from one of the antibodies or antigen-binding fragments described in A above;

[0131] (3) The capture antibody is selected from at least one of the antibodies or antigen-binding fragments described in A and C above, and the detection antibody is selected from one of the antibodies or antigen-binding fragments described in B above.

[0132] (4) the capture antibody is selected from one of the antibodies or antigen-binding fragments described in A above, and the detection antibody is selected from at least one of the antibodies or antigen-binding fragments described in B and C above;

[0133] (5) The capture antibody is selected from one of the antibodies or antigen-binding fragments described in B above, and the detection antibody is selected from at least one of the antibodies or antigen-binding fragments described in A and C above;

[0134] (6) The capture antibody is selected from one of the antibodies or antigen-binding fragments described in C above, and the detection antibody is selected from at least one of the antibodies or antigen-binding fragments described in A and B above.

[0135] As an embodiment, when the capture antibody is selected from two antibodies or antigen-binding fragments of A and B, the detection antibody is selected from one antibody or antigen-binding fragment of C; or, when the capture antibody is selected from two antibodies or antigen-binding fragments of B and C, the detection antibody is selected from the antibody or antigen-binding fragment in A; or, when the capture antibody is selected from two antibodies or antigen-binding fragments of A and C, the detection antibody is selected from the antibody or antigen-binding fragment in B. This allows the recognition of different segments of the C-terminus of the FGF23 protein, avoids missed detection of cFGF23 due to epitope competition of antibodies, and has a higher sensitivity for the detection of cFGF23.

[0136] As an embodiment, when the capture antibody is selected from the antibody or antigen-binding fragment described in A, the detection antibody is selected from two antibodies or antigen-binding fragments of B and C; or, when the capture antibody is selected from the antibody or antigen-binding fragment described in B, the detection antibody is selected from two antibodies or antigen-binding fragments of A and C; or, when the capture antibody is selected from the antibody or antigen-binding fragment described in C, the detection antibody is selected from two antibodies or antigen-binding fragments of A and B.

[0137] Specifically, the kit is a chemiluminescent diagnostic kit, which includes a biotin-labeled capture antibody, an acridinium ester-labeled detection antibody, streptavidin-coupled magnetic beads, an excitation solution, a pre-excitation solution, and a washing concentrate.

[0138] The exciting liquid is hydrogen peroxide with a mass fraction of 0.1-1%; the pre-exciting liquid is a sodium hydroxide solution with a mass fraction of 0.05-1 mol / L.

[0139] The washing concentrated solution is a Tris buffer solution containing Tween 20; the volume fraction of the Tween 20 is 0.5-3%, and the pH of the washing concentrated solution is 7.0-7.8.

[0140] The particle size of the magnetic beads is 0.3-2 μm, and the amount of the magnetic beads is 15-100 μL.

[0141] The final concentration of the biotin-labeled capture antibody is 2-75 μg / mL.

[0142] The final concentration of the acridinium ester-labeled detection antibody is 0.2-7.5 μg / mL.

[0143] 11. The present invention provides an osteoporosis auxiliary diagnosis system, comprising:

[0144] (1) A cFGF23 protein detection module, which uses the antibody or antigen binding fragment, the cFGF23 protein detection reagent or the cFGF23 protein detection kit to detect the cFGF23 protein concentration.

[0145] (2) A result output module, which assists in diagnosing osteoporosis based on the concentration of cFGF23 protein.

[0146] 12. The present invention provides a computer-readable storage medium, which is used to store computer instructions, programs, code sets or instruction sets. When the computer-readable storage medium is run on a computer, the computer executes the functions corresponding to the cFGF23 protein detection module and the result output module in the osteoporosis auxiliary diagnosis system.

[0147] 13. The present invention provides an electronic device, comprising: one or more processors; and a computer-readable storage medium, wherein the computer storage medium is used to store computer instructions, programs, code sets or instruction sets, and when the computer storage medium is run on a computer, the one or more processors implement the functions corresponding to the cFGF23 protein detection module and the result output module in the osteoporosis auxiliary diagnosis system.

[0148] 14. The present invention provides an auxiliary diagnosis method for osteoporosis, comprising: using the antibody or antigen binding fragment, the detection reagent of cFGF23 protein, or the cFGF23 protein detection kit to detect the concentration of cFGF23 protein, and then assisting in diagnosing whether osteoporosis occurs based on the cFGF23 protein concentration.

[0149] 15. The present invention provides a method for determining the concentration of cFGF23 protein, using the antibody or antigen binding fragment, the detection reagent for cFGF23 protein or the detection kit for cFGF23 protein to determine the concentration of cFGF23 protein.

[0150] In one embodiment, the determination method is a chemiluminescence method, comprising the following steps:

[0151] (1) establishing a calibration curve using a cFGF23 protein calibrator; the cFGF23 protein sequence is shown in SEQ ID NO.4;

[0152] (2) Using the antibody or antigen binding fragment, the cFGF23 protein detection reagent or the cFGF23 protein detection kit to determine the luminescence value of the sample to be tested, and calculating the cFGF23 protein concentration of the sample to be tested based on the luminescence value of the sample to be tested using the calibration curve of (1).

[0153] Definition of terms:

[0154] "Antigen-binding fragment" refers to all proteins / protein fragments containing the CDR region, including Fab, F(ab')2, Fd, Fv, ScFv, etc.

[0155] "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins. In the present invention, CDR H1, CDR H2, and CDR H3 represent the three CDRs of the heavy chain variable region, and CDR L1, CDR L2, and CDR L3 represent the three CDRs of the light chain variable region.

[0156] "Identity" refers to the sequence identity between two amino acid sequences or between two nucleic acid sequences. Having an identity of 90% or more means having an identity of, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, etc.

[0157] The beneficial effects of the present invention are:

[0158] (1) The three specific antibodies in the present invention can all specifically recognize the C-terminus of the FGF23 eukaryotic protein, and the capture antibody A, capture antibody B and detection antibody C recognize the C-terminus (180-205), C-terminus (200-229) and C-terminus (222-251) of the FGF23 protein, respectively. When detecting cFGF23 protein, the mixed capture of capture antibody A and capture antibody B can greatly improve the capture performance of the detection system for cFGF23.

[0159] (2) The three specific antibodies in the present invention can respectively recognize different segments of the C-terminus of the FGF23 protein, thereby avoiding missed detection of cFGF23 due to epitope competition among antibodies and achieving higher detection sensitivity for cFGF23.

[0160] (3) The chemiluminescent diagnostic kit involved in the present invention has high sensitivity, lower detection limit, better stability, and the detection limit can be as low as 6.25pg / ml, which can accurately determine the concentration of cFGF23 in osteoporotic and healthy human serum samples. Clinical sample verification shows that the specificity of the cFGF23 detection kit is 88.2% and the sensitivity is 69.4%.

[0161] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0163] Figure 1 It is the SDS-PAGE electrophoresis diagram of iFGF23 (mature FGF23 protein) and cFGF23 protein.

[0164] Figure 2 These are the epitope validation results for 3B11 antibody, 9D3 antibody, and 2D5 antibody.

[0165] Figure 3 This is the SDS-PAGE electrophoresis diagram of cFGF23 antibody.

[0166] Figure 4 For cFGF23 antibody titer detection.

[0167] Figure 5 Comparison of detection performance before and after cFGF23 antibody mixed capture.

[0168] Figure 6 It is the calibration curve of the cFGF23 detection kit, wherein the Y-axis represents the logarithm of the OD value, and the X-axis represents the logarithm of the concentration of the cFGF23 calibrator.

[0169] Figure 7 To establish the detection limit of FGF23 kit.

[0170] Figure 8 This is a scatter plot of the cFGF23 test kit's test results for osteoporosis and normal subjects.

[0171] Fig. 9 ROC analysis results of cFGF23 kit for detecting osteoporosis and normal human samples. DETAILED DESCRIPTION

[0172] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0173] Example 1. Recombinant expression and purification of cFGF23 protein

[0174] 1.1 Protein recombinant expression:

[0175] The amino acid sequence of the mature FGF23 protein from human (amino acids 25-251 in the full length of FGF23) is shown in SEQ ID NO.32 (YPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAG FVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRA KRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRRHTRSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI). DNA encoding the above amino acids was synthesized (see SEQ ID NO.33).

[0176] (As shown in (TACCCCAACGCCTCCCCCCTGCTGGGCTCCTCCTGGGGCGGCCTGATCCACCTGTACA CCGCCACCGCCAGGAACTCCTACCACCTGCAGATCCACAAGAACGGCCACGTGGACGGCGCCCCCCACCAGACCATCTACTCCGCCCTGATGATCAGGTCCGAGGACGCCGGCTTCGTGGTGATCACCGGCGTGATGTCCAGGAGGTACCTGTGCATGGACTTCAGGGGCAACATCTTCGGCTCCCACTACTTCGACCCCGAGAACTGCAGGTTCCAGCACCAGACCCTGGAGAACGGCTACGACGTGTACCACTCCCCCCAGTACCACTTCCTGGTGTCCCTGGGCAGGGCCAAGAGGGCCTTCCTGCCCGGCATGAACCCCCCCCCCTACTCCCAGTTCCTGTCCAGGAGGAACGAGATCCCCCTGATCCACTTCAACACCCCCATCCCCAGGAGGCACACCAGGTCCGCCGAGGACGACTCCGAGAGGGACCCCCTGAACGTGCTGAAGCCCAGGGCCAGGATGACCCCCGCCCCCGCCTCCTGCTCCCAGGAGCTGCCCTCCGCCGAGGACAACTCCCCCATGGCCTCCGACCCCCTGGGCGTGGTGAGGGGCGGCAGGGTGAACACCCACGCCGGCGGCACCGGCCCCGAGGGCTGCAGGCCCTTCGCCAAGTTCATC)), this DNA has been optimized for mammalian expression codons.

[0177] The amino acid sequence of cFGF23 protein (amino acids 180-251 in the full length of FGF23) is shown in SEQ ID No. 4 (SAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI). A DNA encoding the above amino acid sequence (shown in SEQ ID NO. 34 (TCCGCCGAGGACGACTCCGAGAGGGACCCCCTGAACGTGCTGAAGCCCAGGGCCAG GATGACCCCCGCCCCCGCCTCCTGCTCCCAGGAGCTGCCCTCCGCCGAGGACAACTCCC CCATGGCCTCCGACCCCCTGGGCGTGGTGAGGGGCGGCAGGGTGAACACCCACGCCGG CGGCACCGGCCCCGAGGGCTGCAGGCCCTTCGCCAAGTTCATC)) was synthesized, and the DNA was optimized for mammalian expression codons.

[0178] The synthesized DNAs shown in SEQ ID NO.33 and SEQ ID NO.34 were inserted into the pcDNA3.1 vector containing 6×His tags to obtain pcDNA3.1-FGF23 and pcDNA3.1-cFGF23. Then, pcDNA3.1-FGF23 and pcDNA3.1-cFGF23 were transformed into Escherichia coli DH5α competent cells, positive clones were picked and cultured in large quantities, and recombinant plasmids pcDNA3.1-FGF23 and pcDNA3.1-cFGF23 were extracted using a high-purity plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., endotoxin-free plasmid extraction kit (enhanced) (Cat. No. DP120)). The recombinant plasmids were transferred into 293F cells, and the pcDNA3.1 empty vector was transfected as a negative control at the same time. The cells were cultured in expi293 medium at 37°C and 8% CO2 for 72 hours, and the supernatant was collected and filtered using a 0.22μm filter membrane to obtain a filtrate.

[0179] 1.2 Protein purification:

[0180] Take 30 mL of the filtrate obtained in 1.1 and perform Ni-NTA affinity chromatography under non-denaturing conditions. Use equilibrium buffer to equilibrate the chromatography column; then load the sample. After loading, wash the chromatography column with equilibrium buffer; elute with elution buffer, and collect the eluate. Use a 3kD ultrafiltration tube to concentrate the protein solution (i.e., eluate), and store the protein in 50mM PBS (pH7.4) buffer at -80°C. The theoretical molecular weights of FGF23 and cFGF23 proteins are approximately 25kDa and 8kD, respectively. The purified protein was identified by SDS-PAGE electrophoresis purity, and the results showed that it was consistent with the corresponding theoretical size. Grayscale analysis showed that the protein purity reached more than 90%, see Figure 1 The composition of the equilibration buffer was 50 mM PBS, 10 mM imidazole, 150 mM NaCl, pH 7.6, and the composition of the elution buffer was 50 mM PBS, 250 mM imidazole, 150 mM NaCl, pH 7.6.

[0181] Example 2. Preparation of FGF23 mouse monoclonal antibody, screening of parent clones and epitope verification

[0182] 2.1 Preparation of mouse monoclonal antibodies:

[0183] The eukaryotic recombinant FGF23 protein and the eukaryotic recombinant cFGF23 protein purified in Example 1 were used as antigens to immunize 3 batches of BALB / c mice (6-8 weeks old, female), 3 mice each time. Each mouse was immunized with 50 μg of antigen each time. The antigen was mixed with Freund's complete adjuvant in a 1:1 ratio for the first immunization, and then the antigen was mixed with Freund's incomplete adjuvant in a 1:1 ratio. The immunization was performed once every two weeks for a total of 3 times. The tail was cut off and blood was collected ten days after the third immunization to detect the serum titer.

[0184] 2.2 Mother clone screening:

[0185] After hybridoma cell fusion, mother clones were obtained, and C-terminal polypeptides corresponding to three segments of FGF23 were synthesized (synthesized by GenScript Biotech Co., Ltd.) according to the amino acid sequences shown in SEQ ID NO.1-3, and the synthesized C-terminal polypeptides were named C1, C2, and C3, respectively. Mother clones that were reactive with eukaryotic recombinant cFGF23 protein and reactive with different regions of C1, C2, and C3 at the C-terminus were screened, and three mother clone cells that recognized C1, C2, and C3, respectively, were obtained.

[0186] 2.3 Monoclonal antibody acquisition and epitope verification:

[0187] The above mother clones were screened for monoclonal clones, and finally three monoclonal cell lines 3B11, 9D3 and 2D5 were obtained, which were reactive with the C-terminal polypeptides C1, C2 and C3 of FGF23, respectively.

[0188] The 3B11 cell line, 9D3 cell line and 2D5 cell line were respectively sent to the China Center for Type Culture Collection for preservation, and the preservation numbers are:

[0189] Hybridoma cell line 3B11, deposit number is CCTCC NO: C202513;

[0190] Hybridoma cell line 9D3, deposit number is CCTCC NO: C202502;

[0191] The hybridoma cell line 2D5 has the accession number of CCTCC NO: C202514.

[0192] The supernatants of the above 3B11, 9D3 and 2D5 monoclonal cells were collected respectively, and the 3B11, 9D3 and 2D5 antibodies were purified.

[0193] The chemiluminescent plate was coated with 1 μg / mL of 1 μg / mL eukaryotic recombinant cFGF23 protein, C1, C2, and C3 polypeptides in carbonate buffer (pH 9.5) in a volume of 100 μL at 4°C overnight, and gradiently diluted 3B11, 9D3, and 2D5 antibodies (all at a concentration of 0-1 μg / mL) were added, followed by goat anti-mouse IgG-HRP (100 ng / mL) and the luminescence value was detected.

[0194] The results showed that the 3B11 antibody was reactive only with the C1 polypeptide and the eukaryotic recombinant cFGF23 protein, the 9D3 antibody was reactive only with the C2 polypeptide and the eukaryotic recombinant cFGF23 protein, and the 2D5 antibody was reactive only with the C3 polypeptide and the eukaryotic recombinant cFGF23 protein. This indicates that the 3B11 antibody recognizes the C1 segment (180-205 segment) of the FGF23 protein, the 9D3 antibody recognizes the C2 segment (200-229 segment) of the FGF23 protein, and the 2D5 antibody recognizes the C3 segment (222-251 segment) of the FGF23 protein. The results are shown in the attached Figure 2 .

[0195] 2.3 Antibody Sequencing:

[0196] The obtained 3B11 cell line, 9D3 cell line, and 2D5 cell line were sequenced (after obtaining the cDNA of the hybridoma cell line, the primers were designed using existing technology to amplify the sequence, and the base composition of the sequence was obtained by sequencing) to obtain the corresponding antibody variable region sequence, and the sequenced sequence was classified by IMGT to obtain the CDR region sequence.

[0197] The results are as follows:

[0198] The amino acid sequence of the heavy chain variable region of the 3B11 antibody is EVKLEESGGGLVQPGGSMKLSCAASGSTFCDSWMDWVRQSPEKGLEWVAEIRAKATNCATYYAESVKGRFTISRDDSKSSVYLQMSSLRPEDTGIYYCNYCAYASDYWGQGTLVTVSA (SEQ ID NO.5); the heavy chain variable region of the 3B11 antibody includes: CDR H1: GSTFCDSW (SEQ ID NO.6); CDR H2: IRAKATTNCAT (SEQ ID NO.7); CDR H3: NYCAYASDY (SEQ ID NO.8).

[0199] The amino acid sequence of the light chain variable region of the 3B11 antibody is DIVMTQSHKFMSTSVGDRVSITCKASQSVCTAVAWYQQKPGQSPKLLIYIASTRHTGVPDRFTGSGSGTDYILTISSVQAEDLALYYCQSHYCTPPTFSGGTKLEIK (SEQ ID NO.9); the light chain variable region of the 3B11 antibody includes: CDR L1: QSVCTA (SEQ ID NO.10); CDR L2: IAS; CDRL3: QSHYCTPPT (SEQ ID NO.11).

[0200] The amino acid sequence of the heavy chain variable region of the 9D3 antibody is

[0201] QVQLQQPGVDLVRPGASVKLSCKASGYCFTAYWMNWVKQRPGQGLEWIGMIHPADYETRLNQKFKDKATLTVDKTSSTAYMQLSSPTSEDSAVYYCARFSYSVYCTMSYWGQGTSVTVSS (SEQ ID NO. 12); the heavy chain variable region of the 9D3 antibody includes: CDR H1: GYCFTAYW (SEQ ID NO.13); CDR H2: IHPADYET (SEQ ID NO.14); CDR H3: ARFSYSVYCTMSY (SEQ ID NO.15).

[0202] The amino acid sequence of the light chain variable region of the 9D3 antibody is

[0203] DIVMTQAAFSNPVTLGTSASISCRSSKSCLHSNGYTYLYWYLQKPGQSPQLLIYMCSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQCLSLALTFGAGTKLELK (SEQ ID NO.16); The light chain variable region of the 9D3 antibody includes: CDR L1: KSCLHSNGYTY (SEQ ID NO.17); CDR L2: MCS; CDR L3: AQCLSLALT (SEQ ID NO. 18).

[0204] The amino acid sequence of the heavy chain variable region of the 2D5 antibody is DVQLQESGPGLVKPSQSLSLTCTVTGYCITSSYAWNWIRQFPGNKLEWMGYICTYGNTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVAGCSYWGQGTLVTVSA (SEQ ID NO.19); the heavy chain variable region of the 2D5 antibody includes: CDR H1: GYCITSSYA (SEQ ID NO.20); CDRH2: ICTYGNT (SEQ ID NO.21); CDR H3: VAGCSY (SEQ ID NO.22).

[0205] The amino acid sequence of the light chain variable region of the 2D5 antibody is DVVLTQTPLTLSVTIGQPASISCKSSQCLLDSAGCTYLNWLLQRPGQSPKRLIYLYCDLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCYQCTHFSYTFGGGTKLEIK (SEQ ID NO.23); the light chain variable region of the 2D5 antibody includes: CDR L1: QCLLDSAGCTY (SEQ ID NO.24); CDRL2: LYC; CDR L3: YQCTHFSYT (SEQ ID NO.25).

[0206] Example 3: Recombinant expression and titer verification of FGF23 antibody

[0207] 3.1 Antibody recombinant expression:

[0208] The DNA of heavy (H chain) and light chain (L chain) were designed and synthesized according to the variable region sequences of 3B11, 9D3 and 2D5 antibodies, and optimized to mammalian expression codons. Among them, the DNA encoding the heavy chain variable region of 3B11 antibody is GAGGTGAAGCTGGAGGAGAGCGGCGGCGGCCTGGTGCAGCCTGGCGGCAGCATGAAGCTGAGCTGCGCCGCCAGCGGCAGCACCTTCTGCGACAGCTGGATGGACTGGGTGAGACAGAGCCCTGAGAAGGGCCTGGAGTGGGTGGCCGAGATCAGAGCCAAGGCCACCAACTGCGCCACCTACTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAGCAGCGTGTACCTGCAGATGAGCAGCCTGAGACCTGAGGACACCGGCATCTACTACTGCAACTACTGCGCCTACGCCAGCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC (SEQ ID NO.26); The DNA encoding the variable region of the light chain of the 3B11 antibody is GACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGACAGAGTGAGCATCACCTGCAAGGCCAGCCAGAGCGTGTGCACCGCCGTGGCCTGGTACCAGCAGAAGCCTGGCCAGAGCCCTAAGCTGCTGATCTACATC GCCAGCACCAGACACACCGGCGTGCCTGACAGATTCACCGGCAGCGGCAGCGGCACCGACTACATCCTGACCATCAGCAGCGTGCAGGCCGAGGACCTGGCCCTGTACTACTGCCAGAGCCACTACTGCACCCCTCCTACCTTCAGCGGCGGCACCAAGCTGGAGATCAAG(SEQ ID NO.27); The DNA encoding the variable region of the 9D3 antibody heavy chain is CAGGTGCAGCTGCAGCAGCCTGGCGTGGACCTGGTGAGACCTGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACTGCTTCACCGCCTACTGGATGAACTGGGTGAAGCAGAGACCTGGCCAGGGCCTGGAGTGGATCGGCATGATCCACCCTGCCGACTACGAGACCAGACTGAACCAGAAGTTCAAGGACAAGGCCACCCTGACCGTGGACAAGACCAGCAGCACCGCCTACATGCAGCTGAGCAGCCCTACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATTCAGCTACAGCGTGTACTGCACCATGAGCTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC (SEQ ID NO.28); The DNA encoding the variable region of the 9D3 antibody light chain is GACATCGTGATGACCCAGGCCGCCTTCAGCAACCCTGTGACCCTGGGCACCAGCGCCAGCATCAGCTGCAGAAGCAGCAAGAGCTGCCTGCACAGCAACGGCTACACCTACCTGTACTGGTACCTGCAGAAGCCTGGCCAGAGCCCTCAGCTGCTGATCTACATGTGCAGCAACCTGGCCAGCGGCGTGCCTGACAGATTCAGCAGCAGCGGCAGCGGCACCGACTTCACCCTGAGAATCAGCAGAGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGTGCCTGAGCCTGGCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG (SEQ ID NO.29); The DNA encoding the variable region of the 2D5 antibody heavy chain is GACGTGCAGCTGCAGGAGAGCGGCCCTGGCCTGGTGAAGCCTAGCCAGAGCCTGAGCCTGACCTGCACCGTGACCGGCTACTGCATCACCAGCAGCTACGCCTGGAACTGGATCAGACAGTTCCCTGGCAACAAGCTGGAGTGGATGGGCTACATCTGCACCTACGGCAACACCAACTACAACCCTAGCCTGAAGAGCAGAATCAGCATCACCAGAGACACCAGCAAGAACCAGTTCTTCCTGCAGCTGAACAGCGTGACCACCGAGGACACCGCCACCTACTACTGCGTGGCCGGCTGCAGCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC (SEQ ID NO.30); The DNA encoding the variable region of the 2D5 antibody light chain is GACGTGGTGCTGACCCAGACCCCTCTGACCCTGAGCGTGACCATCGGCCAGCCTGCCAGCATCAGCTGCAAGAGCAGCCAGTGCCTGCTGGACAGCGCCGGCTGCACCTACCTGAACTGGCTGCTGCAGAGACCTGGCCAGAGCCCTAAGAGACTGATCTACCTGTACTGCGACCTGGACAGCGGCGTGCCTGACAGATTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTACCAGTGCACCCACTTCAGCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO.31).

[0209] According to the sequences P01868 and P01837 of the mouse IgG1 heavy and light chain constant regions in Uniprot, DNA encoding the heavy chain constant region and light chain constant region of the antibody were designed respectively, and optimized to mammalian expression codons (mammalian expression codons can achieve antibody expression). DNA encoding the heavy chain constant region of the antibody was serially connected to the tail of the DNA encoding the heavy chain variable region of the 3B11 antibody to synthesize the DNA encoding the heavy chain of the 3B11 antibody, and DNA encoding the light chain constant region of the antibody was serially connected to the tail of the DNA encoding the light chain variable region of the 3B11 antibody to synthesize the DNA encoding the light chain of the 3B11 antibody. DNA encoding the heavy chain constant region of the antibody was serially connected to the tail of the DNA encoding the heavy chain variable region of the 9D3 antibody to synthesize the DNA encoding the heavy chain of the 9D3 antibody, and DNA encoding the light chain constant region of the antibody was serially connected to the tail of the DNA encoding the light chain variable region of the 9D3 antibody to synthesize the DNA encoding the light chain of the 9D3 antibody. DNA encoding the heavy chain variable region of the 2D5 antibody was tandemly added to the tail of the DNA encoding the heavy chain constant region of the antibody to synthesize the DNA encoding the heavy chain of the 2D5 antibody. DNA encoding the light chain variable region of the 2D5 antibody was tandemly added to the tail of the DNA encoding the light chain constant region of the antibody to synthesize the DNA encoding the light chain of the 2D5 antibody.

[0210] The DNA encoding the heavy chain of 3B11 antibody, the DNA encoding the light chain of 3B11 antibody, the DNA encoding the heavy chain of 9D3 antibody, the DNA encoding the light chain of 9D3 antibody, the DNA encoding the heavy chain of 2D5 antibody, and the DNA encoding the light chain of 2D5 antibody were respectively inserted into the pcDNA3.1 vector to obtain pcDNA3.1-3B11-H, pcDNA3.1-3B11-L, pcDNA3.1-9D3-H, pcDNA3.1-9D3-L, pcDNA3.1-2D5-H, and pcDNA3.1-2D5-L. The plasmids were then transformed into Escherichia coli DH5α competent cells, positive clones were picked and cultured in large quantities, and the recombinant plasmids were extracted using a high-purity plasmid extraction kit. The recombinant plasmids were mixed and transferred into CHO-S cells according to the heavy chain and light chain molar ratio of 1:2 (pcDNA3.1-3B11-H and pcDNA3.1-3B11-L as a group; pcDNA3.1-9D3-H and pcDNA3.1-9D3-L as a group; pcDNA3.1-2D5-H and pcDNA3.1-2D5-L3 as a group), and the pcDNA3.1 empty vector was transfected as a negative control at the same time. The transfected CHO-S were cultured in expiCHO medium at 37°C and 8% CO2 for 72h, the supernatant was collected, and the supernatant was filtered using a 0.22μm filter membrane to obtain a filtrate.

[0211] 3.2 Antibody purification:

[0212] Add ammonium sulfate solid (final concentration of 50%) to the filtrate collected in 3.1, stir evenly at 4°C, let stand for 1h, collect the precipitate at 12000rpm, and redissolve the precipitate with 50mM PBS solution. Load the crude purified antibody obtained onto the Protein A-Sepharose affinity chromatography column at a flow rate of 1mL / min, wash with 5 column volumes of binding buffer (50mM PBS pH7.0), and then elute the antibody with 0.1M glycine-hydrochloric acid solution pH3.0 (1M pH 9.0 Tris buffer was added to each collection tube in advance for neutralization) to obtain the target antibody, which was dialyzed 3 times with 50mM PBS solution. The dialyzed antibodies were subjected to 10% SDS-PAGE electrophoresis, and the results showed that all three antibodies had bands at 55kD and 25kD, and the grayscale analysis purity was greater than 90%. See attached Figure 3 .

[0213] 3.3 Potency detection:

[0214] 1 μg / mL carbonate buffer (pH 9.5) of eukaryotic recombinant cFGF23 protein was used to coat the ELISA plate at 4°C overnight in a volume of 100 μL. 100 μL of antibodies of different concentrations (0-100 ng / ml) were added, respectively, and incubated at 37°C for 60 min. After washing, 100 μL of HPR-labeled goat anti-mouse antibody with a concentration of 100 ng / mL was added, and incubated at 37°C for 60 min. After washing, TMB substrate and stop solution were added, and the absorbance at 450 nm was detected. The antibody concentration when the OD value was 1.0 was defined as the corresponding antibody titer. From the results, the titers of 9D3 antibody and 3B11 antibody were between 6.25-12.5 ng / mL, while the titer of 2D5 antibody was between 12.5-25 ng / mL. The results are shown in the attached Figure 4 .

[0215] Example 4: Biotin and Acridinium Ester Labeling of FGF23 Antibodies

[0216] 4.1 Biotin antibody labeling:

[0217] Take 2 mg of 3B11 and 9D3 antibodies respectively, stir and dialyze with 0.1 mol / L, pH 9.3 carbonate buffer at 2-8°C for 6-8 hours, and change the solution once in the middle; add biotin solution to the dialyzed antibody solution at a molar ratio of antibody to biotin of 1:20, mix well; oscillate slowly, and react at 37°C in the dark for 2 hours; add 60-80 μL of 3 mol / L ethanolamine solution, and react at room temperature in the dark for 30 minutes; stir and dialyze with 0.01 mol / L PBS solution at 2-8°C, change the solution every 5-6 hours, and change the solution 3-4 times in total; add an equal volume of glycerol to the dialyzed antibody solution, mix well, and dispense to a concentration of 0.5 mg / mL, and store at -20°C.

[0218] 4.2 Acridinium ester antibody labeling:

[0219] Take 50 μL of 5 mg / mL 2D5 antibody, add 150 μL of 0.05M pH 9.3 carbonate buffer, mix well, then add 1 μL of 1 mg / mL acridinium ester and mix well, react at room temperature in the dark, take out after 1-2 hours, and treat with a 50Kd ultrafiltration tube. During the ultrafiltration process, first treat with pure water and PBS buffer respectively, and finally add the obtained acridinium ester-labeled detection antibody solution, collect the liquid in the centrifuge tube into a storage tube, obtain the acridinium ester-labeled detection antibody solution, and store it at -20°C for later use.

[0220] Example 5: Comparison of single capture and mixed capture performance

[0221] 5.1cFGF23 Detection Kit

[0222] The kit includes the following ingredients:

[0223] Streptavidin-coupled magnetic beads, biotin-labeled capture antibodies (3B11 antibody, 9D3 antibody), acridinium ester-labeled detection antibodies (2D5 antibody), chemiluminescent excitation solution, chemiluminescent pre-excitation solution and washing concentrate are also included.

[0224] The chemiluminescence excitation liquid is hydrogen peroxide with a mass fraction of 0.5%; and the pre-excitation liquid is a 0.1 mol / L sodium hydroxide solution.

[0225] The washing concentrated solution is a Tris buffer solution containing Tween 20; the volume fraction of the Tween 20 is 1%, and the pH value of the washing concentrated solution is 7.4.

[0226] The particle size of the magnetic beads is 1 μm.

[0227] The final concentration of biotin-labeled capture antibody was 10 μg / mL.

[0228] The final concentration of the acridinium ester-labeled detection antibody was 1 μg / mL.

[0229] 5.2 Single capture and mixed capture performance comparison:

[0230] The present invention uses a fully automatic chemiluminescence instrument as a detection tool, and sequentially adds 75 μL of capture antibody A (biotin-labeled 3B11 antibody) or capture antibody B (biotin-labeled 9D3 antibody) or mixed capture antibody (wherein the mixed capture antibody ratio is biotin-labeled 3B11 antibody: biotin-labeled 9D3 antibody molar ratio=1:1) and 100 μL of calibrator (eukaryotic recombinant cFGF23 protein prepared in Example 1, with a concentration gradient of 0-6400 pg / mL) into the instrument for 10 minutes of reaction, and then adds 15 μL of streptavidin-coupled magnetic particles for magnetic separation, reacts for 5 minutes, dilutes the washing concentrate and washes 4 times, adds 75 μL of acridinium ester-labeled 2D5 antibody, reacts for 5 minutes, washes 4 times, sends the reaction complex into the darkroom of the instrument, sequentially adds 100 μL of chemiluminescence excitation solution and 100 μL of chemiluminescence pre-excitation solution for luminescence reaction, and finally records the luminescence intensity. The results show that the detection sensitivity of the calibration curve of the mixed capture is much higher than that of the single capture system (the detection sensitivity is increased by 4-20 times). Figure 5 .

[0231] Example 6. Calibration curve of cFGF23 magnetic microparticle system

[0232] 75 μL of mixed capture antibody (biotin-labeled 3B11 antibody: biotin-labeled 9D3 antibody molar ratio = 1:1) and 100 μL of calibration substance (eukaryotic recombinant cFGF23 protein prepared in Example 1) were added to the fully automatic chemiluminescence instrument in turn. After reacting for 10 minutes, 15 μL of streptavidin-coupled magnetic particles were added for magnetic separation. After reacting for 5 minutes and washing 4 times, 75 μL of acridinium ester-labeled 2D5 antibody was added. After reacting for 5 minutes, the reaction complex was washed 4 times. The reaction complex was sent to the darkroom of the instrument, and 100 μL of chemiluminescent excitation solution and 100 μL of chemiluminescent pre-excitation solution were added in turn for luminescent reaction. Finally, the luminescence intensity was recorded, and a calibration curve was made with the luminescence value and the concentration of the cFGF23 protein calibration substance. According to the luminescence value of the sample to be tested, the cFGF23 protein concentration of the sample to be tested can be calculated using the calibration curve. The linear range of the calibration curve is 12.5-6400 pg / mL, with Figure 6 It is a calibration curve of cFGF23 protein, wherein the Y-axis represents the logarithmic value of the luminescence value, and the X-axis represents the logarithmic value of the concentration of the cFGF23 protein calibrator.

[0233] The 12.5pg / mL eukaryotic recombinant FGF23 protein calibrator was further diluted with protein stabilizer II (purchased from Huzhou Yingchuang Biotechnology Co., Ltd.) to determine the lower limit of detection of the system. The results showed that it could still be stably detected when diluted to 6.25pg / mL. Therefore, the lower limit of detection of the FGF23 detection kit can reach 6.25pg / mL, see attached Figure 7 .

[0234] Example 7: Clinical performance verification of cFGF23 kit

[0235] cFGF23 detection kit is used for the diagnosis of osteoporosis samples: 50 plasma samples of osteoporosis patients and 50 samples of normal subjects were collected from the hospital. The cFGF23 detection kit was used to detect the concentration of cFGF23 in the plasma of osteoporosis patients and normal subjects. The sample concentration scatter plot shows that cFGF23 has statistical significance in distinguishing the test results of osteoporosis and normal subjects, see the attached Figure 8 The ROC curve statistical results showed that the area under the curve was 0.797. Taking 112 pg / mL as the detection reference value, the specificity of the cFGF23 detection kit was 88.2% and the sensitivity was 69.4%. Fig. 9 .

[0236] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment that specifically binds to cFGF23 protein, characterized in that: A: The heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.6, CDR H2 shown in SEQ ID NO.7, and CDR H3 shown in SEQ ID NO.8, and the light chain variable region includes CDRL1 shown in SEQ ID NO.10, CDR L2 with sequence IAS, and CDR L3 shown in SEQ ID NO.11; Or C: The heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.20, CDR H2 shown in SEQ ID NO.21, and CDR H3 shown in SEQ ID NO.22, and the light chain variable region includes CDR L1 shown in SEQ ID NO.24, CDR L2 with sequence LYC, and CDR L3 shown in SEQ ID NO.

25.

2. The antibody or antigen-binding fragment that specifically binds to cFGF23 protein according to claim 1, characterized in that: It is characterized in that include: A: heavy chain variable region shown in SEQ ID NO.5 and light chain variable region shown in SEQ ID NO.9; or C: the heavy chain variable region shown in SEQ ID NO.19 and the light chain variable region shown in SEQ ID NO.

23.

3. A hybridoma cell line secreting the antibody or antigen-binding fragment that specifically binds to the cFGF23 protein according to claim 1 or 2.

4. The hybridoma cell line according to claim 3, characterized in that The hybridoma cell line secreting antibody A is named 3B11, and the hybridoma cell line secreting antibody C is named 2D5. The deposit number of 3B11 is CCTCC NO: C202513, and the classification name is hybridoma cell line 3B11; the deposit number of 2D5 is CCTCC NO: C202514; and the classification name is hybridoma cell line 2D5.

5. A nucleic acid encoding the antibody or antigen-binding fragment of claim 1 or 2.

6. A vector comprising the nucleic acid according to claim 5.

7. A host cell containing the vector according to claim 6.

8. A capture reagent for cFGF23 protein, characterized in that: The capture reagent comprises at least one selected from the following antibodies or antigen-binding fragments: (1) The antibody or antigen-binding fragment as described in A of claim 1, (2) The antibody or antigen-binding fragment as described in C of claim 1, (3) The heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.13, CDR H2 shown in SEQ ID NO.14, and CDR H3 shown in SEQ ID NO.15, and the light chain variable region includes CDR L1 shown in SEQ ID NO.17, CDR L2 with the sequence of MCS, and CDR L3 shown in SEQ ID NO.18; (4) The antibody or antigen-binding fragment as described in A of claim 2; (5) The antibody or antigen-binding fragment as described in C of claim 2; (6) The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

16.

9. A detection reagent for cFGF23 protein, characterized in that: The detection reagent comprises at least one selected from the following antibodies or antigen-binding fragments: (1) The antibody or antigen-binding fragment as described in A of claim 1, (2) The antibody or antigen-binding fragment as described in C of claim 1, (3) The heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.13, CDR H2 shown in SEQ ID NO.14, and CDR H3 shown in SEQ ID NO.15, and the light chain variable region includes CDR L1 shown in SEQ ID NO.17, CDR L2 with the sequence of MCS, and CDR L3 shown in SEQ ID NO.18; (4) The antibody or antigen-binding fragment as described in A of claim 2; (5) The antibody or antigen-binding fragment as described in C of claim 2; (6) The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

16.

10. Use of the antibody or antigen-binding fragment of claim 1 or 2, the hybridoma cell line of claim 3 or 4, the nucleic acid of claim 5, the vector of claim 6, the host cell of claim 7, the capture reagent of claim 8, or the detection reagent of claim 9 in any of the following aspects: (1) preparing a cFGF23 protein detection kit; (2) Prepare an osteoporosis auxiliary diagnostic kit.

11. A cFGF23 protein detection kit, characterized in that: The kit comprises: a capture antibody and a detection antibody; The capture antibody and the detection antibody are both selected from at least one of ①-③, and the capture antibody is different from the detection antibody; Wherein: ① the antibody or antigen-binding fragment as shown in A of claim 1 or 2, ② The antibody or antigen-binding fragment as described in C of claim 1 or 2, ③ The heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.13, CDR H2 shown in SEQ ID NO.14, and CDR H3 shown in SEQ ID NO.15, and the light chain variable region includes CDR L1 shown in SEQ ID NO.17, CDR L2 with a sequence of MCS, and CDR L3 shown in SEQ ID NO.18; or, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is as shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

16.

12. Osteoporosis auxiliary diagnosis system, including: (1) a cFGF23 protein detection module, which uses the antibody or antigen-binding fragment of claim 1 or 2, the cFGF23 protein detection reagent of claim 9, or the cFGF23 protein detection kit of claim 11 to detect the cFGF23 protein concentration; (2) A result output module, which assists in diagnosing osteoporosis based on the concentration of cFGF23 protein.

13. A computer-readable storage medium, characterized in that: The computer storage medium is used to store computer instructions, programs, code sets or instruction sets, which, when run on a computer, enable the computer to execute the functions corresponding to the cFGF23 protein detection module and result output module in the osteoporosis auxiliary diagnosis system of claim 12.

14. An electronic device, characterized in that: include: one or more processors; And a computer-readable storage medium, which is used to store computer instructions, programs, code sets or instruction sets, which, when run on a computer, enable the one or more processors to implement the functions corresponding to the cFGF23 protein detection module and the result output module in the osteoporosis auxiliary diagnosis system according to claim 12.

15. A method for determining the concentration of cFGF23 protein, characterized in that: The FGF23 protein concentration is determined using the antibody or antigen binding fragment described in claim 1 or 2, the cFGF23 protein detection reagent described in claim 9, or the cFGF23 protein detection kit described in claim 11.

16. The measuring method according to claim 15, characterized in that The determination method is a chemiluminescence method, comprising: (1) establishing a calibration curve using a cFGF23 protein calibrator; the cFGF23 protein sequence is shown in SEQ ID NO.4; (2) Using the antibody or antigen binding fragment, the cFGF23 protein detection reagent or the cFGF23 protein detection kit to determine the luminescence value of the sample to be tested, and calculating the cFGF23 protein concentration of the sample to be tested based on the luminescence value of the sample to be tested using the calibration curve of (1).

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