IFGF23 specific antibody and application thereof in preparation of chronic kidney disease auxiliary diagnostic reagent

Abstract

The invention relates to an iFGF23 specific antibody and application thereof in preparation of a chronic kidney disease auxiliary diagnosis reagent, and belongs to the technical field of disease auxiliary diagnosis. The specific antibody disclosed by the invention can be used for respectively recognizing an N terminal (25-179) and a C terminal (180-251) of the FGF23 protein, and can be used as a combination of a capture antibody and a detection antibody, so that the problem that the sensitivity is reduced due to site competition in a double-antibody sandwich is avoided, and the sensitivity is enhanced. In a word, the antibody provided by the invention can be used for realizing accurate determination of the iFGF23 concentration in a human body and can be used for auxiliary diagnosis of chronic kidney diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease auxiliary diagnosis, and relates to iFGF23-specific antibodies and applications thereof in the preparation of chronic kidney disease auxiliary diagnosis reagents. Background Art

[0002] Chronic kidney disease (CKD) is a disease that is extremely harmful to human health. In 2002, it was compiled by the American Kidney Foundation and modified and confirmed by the Kidney Disease Improving Global Outcomes (KDIGO). CKD is defined as renal structural and functional disorders caused by various reasons for more than 3 months, including normal and abnormal pathological damage to glomerular filtration rate (GFR), abnormal blood or urine components or abnormal renal imaging examinations, or unexplained GFR decrease (﹤60ml / min.1.73m2) for more than 3 months. With the development of the economy and the aging of the population, the incidence of hypertension, diabetes and other kidney diseases has gradually increased, the incidence of CKD will continue to grow, and more and more CKD patients will progress to end-stage renal disease (ESRD). About 200 million patients are diagnosed with chronic kidney disease worldwide.

[0003] Patients with chronic kidney disease will gradually lose kidney function over months or years. Moreover, this disease has no symptoms in the early stage, and then symptoms such as leg edema, fatigue, vomiting or loss of appetite begin to appear. During this period, heart disease, hypertension, bone disease or anemia may also occur, endangering the patient's life. Almost all CKD patients have varying degrees of calcium and phosphorus metabolism disorders. Long-term calcium and phosphorus metabolism disorders can cause hyperparathyroidism, abnormal mineral and bone metabolism, cardiovascular events, etc., which seriously affect the progression and prognosis of CKD. Clinically, it is recommended to screen people at risk, including patients with diabetes, hypertension, glomerulonephritis, polycystic kidney disease and those with a family history. In the past, active vitamin D and parathyroid hormone (PTH) were generally believed to be the most important regulators of calcium and phosphorus metabolism. In recent years, with the discovery of FGF23, new perspectives have been provided for this field.

[0004] 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 with a relative molecular mass of 26kDa. FGF23 is a powerful calcium-phosphorus regulatory factor that can regulate the balance of blood phosphorus and vitamin D in the body. 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).

[0005] It is currently believed that elevated iFGF23 is an independent risk factor for progression to end-stage renal disease and death in CKD patients. In patients with chronic kidney disease (CKD), the increase in iFGF23 levels precedes the increase in blood phosphorus and parathyroid hormone, indicating that changes in iFGF23 occur in the early stages of CKD. In patients with confirmed chronic kidney disease, higher levels of iFGF23 are associated with an increased risk of progressive renal function loss. In addition, in the general population, higher levels of iFGF23 are associated with poor chronic kidney disease prognosis. Therefore, early monitoring of iFGF23 levels is of great significance for delaying the progression of chronic kidney disease and improving patient prognosis. However, the level of iFGF23 in the plasma of patients with early kidney disease is low, so it is urgent to develop a highly sensitive iFGF23 detection system, and the main problem that needs to be solved is the acquisition of high-affinity paired antibodies. At present, there are two main problems in the preparation technology of iFGF23 antibodies. (1) iFGF23 protein has a high homology among species as an immunogen, and it is not easy to obtain high-affinity antibodies through immunization, especially antibodies that recognize the N-terminus of its sequence; (2) Since the N-terminal structure of the iFGF23 protein is relatively complex, the epitope of the antibody recognition segment is a conformational epitope, so it is difficult to obtain highly specific antibodies. Summary of the invention

[0006] In order to detect iFGF23 protein with high specificity and sensitivity, the present invention provides two monoclonal antibodies that recognize FGF23 protein, provides a tool for auxiliary diagnosis of diseases related to iFGF23 protein, and further provides the use of the antibody or kit in auxiliary diagnosis of chronic kidney disease.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A: The present invention provides an antibody or antigen-binding fragment that specifically binds to FGF23 protein, wherein the recognition epitope or binding epitope of the antibody or antigen-binding fragment is the 180-251 segment of the FGF23 protein (the sequence is shown in SEQ ID NO: 2 [SAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHA GGTGPEGCRPFAKFI], and the antibody or antigen-binding fragment comprises:

[0009] The heavy chain variable region CDRs of at least one of the following:

[0010] (1-1) comprising CDR H1 shown in SEQ ID NO.5; or comprising CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.5;

[0011] (2-1) comprising CDR H2 shown in SEQ ID NO.6; or comprising CDR H2 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.6;

[0012] (3-1) comprising CDR H3 shown in SEQ ID NO.7; or comprising CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.7.

[0013] As an embodiment, the antibody or antigen-binding fragment comprises:

[0014] The heavy chain variable region CDRs of at least one of the following:

[0015] (4-1) CDRH1 shown in SEQ ID NO.5; or CDR H1 having an amino acid sequence with an identity of 90% or more to that shown in SEQ ID NO.5;

[0016] (5-1) CDRH2 shown in SEQ ID NO.6; or CDR H2 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.6;

[0017] (6-1) CDRH3 shown in SEQ ID NO.7; or CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.7.

[0018] The present invention provides an antibody or antigen-binding fragment that specifically binds to FGF23 protein, wherein the antibody or antigen-binding fragment comprises:

[0019] The CDRs of the light chain variable region of at least one of the following:

[0020] (7-1) comprising CDR L1 shown in SEQ ID NO.9; or comprising CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.9;

[0021] (8-1) 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;

[0022] (9-1) comprising CDR L3 shown in SEQ ID NO. 10; or comprising CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 10.

[0023] As an embodiment, the antibody or antigen-binding fragment comprises:

[0024] The CDRs of the light chain variable region of at least one of the following:

[0025] (10-1) CDR L1 shown in SEQ ID NO.9; or CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.9;

[0026] (11-1) CDR L2 having an amino acid sequence of MCS; or a CDR L2 having an amino acid sequence of MCS having an identity of 90% or more;

[0027] (12-1) CDR L3 shown in SEQ ID NO. 10; or CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 10.

[0028] 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).

[0029] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes CDRH1 shown in SEQ ID NO.5, CDR H2 shown in SEQ ID NO.6, and CDR H3 shown in SEQ ID NO.7, and the light chain variable region includes CDR L1 shown in SEQID NO.9, CDR L2 with the sequence of MCS, and CDR L3 shown in SEQ ID NO.10.

[0030] The present invention provides an antibody or antigen-binding fragment that specifically binds to FGF23 protein, wherein the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0031] It comprises the amino acid sequence shown in SEQ ID NO.4; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.4.

[0032] As an embodiment, the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0033] The amino acid sequence shown in SEQ ID NO.4; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.4.

[0034] The present invention provides an antibody or antigen-binding fragment that specifically binds to FGF23 protein, wherein the antibody or antigen-binding fragment comprises the following light chain variable region:

[0035] It comprises the amino acid sequence shown in SEQ ID NO.8; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.8.

[0036] As an embodiment, the antibody or antigen-binding fragment comprises the following light chain variable region:

[0037] The amino acid sequence shown in SEQ ID NO.8; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.8.

[0038] As an embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO.4 and a light chain variable region shown in SEQ ID NO.8.

[0039] Alternatively, B: The present invention provides an antibody or antigen-binding fragment that specifically binds to FGF23 protein, wherein the antibody recognition epitope or binding epitope of the antibody or antigen-binding fragment is segment 25-179 of FGF23 protein (the sequence is shown in SEQ ID NO: 1 [YPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDA GFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGR AKRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRRHTR], and the antibody or antigen-binding fragment comprises:

[0040] The heavy chain variable region CDRs of at least one of the following:

[0041] (1-2) comprising CDR H1 shown in SEQ ID NO. 12; or comprising CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 12;

[0042] (2-2) comprising CDR H2 of SEQ ID NO. 13; or comprising CDR H2 having 90% or more identity with the amino acid sequence of SEQ ID NO. 13;

[0043] (3-2) comprising CDR H3 shown in SEQ ID NO. 14; or comprising CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 14.

[0044] In some embodiments, the antibody or antigen-binding fragment comprises:

[0045] The heavy chain variable region CDRs of at least one of the following:

[0046] (4-2) CDRH1 shown in SEQ ID NO.12; or CDR H1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.12;

[0047] (5-2) CDRH2 shown in SEQ ID NO.13; or CDR H2 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.13;

[0048] (6-2) CDRH3 shown in SEQ ID NO.14; or CDR H3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.14.

[0049] The present invention provides an antibody or antigen-binding fragment that specifically binds to FGF23 protein, wherein the antibody or antigen-binding fragment comprises:

[0050] The CDRs of the light chain variable region of at least one of the following:

[0051] (7-2) comprising CDR L1 shown in SEQ ID NO.16; or comprising CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.16;

[0052] (8-2) comprising a CDR L2 having a sequence of CYS; or comprising a CDR L2 having an amino acid sequence having a sequence of CYS that is 90% or more identical;

[0053] (9-2) comprising CDR L3 shown in SEQ ID NO. 17; or comprising CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 17.

[0054] In some embodiments, the antibody or antigen-binding fragment comprises:

[0055] The CDRs of the light chain variable region of at least one of the following:

[0056] (10-2) CDR L1 shown in SEQ ID NO.16; or CDR L1 having 90% or more identity with the amino acid sequence shown in SEQ ID NO.16;

[0057] (11-2) CDR L2 having a sequence of CYS; or a CDR L2 having an amino acid sequence of CYS that is more than 90% identical;

[0058] (12-2) CDR L3 shown in SEQ ID NO. 17; or CDR L3 having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 17.

[0059] 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).

[0060] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.12, CDR H2 shown in SEQ ID NO.13, and CDR H3 shown in SEQ ID NO.14, and the light chain variable region includes CDR L1 shown in SEQ ID NO.16, CDR L2 with sequence CYS, and CDR L3 shown in SEQ ID NO.17.

[0061] The present invention provides an antibody or antigen-binding fragment that specifically binds to FGF23 protein, wherein the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0062] The amino acid sequence of SEQ ID NO.11 is included; or the amino acid sequence of SEQ ID NO.11 is included which has 90% or more identity with the amino acid sequence of SEQ ID NO.11.

[0063] As an embodiment, the antibody or antigen-binding fragment comprises the following heavy chain variable region:

[0064] The amino acid sequence shown in SEQ ID NO.11; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.11.

[0065] The present invention provides an antibody or antigen-binding fragment that specifically binds to FGF23 protein, wherein the antibody or antigen-binding fragment comprises the following light chain variable region:

[0066] It comprises the amino acid sequence shown in SEQ ID NO.15; or it comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.15.

[0067] As an embodiment, the antibody or antigen-binding fragment comprises the following light chain variable region:

[0068] The amino acid sequence shown in SEQ ID NO.15; or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO.15.

[0069] As an embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO.11 and a light chain variable region shown in SEQ ID NO.15.

[0070] 2. The present invention also provides a hybridoma cell line that secretes antibodies or antigen-binding fragments that specifically bind to FGF23 protein. The hybridoma cell line that secretes antibody A or antigen-binding fragment is named 9D3, and the hybridoma cell line that secretes antibody B or antigen-binding fragment is named 1N23.

[0071] 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;

[0072] Hybridoma cell line 1N23 is deposited in China Center for Type Culture Collection, with deposit number CCTCC NO: C202515, deposit date December 19, 2024, deposit address: Wuhan University, Wuhan, China, classification name: Hybridoma cell line 1N23.

[0073] 3. The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment.

[0074] In a specific embodiment, the DNA encoding the heavy chain variable region of the 9D3 antibody is shown as SEQ ID NO.18, the DNA encoding the light chain variable region of the 9D3 antibody is shown as SEQ ID NO.19, the DNA encoding the heavy chain variable region of the 1N23 antibody is shown as SEQ ID NO.20, and the DNA encoding the light chain variable region of the 1N23 antibody is shown as SEQ ID NO.21.

[0075] 4. The present invention also provides a vector containing the above nucleic acid.

[0076] In a specific embodiment, the vector is a pcDNA3.1 vector.

[0077] 6. The present invention provides a host cell containing the above-mentioned vector.

[0078] In a specific embodiment, the host cell is an Escherichia coli DH5α competent cell, a 293F cell or a CHO-S cell.

[0079] 7. The present invention provides a capture reagent for iFGF23 protein, wherein the capture reagent comprises at least one of the antibodies or antigen-binding fragments shown in A or B above.

[0080] 8. The present invention further provides a detection reagent for iFGF23 protein, which comprises at least one of the antibodies or antigen-binding fragments shown in A or B above.

[0081] As an embodiment, when used to detect iFGF23 protein, the detection reagent comprises an antibody or antigen-binding fragment in A, and an antibody or antigen-binding fragment in B, so as to simultaneously recognize the N-terminus and C-terminus of the iFGF23 protein.

[0082] 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:

[0083] (1) preparing an iFGF23 protein detection kit;

[0084] (2) Preparation of a diagnostic kit for chronic kidney disease;

[0085] (3) Assist in the diagnosis of chronic kidney disease.

[0086] 10. The present invention provides an iFGF23 protein detection kit, the kit comprising: a capture antibody and a detection antibody;

[0087] (1) The capture antibody is selected from at least one of the antibodies or antigen-binding fragments shown in A above, and the detection antibody is selected from at least one of the antibodies or antigen-binding fragments shown in B above;

[0088] Or (2) the capture antibody is selected from at least one of the antibodies or antigen-binding fragments shown in B above, and the detection antibody is selected from at least one of the antibodies or antigen-binding fragments shown in A above.

[0089] When the capture antibody is selected from an antibody or antigen-binding fragment described in A, the detection antibody is selected from an antibody or antigen-binding fragment in B; or, when the capture antibody is selected from an antibody or antigen-binding fragment in B, the detection antibody is selected from an antibody or antigen-binding fragment in A, which enables the capture antibody and the detection antibody to recognize the N-terminus and C-terminus of the iFGF23 protein, and the detection sensitivity is high.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] The particle size of the magnetic beads is 0.3-2 μm, and the amount of the magnetic beads is 15-100 μL.

[0094] The final concentration of the biotin-labeled capture antibody is 2-75 μg / mL.

[0095] The final concentration of the acridinium ester-labeled detection antibody is 0.2-7.5 μg / mL.

[0096] 11. The present invention provides a chronic kidney disease auxiliary diagnosis system, comprising:

[0097] (1) an iFGF23 protein detection module, which uses the antibody or antigen-binding fragment, the iFGF23 protein detection reagent or the iFGF23 protein detection kit to detect the iFGF23 protein concentration;

[0098] (2) A result output module, which assists in diagnosing chronic kidney disease based on the iFGF23 protein concentration.

[0099] 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, it enables the computer to execute the functions corresponding to the iFGF23 protein detection module and the result output module in the chronic kidney disease auxiliary diagnosis system.

[0100] 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 iFGF23 protein detection module and the result output module in the chronic kidney disease auxiliary diagnosis system.

[0101] 14. The present invention provides a method for auxiliary diagnosis of chronic kidney disease, comprising: using the antibody or antigen binding fragment, the detection reagent of iFGF23 protein, or the iFGF23 protein detection kit to detect the concentration of iFGF23 protein, and then assisting in diagnosing whether chronic kidney disease exists based on the concentration of iFGF23 protein.

[0102] 15. The present invention provides a method for determining the concentration of iFGF23 protein, using the antibody or antigen-binding fragment, the iFGF23 protein detection reagent or the iFGF23 protein detection kit to determine the concentration of iFGF23 protein.

[0103] In one embodiment, the determination method is a chemiluminescence method, comprising the following steps:

[0104] (1) establishing a calibration curve using an FGF23 protein calibrator; the FGF23 protein sequence is shown in SEQ ID NO.3;

[0105] (2) Using the antibody or antigen binding fragment, the iFGF23 protein detection reagent or the iFGF23 protein detection kit to measure the luminescence value of the sample to be tested, and calculating the iFGF23 protein concentration in the sample to be tested based on the luminescence value of the sample to be tested using the calibration curve of (1).

[0106] Definition of terms:

[0107] "Antigen-binding fragment" refers to all proteins / protein fragments containing the CDR region, including Fab, F(ab')2, Fd, Fv, ScFv, etc.

[0108] "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.

[0109] "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.

[0110] The beneficial effects of the present invention are:

[0111] (1) The two monoclonal antibodies that recognize FGF23 protein in the present invention can both specifically recognize FGF23 eukaryotic protein, and recognize the N-terminus (25-179) and C-terminus (180-251) of the FGF23 protein, respectively.

[0112] (2) When detecting iFGF23 protein, the two monoclonal antibodies in the present invention that recognize the N-terminus and C-terminus of the FGF23 protein are used in combination as capture antibody and detection antibody, respectively, avoiding the problem of decreased sensitivity caused by site competition in the double antibody sandwich and enhancing sensitivity.

[0113] (3) The 1N23 antibody of the present invention can specifically recognize the conformational epitope at the N-terminus of the FGF23 protein, thereby avoiding missed detection caused by incomplete capture of the iFGF23 protein in serum by conventional linear epitope antibodies, and achieving higher detection sensitivity for the iFGF23 protein.

[0114] (4) The chemiluminescent diagnostic kit involved in the present invention has high sensitivity, a wide detection linear range and a lower limit, and the detection limit can be as low as 1pg / mL, which can accurately determine the concentration of iFGF23 in serum samples of early chronic kidney disease and healthy people. Small sample clinical sample verification shows that the specificity of the iFGF23 detection kit is 88% and the sensitivity is 78%.

[0115] 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

[0116] 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:

[0117] Figure 1 This is the SDS-PAGE electrophoresis diagram of FGF23 protein. In the figure, M represents protein marker, R represents protein under reducing conditions, and NR represents protein under non-reducing conditions.

[0118] Figure 2 These are the results of epitope validation for the 1N23 and 9D3 antibodies.

[0119] Figure 3 This is the SDS-PAGE electrophoresis diagram of FGF23 antibody. In the figure, M represents protein marker, 9D3 and 1N23 represent 9D3 antibody and 1N23 antibody respectively.

[0120] Figure 4 For titer detection and specificity analysis of FGF23 antibodies.

[0121] Figure 5 This is the validation result of the recognition epitope of FGF23 antibody.

[0122] Figure 6 It is the calibration curve of the FGF23 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 FGF23 calibrator.

[0123] Figure 7 To establish the detection limit of FGF23 kit.

[0124] Figure 8 This is a scatter plot of the test results of the FGF23 test kit for chronic kidney disease and normal people.

[0125] Fig. 9 ROC analysis results of FGF23 kit for detecting chronic kidney disease and normal human samples. DETAILED DESCRIPTION

[0126] 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.

[0127] Experimental methods for which specific conditions are not specified in the examples are generally performed under conventional conditions, such as those described in Molecular Cloning Laboratory Manual (3rd edition, by J. Sambrook et al.), or under conditions recommended by manufacturers.

[0128] Example 1. Recombinant expression and purification of FGF23 protein

[0129] 1.1 Recombinant expression of FGF23 protein

[0130] 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.3 (YPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGF VVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRAK RAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRRHTRSAEDDSERDPLNVLKPRARMTPAPAS CSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI). DNA encoding the above amino acids was synthesized (see SEQ ID NO.22).

[0131] (TACCCCAACGCCTCCCCCCTGCTGGGCTCCTCCTGGGGCGGCCTGATCCACCTGTACA )), which has been optimized for mammalian expression codons. The DNA shown in SEQ ID NO.22 was inserted into the pcDNA3.1 vector containing a 6×His tag to obtain pcDNA3.1-FGF23. Then pcDNA3.1-FGF23 was transformed into Escherichia coli DH5α competent cells, positive clones were picked and cultured in large quantities, and the recombinant plasmid pcDNA3.1-FGF23 was 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 plasmid was transferred into 293F cells, and the pcDNA3.1 empty vector was transfected at the same time as a negative control. The transfected 293F cells were cultured in expi293 medium at 37°C and 8% CO2 for 72 h, the supernatant was collected, and the supernatant was filtered using a 0.22 μm filter membrane to obtain a filtrate.

[0132] 1.2 Protein purification

[0133] 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 (pH 7.4) buffer at -80°C. The purified protein was subjected to SDS-PAGE electrophoresis purity identification, 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 is 50mM PBS, 10mM imidazole, 150mM NaCl, pH 7.6, and the composition of the elution buffer is 50mM PBS, 250mM imidazole, 150mM NaCl, pH 7.6. The theoretical molecular weight of FGF23 protein is about 25kD.

[0134] Example 2: Preparation of FGF23 mouse monoclonal antibody and confirmation of conformational epitope

[0135] 2.1 Preparation of mouse monoclonal antibodies

[0136] The eukaryotic recombinant FGF23 protein purified in Example 1 was used as an antigen 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 first immunization FGF23 antigen was mixed with Freund's complete adjuvant in a 1:1 ratio, and then FGF23 was mixed with Freund's incomplete adjuvant in a 1:1 ratio. Immunization was performed once every two weeks for a total of 3 times. Blood was collected by tail cutting ten days after the third immunization to detect serum titer. After hybridoma cell fusion and monoclonal screening, cloned cell strains were obtained, and antibody strains that were reactive with eukaryotic recombinant FGF23 protein and non-reactive with denatured FGF23 protein were screened to finally obtain 1N23 cell strains. Antibody strains that were reactive with eukaryotic recombinant FGF23 protein and reactive with FGF23 C-terminus (180-251 segment) were screened to finally obtain 9D3 cell strains.

[0137] The 9D3 cell line and 1N23 cell line were respectively sent to the China Center for Type Culture Collection for preservation, and the preservation numbers are:

[0138] Hybridoma cell line 9D3, deposit number is CCTCC NO: C202502;

[0139] The hybridoma cell line 1N23 has a deposit number of CCTCC NO: C202515.

[0140] 2.2 Validation of epitope recognition

[0141] The supernatants of the 9D3 cell line and 1N23 cell line were collected respectively, and the 9D3 antibody and 1N23 antibody were purified.

[0142] The chemiluminescent plate was coated with 1 μg / mL carbonate buffer (pH 9.5) containing eukaryotic recombinant FGF23 protein and high-temperature denatured eukaryotic recombinant FGF23 protein, respectively, in a volume of 100 μL at 4°C overnight, and gradiently diluted 9D3 antibody (concentration of 0-1 μg / mL) and gradiently diluted 1N23 antibody (concentration of 0-1 μg / mL) were added, respectively, and then goat anti-mouse IgG-HRP (100 ng / mL) was added, and the luminescence value was detected.

[0143] The results showed that the 9D3 antibody reacted with both denatured and undenatured eukaryotic recombinant FGF23 proteins, while the 1N23 antibody had a reactive activity with undenatured eukaryotic recombinant FGF23 proteins but had no reaction with heat-denatured eukaryotic recombinant FGF23 proteins, indicating that the 9D3 antibody recognized the linear epitope of the FGF23 protein, while the 1N23 antibody recognized the conformational epitope of the FGF23 protein. The results are shown in the attached Figure 2 .

[0144] 2.3 Antibody Sequencing

[0145] The monoclonal cell lines 9D3 and 1N23 were sequenced (after obtaining the cDNA of the hybridoma cell lines, the primers were designed using existing technology to amplify the sequences, and the base composition of the sequences was obtained by sequencing) to obtain the corresponding antibody variable region sequences, and the sequenced sequences were classified by IMGT to obtain the CDR region sequences.

[0146] The results are as follows: The amino acid sequence of the heavy chain variable region of the 9D3 antibody is

[0147] QVQLQQPGVDLVRPGASVKLSCKASGYCFTAYWMNWVKQRPGQGLEWIGMIHPADYETRLNQKFKDKATLTVDKTSSTAYMQLSSPTSEDSAVYYCARFSYSVYCTMSYWGQGTSVTVSS (SEQ ID NO.4); the heavy chain variable region of the 9D3 antibody includes: CDR H1: GYCFTAYW (SEQ ID NO.5); CDRH2: IHPADYET (SEQ ID NO.6); CDR H3: ARFSYSVYCTMSY (SEQ ID NO.7).

[0148] The amino acid sequence of the light chain variable region of the 9D3 antibody is

[0149] DIVMTQAAFSNPVTLGTSASISCRSSKSCLHSNGYTYLYWYLQKPGQSPQLLIYMCSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQCLSLALTFGAGTKLELK (SEQ ID NO.8); The light chain variable region of the 9D3 antibody includes: CDR L1: KSCLHSNGYTY (SEQ ID NO.9); CDR L2: MCS; CDR L3: AQCLSLALT (SEQ ID NO. 10).

[0150] The amino acid sequence of the heavy chain variable region of the 1N23 antibody is DVHLQESGPGLVKPSQSLSLICTVTGHAITCSFAWNWIRQFPGSKLQWMGYIRYCGTTTYNPSLKSRISITRDTSENQFFLHLHSVTTEDTATYYCARWAYIGCSPSYWGQGTTLTVSS (SEQ ID NO.11); the heavy chain variable region of the 1N23 antibody includes: CDR H1: GHAITCSFA (SEQ ID NO.12); CDR H2: IRYCGTT (SEQ ID NO.13); CDR H3: ARWAYIGCSPSY (SEQ ID NO.14).

[0151] The amino acid sequence of the light chain variable region of the 1N23 antibody is

[0152] DIKMTQSPSSMYASLGERVTITCKASQSINCYLSWFQQKPGKSPQTLIYCYSRMIDGVPSRFSGSGSGQDYSLTISSLEYEDLGIYYCLQSDEFAYMFGGGTKLEIK (SEQ ID NO.15): The light chain variable region of the 1N23 antibody includes: CDR L1: QSINCY (SEQ ID NO.16); CDR L2: CYS; CDR L3: LQSDEFAYM (SEQ ID NO. 17).

[0153] Example 3: Recombinant expression and purification of FGF23 antibody

[0154] 3.1 Recombinant expression of antibodies

[0155] DNA encoding the heavy chain (H chain) variable region and light chain (L chain) variable region was designed according to the variable region sequences of 9D3 and 1N23 antibodies, and optimized to mammalian expression codons. Among them, the DNA encoding the heavy chain variable region of 9D3 antibody is CAGGTGCAGCTGCAGCAGCCTGGCGTGGACCTGGTGAGACCTGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACTGCTTCACCGCCTACTGGATGAACTGGGTGAAGCAGAGACCTGGCCAGGGCCTGGAGTGGATCGGCATGATCCACCCTGCCGACTACGAGACCAGACTGAACCAGAAGTTCAAGGACAAGGCCACCCTGACCGTGGACAAGACCAGCAGCACCGCCTACATGCAGCTGAGCAGCCCTACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATTCAGCTACAGCGTGTACTGCACCATGAGCTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC (SEQ ID NO.18); The DNA encoding the variable region of the light chain of the 9D3 antibody is GACATCGGTGATGACCCAGGCCGCCTTCAGCAACCCTGTGACCCTGGGCACCAGCGCCAGCATCAGCTGCAGAAGCAGCAAGAGCTGCCTGCACAGCAACGGCTACACCTACCTGTACTGGTACCTGCAGAAGCCTGGCCAGAGCCCTCAGCTGCTGAT CTACATGTGCAGCAACCTGGCCAGCGGCGTGCCTGACAGATTCAGCAGCAGCGGCAGCGGCACCGACTTCACCCTGAGAATCAGCAGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGTGCCTGAGCCTGGCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG(SEQ ID NO.19); The DNA encoding the variable region of the heavy chain of the 1N23 antibody is GACGTGCACCTGCAGGAGAGCGGCCCTGGCCTGGTGAAGCCTAGCCAGAGCCTGAGCCTGATCTGCACCGTGACCGGCCACGCCATCACCTGCAGCTTCGCCTGGAACTGGATCAGACAGTTCCCTGGCAGCAAGCTGCAGTGGATGGGCTACATCAGATACTGCGGCACCACCACCTACAACCCTAGCCTGAAGAGCAGAATCAGCATCACCAGAGACACCAGCGAGAACCAGTTCTTCCTGCACCTGCACAGCGTGACCACCGAGGACACCGCCACCTACTACTGCGCCAGATGGGCCTACATCGGCTGCAGCCCTAGCTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC (SEQ ID NO.20); The DNA encoding the variable region of the light chain of the 1N23 antibody is GACATCAAGATGACCCAGAGCCCTAGCAGCATGTACGCCAGCCTGGGCGAGAGAGTGACCATCACCTGCAAGGCCAGCCAGAGCATCAACTGCTACCTGAGCTGGTTCCAGCAGAAGCCTGGCAAGAGCCCTCAGACCCTGATCTACTGCTACAGCAGAATGATCGACGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCCAGGACTACAGCCTGACCATCAGCAGCCTGGAGTACGAGGACCTGGGCATCTACTACTGCCTGCAGAGCGACGAGTTCGCCTACATGTTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO.21).

[0156] 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 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 constant region of the antibody was serially connected to the tail of the DNA encoding the heavy chain variable region of the 1N23 antibody to synthesize the DNA encoding the heavy chain of the 1N23 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 1N23 antibody to synthesize the DNA encoding the light chain of the 1N23 antibody.

[0157] The DNA encoding the heavy chain of the 9D3 antibody, the DNA encoding the light chain of the 9D3 antibody, the DNA encoding the heavy chain of the 1N23 antibody, and the DNA encoding the light chain of the 1N23 antibody were respectively inserted into the pcDNA3.1 vector to obtain pcDNA3.1-9D3-H, pcDNA3.1-9D3-L, pcDNA3.1-1N23-H, and pcDNA3.1-1N23-L, respectively. 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 (pcDNA3.1-9D3-H and pcDNA3.1-9D3-L as one group; pcDNA3.1-1N23-H and pcDNA3.1-1N23-L as one group), and the pcDNA3.1 empty vector was transfected at the same time as a negative control. The transfected CHO-S cells were cultured in expiCHO medium at 37°C and 8% CO2 for 72 h, the supernatant was collected, and the supernatant was filtered using a 0.22 μm filter membrane to obtain a filtrate.

[0158] 3.2 Antibody purification

[0159] Add ammonium sulfate solid (final concentration is 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 onto the ProteinA-Sepharose affinity chromatography column at a flow rate of 1mL / min, wash with 5 column volumes of binding buffer (50mMPBS 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 antibody was subjected to 10% SDS-PAGE electrophoresis, and the results showed that both antibodies (9D3, 1N23) had bands at 55kD and 25kD, and the grayscale analysis purity was greater than 90%. See attached Figure 3 .

[0160] Example 4. FGF23 antibody titer detection and specificity analysis

[0161] 4.1 Potency detection and specificity analysis

[0162] 1 μg / mL of carbonate buffer (pH 9.5) containing recombinant FGF19 (purchased from Beijing Sino Biological Technology Co., Ltd.), FGF21 (purchased from Beijing Sino Biological Technology Co., Ltd.), and eukaryotic recombinant FGF23 protein (Example 1, the sequence is shown in SEQ ID NO.3) was respectively coated on the ELISA plate at 4°C overnight in a volume of 100 μL, and 100 μL of antibodies of different concentrations (0-100 ng / mL) were added respectively, incubated at 37°C for 60 min, and 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 titer of 9D3 antibody is between 6.25 and 12.5 ng / mL, while that of 1N23 antibody is between 25 and 50 ng / mL; at the same time, both 9D3 antibody and 1N23 antibody can specifically recognize FGF23 protein without cross-reaction with FGF19 and FGF21, and can be used to construct FGF23 specific detection system. Figure 4 .

[0163] 4.2 Antibody epitope verification

[0164] The chemiluminescent plate was coated with 1 μg / mL of carbonate buffer (pH 9.5) of recombinant FGF23-N truncated protein (25-179) (purchased from Jiangsu Dongkang Biopharmaceutical Technology Co., Ltd.), FGF23-C truncated protein (180-251) (purchased from Jiangsu Dongkang Biopharmaceutical Technology Co., Ltd.), and eukaryotic recombinant FGF23 protein (25-251, the sequence is shown in SEQ ID NO.3, Example 1) at 4°C overnight, and gradient dilutions of 9D3 antibody and 1N23 antibody (concentration of 0-1 μg / mL) were added respectively, and goat anti-mouse IgG-HRP (100 ng / mL) was added, and the luminescence value was detected.

[0165] The results showed that the 9D3 antibody reacted with the eukaryotic recombinant FGF23 protein and the FGF23-C truncated protein but not with the FGF23-N truncated protein, and the 1N23 antibody reacted with the eukaryotic recombinant FGF23 protein and the FGF23-N truncated protein but not with the FGF23-C truncated protein, indicating that the 9D3 antibody recognized the C-terminus of the FGF23 protein, while the 1N23 antibody recognized the N-terminus of the FGF23 protein. The results are shown in the attached Figure 5 .

[0166] Example 5: Biotin and Acridinium Ester Labeling of FGF23 Antibodies

[0167] 5.1 Biotinylated Antibodies

[0168] Take 2 mg of 9D3 antibody, 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 molecule of 1:20, mix well; oscillate slowly, react at 37°C in the dark for 2 hours; add 60-80 μL of 3 mol / L ethanolamine solution, 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, aliquot, the concentration is 0.5 mg / mL, and store at -20°C.

[0169] 5.2 Acridinium ester labeled antibodies:

[0170] Take 50 μL of 5 mg / mL 1N23 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.

[0171] Example 6. Establishment of iFGF23 magnetic microparticle chemiluminescence detection system

[0172] 6.1FGF23 Detection Kit

[0173] The kit includes the following components: streptavidin-coupled magnetic beads, biotin-labeled capture antibody (9D3 antibody), acridinium ester-labeled detection antibody (1N23 antibody), chemiluminescent excitation solution, chemiluminescent pre-excitation solution and washing concentrate.

[0174] 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.

[0175] 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.

[0176] The particle size of the magnetic beads is 1 μm.

[0177] The final concentration of biotin-labeled capture antibody was 10 μg / mL.

[0178] The final concentration of the acridinium ester-labeled detection antibody was 1 μg / mL.

[0179] 6.2 Calibration curve establishment

[0180] The present invention uses a fully automatic chemiluminometer as a detection tool, and adds 75 μL of biotin-labeled 9D3 antibody and 100 μL of FGF23 protein calibrator (eukaryotic recombinant FGF23 protein, Example 1) to the instrument in sequence. After reacting for 10 minutes, 15 μL of streptavidin-coupled magnetic particles are added for magnetic separation, reacting for 5 minutes, and the washing concentrate is diluted and washed 4 times. 75 μL of acridinium ester-labeled 1N23 antibody is added, and after reacting for 5 minutes, it is washed 4 times, and the reaction complex is sent to the darkroom of the instrument, and 100 μL of chemiluminescent excitation solution and 100 μL of chemiluminescent pre-excitation solution are added in sequence for luminescent reaction. Finally, the luminescence intensity is recorded, and a calibration curve is made with the luminescence value and the concentration of the FGF23 protein calibrator. According to the luminescence value of the sample to be tested, the iFGF23 protein concentration of the sample to be tested can be calculated using the calibration curve. The linear range of the calibration curve is 5 to 5000 pg / mL, and the attached Figure 6 It is a calibration curve of FGF23 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 FGF23 protein calibrator.

[0181] The 5pg / mL 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 1pg / mL. Therefore, the lower limit of detection of the FGF23 detection kit can reach 1pg / mL, see attached Figure 7 .

[0182] Example 7: Clinical performance verification of FGF23 detection kit

[0183] FGF23 detection kit is used for auxiliary diagnosis of chronic kidney disease samples: 50 plasma samples of chronic kidney disease patients and 50 samples of normal subjects were collected from the hospital. FGF23 detection kit was used to detect the concentration of FGF23 in the plasma of chronic kidney disease patients and normal subjects.

[0184] The sample concentration scatter plot shows that FGF23 has statistical significance in distinguishing the test results of chronic kidney disease and normal people, see the attached Figure 8 The ROC curve statistical results showed that the area under the curve was 0.806. Taking 32pg / mL as the detection reference value, the specificity of the FGF23 detection kit was 88% and the sensitivity was 78%. See attached Fig. 9 .

[0185] In addition, the clinical detection performance of the kit obtained by using 1N23 antibody as the capture antibody and 9D3 antibody as the detection antibody is basically the same.

[0186] 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 FGF23 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.5, CDR H2 shown in SEQ ID NO.6, and CDRH3 shown in SEQ ID NO.7, and the light chain variable region includes CDRL1 shown in SEQ ID NO.9, CDR L2 with the sequence of MCS, and CDR L3 shown in SEQ ID NO.10; Or B: The heavy chain variable region of the antibody or antigen-binding fragment includes CDR H1 shown in SEQ ID NO.12, CDR H2 shown in SEQ ID NO.13, and CDR H3 shown in SEQ ID NO.14, and the light chain variable region includes CDR L1 shown in SEQ ID NO.16, CDR L2 with sequence CYS, and CDR L3 shown in SEQ ID NO.

17.

2. The antibody or antigen-binding fragment that specifically binds to FGF23 protein according to claim 1, characterized in that: The antibody or antigen-binding fragment comprises: A: heavy chain variable region shown in SEQ ID NO.4 and light chain variable region shown in SEQ ID NO.8; or B: the heavy chain variable region shown in SEQ ID NO.11 and the light chain variable region shown in SEQ ID NO.

15.

3. A hybridoma cell line that secretes the antibody or antigen-binding fragment that specifically binds to the FGF23 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 or antigen-binding fragment is named 9D3, and the hybridoma cell line secreting antibody B or antigen-binding fragment is named 1N23. The deposit numbers of hybridoma cell line 9D3 are CCTCC NO: C202502, and the classification name is hybridoma cell line 9D3; the deposit number of hybridoma cell line 1N23 is CCTCC NO: C202515, and the classification name is hybridoma cell line 1N23.

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 iFGF23 protein, characterized in that: The capture reagent comprises at least one selected from the antibodies or antigen-binding fragments shown in A or B in any one of claims 1-2.

9. A detection reagent for iFGF23 protein, characterized in that: The detection reagent comprises at least one selected from the antibodies or antigen-binding fragments shown in A or B in any one of claims 1-2.

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 an iFGF23 protein detection kit; (2) Prepare a chronic kidney disease auxiliary diagnosis kit.

11. iFGF23 protein detection kit, characterized in that, The kit comprises: a capture antibody and a detection antibody; (1) The capture antibody is selected from at least one of the antibodies or antigen-binding fragments shown in A of claim 1 or 2, and the detection antibody is selected from at least one of the antibodies or antigen-binding fragments shown in B of claim 1 or 2; Or (2) the capture antibody is selected from at least one of the antibodies or antigen-binding fragments shown in B of claim 1 or 2, and the detection antibody is selected from at least one of the antibodies or antigen-binding fragments shown in A of claim 1 or 2.

12. Chronic kidney disease auxiliary diagnosis system, including: (1) an iFGF23 protein detection module, which uses the antibody or antigen-binding fragment of claim 1 or 2, the iFGF23 protein detection reagent of claim 9, or the iFGF23 protein detection kit of claim 11 to detect the iFGF23 protein concentration; (2) A result output module, which assists in diagnosing chronic kidney disease based on the iFGF23 protein concentration.

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 iFGF23 protein detection module and the result output module in the chronic kidney disease auxiliary diagnosis system according to 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 iFGF23 protein detection module and the result output module in the chronic kidney disease auxiliary diagnosis system according to claim 12.

15. A method for determining the concentration of iFGF23 protein, characterized in that: The iFGF23 protein concentration is determined using the antibody or antigen binding fragment described in claim 1 or 2, the iFGF23 protein detection reagent described in claim 9, or the iFGF23 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 an FGF23 protein calibrator; the FGF23 protein sequence is shown in SEQ ID NO.3; (2) Using the antibody or antigen binding fragment, the iFGF23 protein detection reagent or the iFGF23 protein detection kit to measure the luminescence value of the sample to be tested, and calculating the iFGF23 protein concentration in 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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