iFGF23 specific antibody and its application in preparing auxiliary diagnostic reagent for chronic kidney disease

Abstract

The present application relates to iFGF23 specific antibodies and their use in the preparation of chronic kidney disease auxiliary diagnostic reagents, belonging to the technical field of disease auxiliary diagnosis. The specific antibodies in the present application can recognize the N terminal (25-179) and C terminal (180-251) of FGF23 protein respectively, which can be used as a combination of capture antibodies and detection antibodies, avoiding the problem of sensitivity decline caused by site competition in double antibody sandwich, and enhancing the sensitivity. In summary, the antibodies of the present application can be used to realize the accurate determination of iFGF23 concentration in human body, and can be used for auxiliary diagnosis of chronic kidney disease.

Description

Technical Field

[0001] This invention belongs to the field of auxiliary diagnostic technology for diseases, and relates to iFGF23 specific antibodies and their application in the preparation of auxiliary diagnostic reagents for chronic kidney disease. Background Technology

[0002] Chronic kidney disease (CKD) is a disease that poses a significant threat to human health. Developed in 2002 by the Kidney Disease Foundation and revised and confirmed by the Kidney Disease Improving Global Outcomes (KDIGO), CKD is defined as structural and functional impairment of the kidneys lasting more than 3 months due to various causes. This includes pathological damage with normal or abnormal glomerular filtration rate (GFR), abnormal blood or urine components, abnormal renal imaging findings, or an unexplained decrease in GFR (<60 ml / min / 1.73 m²) for more than 3 months. With economic development and population aging, the incidence of hypertension, diabetes, and other kidney diseases is gradually increasing, and the incidence of CKD will continue to rise. More and more CKD patients will progress to end-stage renal disease (ESRD). Approximately 200 million people worldwide are diagnosed with chronic kidney disease.

[0003] In patients with chronic kidney disease (CKD), kidney function gradually deteriorates over months or years. The disease is often asymptomatic in its early stages, later developing symptoms such as leg edema, fatigue, vomiting, or loss of appetite. Complications may also include heart disease, hypertension, bone disease, or anemia, threatening the patient's life. Almost all CKD patients exhibit varying degrees of calcium and phosphorus metabolism disorders. Long-term calcium and phosphorus metabolism disorders can lead to hyperparathyroidism, mineral and bone metabolism abnormalities, and cardiovascular events, severely impacting the progression and prognosis of CKD. Clinically, screening at-risk individuals is recommended, including those with diabetes, hypertension, glomerulonephritis, polycystic kidney disease, and those with a family history of CKD. Previously, active vitamin D and parathyroid hormone (PTH) were generally considered the most important regulators of calcium and phosphorus metabolism; however, the recent discovery of FGF23 has provided new insights into this field.

[0004] Fibroblast growth factor 23 (FGF23) is a member of the fibroblast growth factor (FGFs) family, primarily secreted by osteoblasts and osteocytes. The FGF23 gene is located on human chromosome 12, containing three exons and two introns, with a relative molecular mass of 26 kDa. FGF23 is a potent calcium and phosphorus regulator, capable of regulating the balance of phosphorus and vitamin D in the body. FGF23 exists in three forms in the blood: full-length FGF23 polypeptide (iFGF23), C-terminal FGF23 polypeptide (cFGF23), and N-terminal FGF23P polypeptide (nFGF23).

[0005] Currently, elevated iFGF23 is considered an independent risk factor for progression to end-stage renal disease and death in CKD patients. In CKD patients, elevated iFGF23 levels precede increases in serum phosphorus and parathyroid hormone, indicating that changes in iFGF23 occur early in CKD. In diagnosed CKD patients, higher levels of iFGF23 are associated with an increased risk of progressive renal function loss. Furthermore, in the general population, higher levels of iFGF23 are associated with poor prognosis in CKD. Therefore, early monitoring of iFGF23 levels is crucial for delaying the progression of CKD and improving patient outcomes. However, plasma iFGF23 levels are low in patients with early-stage renal disease, necessitating the development of highly sensitive iFGF23 detection systems. A key challenge is obtaining high-affinity paired antibodies. Currently, there are two main problems with iFGF23 antibody preparation technology. (1) The iFGF23 protein has high homology among species as an immunogen, making it difficult to obtain high-affinity antibodies, especially those that recognize the N-terminus of its sequence; (2) Due to the complex three-dimensional structure of the N-terminus of the iFGF23 protein, the antibody recognition epitope is a conformational epitope, making it difficult to obtain highly specific antibodies. Summary of the Invention

[0006] In order to perform highly specific and sensitive detection of iFGF23 protein, the present invention provides two monoclonal antibodies that recognize FGF23 protein, providing a tool for the auxiliary diagnosis of diseases related to iFGF23 protein, and further providing the application of the antibody or kit in the auxiliary diagnosis of chronic kidney disease.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. A: This 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 region of the FGF23 protein (sequence shown in SEQ ID NO:2 [SAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHA GGTGPEGCRPFAKFI]), and the antibody or antigen-binding fragment comprises:

[0009] CDR of heavy chain variable regions of at least one of the following:

[0010] (1-1) Contains CDR H1 as shown in SEQ ID NO.5; or contains CDR H1 with more than 90% identity to the amino acid sequence shown in SEQ ID NO.5;

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

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

[0013] As one implementation, the antibody or antigen-binding fragment includes:

[0014] CDR of heavy chain variable regions of at least one of the following:

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

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

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

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

[0019] CDR of at least one of the following light chain variable regions:

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

[0021] (8-1) Contains a CDR L2 with the MCS sequence; or contains a CDR L2 with more than 90% identity to an amino acid sequence with the MCS sequence;

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

[0023] As one implementation, the antibody or antigen-binding fragment includes:

[0024] CDR of at least one of the following light chain variable regions:

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

[0026] (11-1) CDR L2 with the MCS sequence; or CDR L2 with more than 90% identity to the amino acid sequence with the MCS sequence;

[0027] (12-1) CDR L3 shown in SEQ ID NO.10; or CDR L3 having more than 90% 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 (1-1) to (6-1) above, and the light chain variable region includes at least one of (7-1) to (12-1) above.

[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 SEQ ID NO.9, CDR L2 with the sequence MCS, and CDR L3 shown in SEQ ID NO.10.

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

[0031] It contains the amino acid sequence shown in SEQ ID NO.4; or contains an amino acid sequence that is more than 90% identical to the amino acid sequence shown in SEQ ID NO.4.

[0032] As one implementation, the antibody or antigen-binding fragment includes the following heavy chain variable region:

[0033] The amino acid sequence shown in SEQ ID NO.4; or an amino acid sequence that is more than 90% identical to the amino acid sequence shown in SEQ ID NO.4.

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

[0035] It contains the amino acid sequence shown in SEQ ID NO.8; or contains an amino acid sequence that is more than 90% identical to the amino acid sequence shown in SEQ ID NO.8.

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

[0037] The amino acid sequence shown in SEQ ID NO.8; or an amino acid sequence that is more than 90% identical to the amino acid sequence shown in SEQ ID NO.8.

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

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

[0040] CDR of heavy chain variable regions of at least one of the following:

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

[0042] (2-2) Contains CDR H2 as shown in SEQ ID NO.13; or contains CDR H2 with more than 90% identity to the amino acid sequence shown in SEQ ID NO.13;

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

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

[0045] CDR of heavy chain variable regions of at least one of the following:

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

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

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

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

[0050] CDR of at least one of the following light chain variable regions:

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

[0052] (8-2) Contains a CDR L2 with the sequence CYS; or contains a CDR L2 with more than 90% identity to an amino acid sequence with the sequence CYS;

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

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

[0055] CDR of at least one of the following light chain variable regions:

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

[0057] (11-2) CDR L2 with the sequence CYS; or CDR L2 with more than 90% identity with the amino acid sequence of CYS;

[0058] (12-2) CDR L3 shown in SEQ ID NO.17; or CDR L3 having more than 90% 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 (1-2) to (6-2) above, and the light chain variable region includes at least one of (7-2) to (12-2) above.

[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 the sequence CYS, and CDR L3 shown in SEQ ID NO.17.

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

[0062] It contains the amino acid sequence shown in SEQ ID NO.11; or contains an amino acid sequence that is more than 90% identical to the amino acid sequence shown in SEQ ID NO.11.

[0063] As one implementation, the antibody or antigen-binding fragment includes the following heavy chain variable region:

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

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

[0066] It contains the amino acid sequence shown in SEQ ID NO.15; or contains an amino acid sequence that is more than 90% identical to the amino acid sequence shown in SEQ ID NO.15.

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

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

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

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

[0071] Hybridoma cell line 9D3 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: C202502, deposit date December 19, 2024, deposit address: Wuhan University, Wuhan, China, classification and name: Hybridoma cell line 9D3.

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

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

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

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

[0076] In one 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 one specific embodiment, the host cell is Escherichia coli DH5α competent cells, 293F cells, or CHO-S cells.

[0079] 7. The present invention provides a capture reagent for the iFGF23 protein. The capture reagent comprises at least one antibody or antigen-binding fragment selected from those shown in A or B above.

[0080] 8. The present invention further provides a detection reagent for iFGF23 protein. The detection reagent comprises at least one antibody or antigen-binding fragment selected from those shown in A or B above.

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

[0082] 9. The present invention provides the application 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) Preparation of iFGF23 protein detection kit;

[0084] (2) Preparation of a reagent kit for auxiliary diagnosis of chronic kidney disease;

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

[0086] 10. This 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 one of the antibodies or antigen-binding fragments described in A, the detection antibody is selected from one of the antibodies or antigen-binding fragments described in B; or, when the capture antibody is selected from one of the antibodies or antigen-binding fragments described in B, the detection antibody is selected from one of the antibodies or antigen-binding fragments described in A. This enables the capture antibody and the detection antibody to recognize the N-terminus and C-terminus of the iFGF23 protein, resulting in high detection sensitivity.

[0090] Specifically, the kit is a chemiluminescence diagnostic kit, which includes a biotin-labeled capture antibody, an acridinium-labeled detection antibody, streptavidin-conjugated magnetic beads, an activation solution, a pre-activation solution, and a washing concentrate.

[0091] The activating solution is hydrogen peroxide with a mass fraction of 0.1-1%; the pre-activating solution is sodium hydroxide solution with a mass fraction of 0.05-1 mol / L.

[0092] The washing concentrate is a Tris buffer solution containing Tween 20; the volume fraction of Tween 20 is 0.5-3%, and the pH of the washing concentrate is 7.0-7.8.

[0093] The magnetic beads have a particle size of 0.3-2μm and a dosage of 15-100μL.

[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. This invention provides an auxiliary diagnostic system for chronic kidney disease, comprising:

[0097] (1) 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) Results output module, which assists in the diagnosis of chronic kidney disease based on the concentration of iFGF23 protein.

[0099] 12. The present invention provides a computer-readable storage medium for storing computer instructions, programs, code sets or instruction sets, which, when run on a computer, cause the computer to perform the functions corresponding to the iFGF23 protein detection module and the result output module in the chronic kidney disease auxiliary diagnostic system.

[0100] 13. The present invention provides an electronic device, comprising: one or more processors; and a computer-readable storage medium for storing computer instructions, programs, code sets or instruction sets, which, when executed on a computer, cause the one or more processors to perform the functions corresponding to the iFGF23 protein detection module and result output module in the chronic kidney disease auxiliary diagnostic system.

[0101] 14. The present invention provides an auxiliary diagnostic method for chronic kidney disease, comprising: using the antibody or antigen-binding fragment, the iFGF23 protein detection reagent, or the iFGF23 protein detection kit to detect the concentration of iFGF23 protein, and then using the iFGF23 protein concentration to assist in the diagnosis of whether or not chronic kidney disease is present.

[0102] 15. This invention provides a method for determining the concentration of iFGF23 protein, which uses 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) A calibration curve was established using the FGF23 protein calibrator; the FGF23 protein sequence is shown in SEQ ID NO.3;

[0105] (2) The fluorescence value of the sample to be tested is determined by the antibody or antigen binding fragment, the detection reagent of iFGF23 protein, or the iFGF23 protein detection kit. Based on the fluorescence value of the sample to be tested, the concentration of iFGF23 protein in the sample to be tested is calculated using the calibration curve of (1).

[0106] Definition of noun:

[0107] "Antigen-binding fragments" refer to all proteins / protein fragments containing CDR regions, 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 this invention, CDR H1, CDR H2, and CDRH3 represent three CDRs of the heavy chain variable region, and CDR L1, CDR L2, and CDR L3 represent three CDRs of the light chain variable region.

[0109] "Identity" refers to the sequence identity between two amino acid sequences or two nucleic acid sequences. A identity of 90% or more indicates an identity such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, etc.

[0110] The beneficial effects of this invention are as follows:

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

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

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

[0114] (4) The chemiluminescence diagnostic kit involved in this invention has high sensitivity, a wide detection linear range, and a lower detection limit, with the lowest detection limit reaching 1 pg / mL. It can accurately determine the concentration of iFGF23 in serum samples from early-stage chronic kidney disease and healthy individuals. Small-sample clinical validation showed that the iFGF23 detection kit had a specificity of 88% and a sensitivity of 78%.

[0115] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0116] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

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

[0118] Figure 2 The results validate the recognition epitopes of the 1N23 and 9D3 antibodies.

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

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

[0121] Figure 5 The results validate the recognition epitope of the FGF23 antibody.

[0122] Figure 6 The calibration curve for the FGF23 detection kit is shown, where the Y-axis represents the logarithmic value of the OD value and the X-axis represents the logarithmic value of the concentration of the FGF23 calibrator.

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

[0124] Figure 8 This is a scatter plot showing the results of the FGF23 kit in detecting chronic kidney disease and healthy individuals.

[0125] Figure 9 ROC analysis results for FGF23 kit detection of samples from chronic kidney disease and healthy individuals. Detailed Implementation

[0126] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0127] Experimental methods not specified in the examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (3rd Edition, by J. Sambrook et al.), or as recommended by the manufacturer.

[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 human FGF23 protein (amino acids 25-251 of the full-length FGF23) is shown in SEQ ID NO. 3 (YPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGF VVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRAK RAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRRHTRSAEDDSERDPLNVLKPRARMTPAPAS CSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI). DNA encoding the above amino acids was synthesized (as shown in SEQ ID NO. 22).

[0131] (as shown in TACCCCAACGCCTCCCCCCTGCTGGGCTCCTCCTGGGGCGGCCTGATCCACCTGTACA) This DNA 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 E. coli DH5α competent cells. Positive clones were picked and cultured in large quantities. The recombinant plasmid pcDNA3.1-FGF23 was then extracted using a high-purity plasmid extraction kit (Tiangen Biotech (Beijing) Co., Ltd., Endotoxin-Free Plasmid Large-Scale Extraction Kit (Enhanced) (Catalog No. DP120)). The recombinant plasmid was transferred into 293F cells, and the empty pcDNA3.1 vector was simultaneously transfected 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 filtered through a 0.22 μm filter membrane to obtain the filtrate.

[0132] 1.2 Protein purification

[0133] Take 30 mL of the filtrate obtained in step 1.1 and perform Ni-NTA affinity chromatography under non-denaturing conditions. Equilibrate the column with equilibration buffer; then load the sample; after loading, wash the column with equilibration buffer; elute with elution buffer and collect the eluent. Concentrate the protein solution (i.e., the eluent) using a 3 kDa ultrafiltration tube and store the protein in 50 mM PBS (pH 7.4) buffer at -80°C. The purified protein was analyzed for purity by SDS-PAGE electrophoresis. The results showed that it was consistent with the theoretical size, and grayscale analysis indicated that the protein purity reached over 90%. Figure 1 The equilibration buffer consisted of 50 mM PBS, 10 mM imidazole, and 150 mM NaCl, pH 7.6, while the elution buffer consisted of 50 mM PBS, 250 mM imidazole, and 150 mM NaCl, pH 7.6. The theoretical molecular weight of FGF23 protein is approximately 25 kDa.

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

[0135] 2.1 Preparation of mouse monoclonal antibodies

[0136] Three batches of BALB / c mice (6-8 weeks old, female) were immunized with the eukaryotic recombinant FGF23 protein purified in Example 1 as antigen, three mice per immunization. Each mouse received 50 μg of antigen per immunization. For the first immunization, the FGF23 antigen was mixed 1:1 with Freund's complete adjuvant, followed by a 1:1 mixture of FGF23 and Freund's incomplete adjuvant, immunized every two weeks for a total of three immunizations. Ten days after the third immunization, blood was collected by tail amputation to detect serum titer. Cloned cell lines were obtained through hybridoma cell fusion and monoclonal screening. Antibody lines that were reactive to eukaryotic recombinant FGF23 protein but not to denatured FGF23 protein were then screened, ultimately yielding the 1N23 cell line. Antibody lines that were reactive to both eukaryotic recombinant FGF23 protein and the FGF23 C-terminus (180-251 region) were then screened, ultimately yielding the 9D3 cell line.

[0137] The 9D3 and 1N23 cell lines were deposited at the China Center for Type Culture Collection, with accession numbers as follows:

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

[0139] Hybridoma cell line 1N23, accession number CCTCC NO:C202515.

[0140] 2.2 Identification and Verification of Epitopes

[0141] The supernatants of the 9D3 and 1N23 cell lines were collected and purified to obtain 9D3 and 1N23 antibodies, respectively.

[0142] Chemiluminescent plates were coated with 100 μL of carbonate buffer (pH 9.5) containing 1 μg / mL eukaryotic recombinant FGF23 protein and high-temperature denatured eukaryotic recombinant FGF23 protein, respectively, and incubated overnight at 4°C. Serially diluted 9D3 antibody (concentration 0–1 μg / mL) and serially diluted 1N23 antibody (concentration 0–1 μg / mL) were added, respectively. 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 protein, while the 1N23 antibody was reactive with undenatured eukaryotic recombinant FGF23 protein but not with heat-denatured eukaryotic recombinant FGF23 protein. This indicates that the 9D3 antibody recognizes the linear epitope of FGF23 protein, while the 1N23 antibody recognizes the conformational epitope of FGF23 protein. See attached results. 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, primers were designed using existing technology to amplify the sequences, and the base composition of the sequences was obtained through sequencing) to obtain the corresponding antibody variable region sequences. The sequencing sequences were then classified using 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 as follows:

[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 as follows:

[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 as follows:

[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 Antibody Recombinant Expression

[0155] DNA encoding the heavy chain (H chain) and light chain (L chain) variable regions was designed based on the variable region sequences of the 9D3 and 1N23 antibodies, and optimized for mammalian expression. Specifically, the DNA encoding the heavy chain variable region of the 9D3 antibody is: CAGGTGCAGCTGCAGCAGCCTGGCGTGGACCTGGTGAGACCTGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACTGCTTCACCGCCTACTGGATGAACTGGGTGAAGCAGAGACCTGGCCAGGGCCTGGAGTGGATCGGCATGATCCACCCTGCCGACTACGAGACCAGACTGAACCAGAAGTTCAAGGACAAGGCCACCCTGACCGTGGACAAGACCAGCAGCACCGCCTACATGCAGCTGAGCAGCCCTACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATTCAGCTACAGCGTGTACTGCACCATGAGCTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC (SEQ ID). NO.18); The DNA encoding the variable region of the light chain of the 9D3 antibody is GACATCGTGATGACCCAGGCCGCCTTCAGCAACCCTGTGACCCTGGGCACCAGCGCCAGCATCAGCTGCAGAAGCAGCAAGAGCTGCCTGCACAGCAACGGCTACACCTACCTGTACTGGTACCTGCAGAAGCCTGGCCAGAGCCCTCAGCTGCTGAT 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] DNA sequences encoding the antibody heavy and light chain constant regions (P01868 and P01837) of the mouse IgG1 heavy and light chains, respectively, were designed and optimized for mammalian expression codons (antibody expression can be achieved by using mammalian expression codons). DNA encoding the antibody heavy chain constant region was tandemly encoded at the tail of the DNA encoding the 9D3 antibody heavy chain variable region to synthesize DNA. Similarly, DNA encoding the 1N23 antibody light chain was synthesized by tandemly encoding the antibody heavy chain constant region at the tail of the DNA encoding the 1N23 antibody heavy chain variable region.

[0157] DNA encoding the 9D3 antibody heavy chain, the 9D3 antibody light chain, the 1N23 antibody heavy chain, and the 1N23 antibody light chain were inserted into the pcDNA3.1 vector, yielding pcDNA3.1-9D3-H, pcDNA3.1-9D3-L, pcDNA3.1-1N23-H, and pcDNA3.1-1N23-L, respectively. The plasmids were then transformed into *E. coli* DH5α competent cells. Positive clones were selected and cultured in large quantities. Recombinant plasmids were then extracted using a high-purity plasmid extraction kit. Recombinant plasmids were mixed at a heavy to light chain molar ratio of 1:2 (pcDNA3.1-9D3-H and pcDNA3.1-9D3-L were used as one group; pcDNA3.1-1N23-H and pcDNA3.1-1N23-L were used as another group) and transfected into CHO-S cells. Simultaneously, empty pcDNA3.1 vector was transfected 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 filtered through a 0.22 μm filter membrane to obtain the filtrate.

[0158] 3.2 Antibody purification

[0159] The filtrate collected in step 3.1 was added to ammonium sulfate solid (final concentration 50%), stirred thoroughly at 4°C, and allowed to stand for 1 hour. The precipitate was collected at 12,000 rpm and redissolved with 50 mM PBS solution. The obtained crude purified antibody was loaded into a Protein A-Sepharose affinity chromatography column at a flow rate of 1 mL / min, washed with 5 column volumes of binding buffer (50 mM PBS pH 7.0), and then eluted with 0.1 M glycine-hydrochloric acid solution pH 3.0 (each collection tube was pre-neutralized with 1 M pH 9.0 Tris buffer) to obtain the target antibody. The antibody was dialyzed three times with 50 mM PBS solution. The dialyzed antibody was subjected to 10% SDS-PAGE electrophoresis. The results showed that both antibodies (9D3 and 1N23) had bands at 55 kDa and 25 kDa, and the purity of both was greater than 90% according to grayscale analysis. See Appendix. Figure 3 .

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

[0161] 4.1 Potency detection and specificity analysis

[0162] ELISA plates were coated overnight at 4°C with 100 μL of carbonate buffer (pH 9.5) containing 1 μg / mL of recombinant FGF19 (purchased from Beijing E.S. Biotechnology Co., Ltd.), FGF21 (purchased from Beijing E.S. Biotechnology Co., Ltd.), and eukaryotic recombinant FGF23 protein (Example 1, sequence shown in SEQ ID NO.3). 100 μL of antibodies at different concentrations (0–100 ng / mL) were added, and the plates were incubated at 37°C for 60 min. After washing, 100 μL of HPR-labeled goat anti-mouse antibody at a concentration of 100 ng / mL was added, and the plates were incubated at 37°C for 60 min. After washing, TMB substrate and stop solution were added, and the absorbance at 450 nm was measured. The antibody concentration at which the OD value was 1.0 was defined as the corresponding antibody titer. The results showed that the titers of 9D3 antibody ranged from 6.25 to 12.5 ng / mL, while those of 1N23 antibody ranged from 25 to 50 ng / mL. Both 9D3 and 1N23 antibodies specifically recognized FGF23 protein without cross-reactivity with FGF19 and FGF21, making them suitable for constructing specific detection systems for FGF23. See attached results. Figure 4 .

[0163] 4.2 Antibody Epitope Validation

[0164] Chemiluminescent plates were coated with 100 μL of carbonate buffer (pH 9.5) containing 1 μg / mL of recombinant FGF23-N truncated protein (25-179) (purchased from Jiangsu Dongkang Biomedical Technology Co., Ltd.), FGF23-C truncated protein (180-251) (purchased from Jiangsu Dongkang Biomedical Technology Co., Ltd.), and eukaryotic recombinant FGF23 protein (25-251, sequence as shown in SEQ ID NO.3, Example 1) incubated overnight at 4°C. Serially diluted 9D3 antibody and 1N23 antibody (concentrations of 0–1 μg / mL) were added, along with goat anti-mouse IgG-HRP (100 ng / mL), and the luminescence values ​​were detected.

[0165] The results showed that the 9D3 antibody reacted with eukaryotic recombinant FGF23 protein and FGF23-C truncated protein, but not with FGF23-N truncated protein. Similarly, 1N23 reacted with eukaryotic recombinant FGF23 protein and FGF23-N truncated protein, but not with FGF23-C truncated protein. This indicates that the 9D3 antibody recognizes the C-terminus of the FGF23 protein, while 1N23 recognizes the N-terminus. See attached results. Figure 5 .

[0166] Example 5: Biotin and acridine ester labeling of FGF23 antibody

[0167] 5.1 Biotin-labeled antibody

[0168] Take 2 mg of 9D3 antibody and dialyze it with 0.1 mol / L, pH 9.3 carbonate buffer at 2–8 °C with stirring for 6–8 hours, changing the buffer once in between. Add biotin solution to the dialyzed antibody solution at a molar ratio of antibody to biotin of 1:20 and mix well. Shake 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. Dialyze with 0.01 mol / L PBS solution at 2–8 °C with stirring, changing the buffer every 5–6 hours, for a total of 3–4 times. Add an equal volume of glycerol to the dialyzed antibody solution, mix well, aliquot, and store at -20 °C to a concentration of 0.5 mg / mL.

[0169] 5.2 Acridinium ester-labeled antibody:

[0170] Take 50 μL of 5 mg / mL 1N23 antibody, add 150 μL of 0.05 M pH 9.3 carbonate buffer, mix well, then add 1 μL of 1 mg / mL acridine ester and mix well. Incubate at room temperature in the dark. After 1-2 hours, remove the solution and process it with a 50 kDa ultrafiltration tube. During ultrafiltration, first treat with purified water and PBS buffer, respectively. Finally, add the obtained acridine ester-labeled detection antibody solution, collect the liquid in the centrifuge tube into a storage tube, and obtain the acridine ester-labeled detection antibody solution. Aliquot and store at -20℃ for later use.

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

[0172] 6.1 FGF23 Detection Kit

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

[0174] The chemiluminescent excitation solution is 0.5% hydrogen peroxide by mass; the pre-excitation solution is a 0.1 mol / L sodium hydroxide solution.

[0175] The washing concentrate is a Tris buffer containing Tween 20; the volume fraction of Tween 20 is 1%, and the pH of the washing concentrate is 7.4.

[0176] The magnetic beads have a particle size of 1 μm.

[0177] The final concentration of the 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] This invention uses a fully automated chemiluminescence analyzer as the detection tool. 75 μL of biotin-labeled 9D3 antibody and 100 μL of FGF23 protein calibrator (eukaryotic recombinant FGF23 protein, Example 1) are added sequentially to the instrument. After reacting for 10 min, 15 μL of streptavidin-conjugated magnetic microparticles are added for magnetic separation, and the reaction is continued for 5 min. The washing concentrate is diluted and washed four times. 75 μL of acridil ester-labeled 1N23 antibody is added, and the reaction is continued for 5 min. After washing four times, the reaction complex is placed in the instrument's dark chamber, and 100 μL of chemiluminescence excitation solution and 100 μL of chemiluminescence pre-excitation solution are added sequentially to initiate the luminescence reaction. Finally, the luminescence intensity is recorded, and a calibration curve is plotted using the luminescence value and the FGF23 protein calibrator concentration. Based on the luminescence value of the sample, the iFGF23 protein concentration of the sample can be calculated using this calibration curve. The linear range of the calibration curve is 5–5000 pg / mL. Figure 6 The calibration curve for FGF23 protein is shown, where 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 5 pg / mL FGF23 protein calibrator was further serially diluted with protein stabilizer II (purchased from Huzhou Yingchuang Biotechnology Co., Ltd.) to determine the limit of detection. The results showed that it remained stable even at a concentration of 1 pg / mL. Therefore, the limit of detection for the FGF23 assay kit can reach 1 pg / mL. (See attached image.) Figure 7 .

[0182] Example 7: Clinical Performance Validation of the FGF23 Detection Kit

[0183] The FGF23 assay kit was used for the auxiliary diagnosis of chronic kidney disease samples: plasma samples from 50 patients with chronic kidney disease and 50 healthy individuals were collected from a hospital. The concentration of FGF23 in the plasma of both patients and healthy individuals was detected using the FGF23 assay kit.

[0184] The scatter plot of sample concentrations shows that FGF23 is statistically significant in differentiating between test results in patients with chronic kidney disease and healthy individuals (see attached figure). Figure 8 ROC curve statistics showed an area under the curve of 0.806. Using 32 pg / mL as the detection reference value, the FGF23 detection kit exhibited a specificity of 88% and a sensitivity of 78%. (See attached image). Figure 9 .

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

[0186] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment that specifically binds to the 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 CDR H3 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 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 the 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 includes: A: The heavy chain variable region shown in SEQ ID NO.4 and the 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 an antibody or antigen-binding fragment that specifically binds to the FGF23 protein as described in claim 1 or 2, characterized in that, The hybridoma cell line that secretes A antibody or antigen-binding fragment is named 9D3, and the hybridoma cell line that secretes B antibody or antigen-binding fragment is named 1N23. The accession number of hybridoma cell line 9D3 is CCTCC NO:C202502, and its classification name is hybridoma cell line 9D3. The accession number of hybridoma cell line 1N23 is CCTCC NO:C202515, and its classification name is hybridoma cell line 1N23.

4. A nucleic acid encoding the antibody or antigen-binding fragment as described in claim 1 or 2.

5. A vector containing the nucleic acid of claim 4.

6. A host cell containing the vector of claim 5.

7. A capture reagent for iFGF23 protein, characterized in that, The capture reagent comprises at least one antibody or antigen-binding fragment selected from A or B of any one of claims 1-2.

8. A reagent for detecting iFGF23 protein, characterized in that, The detection reagent includes at least one antibody or antigen-binding fragment selected from A or B of any one of claims 1-2.

9. The use of the antibody or antigen-binding fragment of claim 1 or 2, the hybridoma cell line of claim 3, the nucleic acid of claim 4, the vector of claim 5, the host cell of claim 6, the capture reagent of claim 7, or the detection reagent of claim 8 in any of the following aspects: (1) Preparation of iFGF23 protein detection kit; (2) Prepare a diagnostic kit for chronic kidney disease.

10. iFGF23 protein detection kit, characterized in that, The kit includes: capture antibody and 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.

11. A diagnostic system for chronic kidney disease, including: (1) iFGF23 protein detection module, wherein the iFGF23 protein concentration is detected by using the antibody or antigen binding fragment as described in claim 1 or 2, the iFGF23 protein detection reagent as described in claim 8, or the iFGF23 protein detection kit as described in claim 10; (2) Results output module, which assists in the diagnosis of chronic kidney disease based on the concentration of iFGF23 protein.

12. 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 perform the functions corresponding to the iFGF23 protein detection module and result output module in the chronic kidney disease auxiliary diagnostic system of claim 11.

13. An electronic device, characterized in that, include: One or more processors; And a computer-readable storage medium for storing computer instructions, programs, code sets or instruction sets, which, when run on a computer, cause the one or more processors to perform the functions corresponding to the iFGF23 protein detection module and the result output module in the chronic kidney disease auxiliary diagnostic system of claim 11.

14. A method for determining the concentration of iFGF23 protein, characterized in that, The concentration of iFGF23 protein was determined using the antibody or antigen-binding fragment as described in claim 1 or 2, the detection reagent for iFGF23 protein as described in claim 8, or the iFGF23 protein detection kit as described in claim 10. The assay method is used for purposes other than disease diagnosis and treatment.

15. The determination method according to claim 14, characterized in that, The determination method is chemiluminescence immunoassay, including: (1) A calibration curve was established using the FGF23 protein calibrator; the FGF23 protein sequence is shown in SEQ ID NO.3; (2) The fluorescence value of the sample to be tested is determined by using the antibody or antigen binding fragment, the detection reagent of iFGF23 protein, or the iFGF23 protein detection kit. Based on the fluorescence value of the sample to be tested, the concentration of iFGF23 protein in the sample to be tested is calculated using the calibration curve of (1).

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