WO2017159473A1

WO2017159473A1 - Method for assessing likelihood of chronic kidney disease patient developing hyperphosphatemia

Info

Publication number: WO2017159473A1
Application number: PCT/JP2017/009145
Authority: WO
Inventors: 豪秀木村; 達也篠田
Original assignee: 協和メデックス株式会社
Priority date: 2016-03-14
Filing date: 2017-03-08
Publication date: 2017-09-21
Also published as: JP2019082329A

Abstract

Provided is a method for assessing the likelihood of a chronic kidney disease (CKD) patient developing hyperphosphatemia by measuring the fibroblast growth factor 23 (FGF-23) in samples collected from the CKD patient and comparing the measurement with the standard that if the FGF-23 concentration in the sample is equal to or above a reference value, the CKD patient is likely to develop hyperphosphatemia, and if the concentration is below the reference value, the CKD patient is unlikely to develop hyperphosphatemia. The method for assessing the likelihood of a CKD patient developing hyperphosphatemia according to the present invention is useful in clinical diagnosis.

Description

A method to test the susceptibility to hyperphosphatemia in patients with chronic kidney disease

The present invention relates to a method for testing the likelihood of developing hyperphosphatemia in patients with chronic kidney disease (hereinafter referred to as CKD), and to test the ease of developing hyperphosphatemia in patients with CKD. It relates to a reagent.

Fibroblast growth factor-23 (hereinafter referred to as FGF-23) is a member of the fibroblast growth factor (FGF) family, which is produced mainly in bone tissue, acts on the kidney, and phosphorus in the renal tubule Inhibits reabsorption.

FGF-23 is a protein consisting of 251 amino acids, of which 24 amino acids at the N-terminus are signal peptides, and secreted FGF-23 is considered to consist of 227 amino acids. Some FGF-23s are processed between the 179th amino acid Arg and the 180th amino acid Ser by furin or the like, which is a kind of protease (see Non-Patent Documents 1 and 2). The full length FGF-23 consisting of 227 amino acids, which is not processed, has biological activities such as a decrease in blood phosphorous concentration, whereas the N-terminal and C-terminal fragments after this processing are It is reported to be inactive (see Non-Patent Documents 1 and 2).

CKD refers to a state in which one or both of the following (1) and (2) continues for 3 months or more.
(1) Urinary abnormalities such as proteinuria (microalbuminuria), conditions in which renal damage is evident by diagnostic imaging, blood tests, pathological findings, etc .;
(2) A state where the estimated glomerular filtration rate (eGRF) estimated based on the serum creatinine level is less than 60 mL / min / 1.73 m ² .

EGFR is used as an index representing the function of the kidney. GFR shows the amount of urine that glomeruli can make by filtering blood per minute, and eGFR in healthy people is about 100 mL / min / 1.73 m ² . CKD has stages classified into stages 1 to 5, and the symptoms become more serious as the stage goes up. In particular, in stage 3-5 CKD patients, eGFR is less than 60 mL / min / 1.73 m ² , and medical treatment in cooperation with specialists is required. When CKD worsens, it shifts to end-stage renal failure, and hyperphosphatemia, which is a condition in which phosphorus accumulates in the body without being excreted outside the body due to a decrease in renal function, appears.

Blood phosphorus levels are regulated by phosphorus absorption from the intestinal tract, phosphorus reabsorption by renal tubules, and dynamic equilibrium with bone and intracellular phosphorus. Absorption is considered the most important for the regulation of chronic phosphorus levels. FGF-23 reduces blood phosphorus levels by suppressing phosphorus reabsorption in renal tubules and by suppressing phosphorus absorption in the intestinal tract through lowering blood 1,25-hydroxyvitamin D levels. It has been reported that the FGF-23 concentration increases in the early stage of CKD, and then the phosphorus concentration increases in the later stage of CKD (see Non-Patent Document 3).

As described above, FGF-23 concentration and phosphorus concentration variation due to CKD stage classification has been reported. On the other hand, in a specific CKD patient, from the FGF-23 concentration in the specimen derived from the CKD patient, It is not known to test whether the CKD patient is likely to develop hyperphosphatemia in the future. An object of the present invention is to provide a method for testing the likelihood of developing hyperphosphatemia in a CKD patient, and a reagent for testing the ease of developing hyperphosphatemia in a CKD patient. is there.

Now, the inventors have found that it is possible to test whether or not the CKD patient is likely to develop hyperphosphatemia in the future from the FGF-23 concentration in the specimen collected from the CKD patient. Was completed. That is, the present invention relates to the following [1] to [10].
[1] FGF-23 in a sample collected from a CKD patient is measured, and if the FGF-23 concentration in the sample is greater than or equal to a reference value, the CKD patient is likely to develop hyperphosphatemia and the reference value A method of testing the likelihood of developing hyperphosphatemia in the CKD patient by comparing with a criterion that the CKD patient is less likely to develop hyperphosphatemia in the case of less than
[2] The method according to [1], comprising the following steps.
(1) collecting a specimen from a CKD patient;
(2) a step of measuring FGF-23 in the sample collected in step (1) and obtaining a measurement value;
(3) A step of creating a calibration curve showing the relationship between the FGF-23 concentration and the measured value by the same method as in step (2) using FGF-23 having a known concentration as a specimen;
(4) From the measurement value obtained in step (2) and the calibration curve showing the relationship between the FGF-23 concentration and the measurement value prepared in step (3), the FGF-23 concentration in the sample is determined. Determining step;
(5) If the FGF-23 concentration in the sample determined in step (4) is higher than the reference value, the CKD patient is likely to develop hyperphosphatemia. Comparing with a criterion that if the CKD patient is less likely to develop hyperphosphatemia if less than a reference value;
(6) As a result of the comparison in step (5), if the FGF-23 concentration in the sample determined in step (4) is above the reference value, the CKD patient is likely to develop hyperphosphatemia. If the value is less than the reference value, determining that the CKD patient is less likely to develop hyperphosphatemia.
[3] The reference value is the upper limit value of the 95% confidence interval of the FGF-23 concentration in the subject whose estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m ² or more, [1] or [2] The method described.
[4] The upper limit of the 95% confidence interval for the FGF-23 concentration in subjects whose estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m ² or more is 50 to 150 pg / mL [ 3] The method described.
[5] The method according to any one of [1] to [4], wherein the CKD patient is a stage 3-5 CKD patient.

[6] When the FGF-23 concentration in the FGF-23 measurement reagent and the sample collected from the CKD patient is higher than the reference value, the CKD patient is likely to develop hyperphosphatemia and is lower than the reference value A reagent for testing the ease of onset of hyperphosphatemia in CKD patients, including a reference table in which the criteria that the CKD patient is less likely to develop hyperphosphatemia is described.
[7] The reagent according to [6], wherein the likelihood of developing hyperphosphatemia in a CKD patient is tested by a method comprising the following steps.
(1) collecting a specimen from a CKD patient;
(2) A step of measuring FGF-23 in the sample collected in step (1) using an FGF-23 measuring reagent and obtaining a measurement value;
(3) A step of creating a calibration curve showing the relationship between the FGF-23 concentration and the measured value by the same method as in step (2) using FGF-23 having a known concentration as a specimen;
(4) From the measurement value obtained in step (2) and the calibration curve showing the relationship between the FGF-23 concentration and the measurement value prepared in step (3), the FGF-23 concentration in the sample is determined. Determining step;
(5) If the FGF-23 concentration in the sample determined in step (4) is higher than the reference value, the CKD patient is likely to develop hyperphosphatemia. Comparing the criteria described in the criteria table that if the CKD patient is less likely to develop hyperphosphatemia if below the reference value;
(6) As a result of the comparison in step (5), if the FGF-23 concentration in the sample determined in step (4) is above the reference value, the CKD patient is likely to develop hyperphosphatemia. If the value is less than the reference value, determining that the CKD patient is less likely to develop hyperphosphatemia.
[8] The reference value is the upper limit value of the 95% confidence interval of the FGF-23 concentration in the subject whose estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m ² or more, [6] or [7] The reagent according to [7].
[9] The upper limit of the 95% confidence interval for the FGF-23 concentration in subjects with an estimated glomerular filtration rate (eGFR) of 60 mL / min / 1.73 m ² or more is 50 to 150 pg / mL [ 8] The reagent according to the above.
[10] The reagent according to any one of [6] to [9], wherein the CKD patient is a stage 3-5 CKD patient.

The present invention provides a method for testing the likelihood of developing hyperphosphatemia in CKD patients and a reagent for testing the ease of developing hyperphosphatemia in CKD patients.

Hyperphosphatemia non-incidence rate created using the Kaplan-Meier method for FGF-23 concentrations in serum collected from patients with CKD less than 60 pg / mL and those with ≥23 pg / mL Represents a curve. The horizontal axis represents the number of days (elapsed time) from the day (reference day) when the FGF-23 concentration was determined, and the vertical axis represents the hyperphosphatemia non-incidence rate. A solid line represents a CKD patient group having an FGF-23 concentration of less than 60 pg / mL, and a dotted line represents a CKD patient group having an FGF-23 concentration of 60 pg / mL or more.

1. 2. Method for Testing Ease of Onset of Hyperphosphatemia in CKD Patient The method of testing the easiness of onset of hyperphosphatemia according to the present invention measures FGF-23 in a sample collected from a CKD patient. When the FGF-23 concentration in the sample is higher than the reference value, the CKD patient is likely to develop hyperphosphatemia, and when the concentration is less than the reference value, the CKD patient is less likely to develop hyperphosphatemia. This is a method characterized by testing the ease of onset of hyperphosphatemia in the CKD patient by comparing with the above criteria.

In the present invention, a CKD patient refers to a patient in which one or both of the following (1) and (2) is continued for 3 months or longer as described above.
(1) Urinary abnormalities such as proteinuria (microalbuminuria), conditions in which renal damage is evident by diagnostic imaging, blood tests, pathological findings, etc .;
(2) eGRF is less than 60 mL / min / 1.73 m ² .

In the present invention, CKD patients include CKD patients in stages 1 to 5, and CKD patients in stages 3 to 5 are preferable.

In the present invention, a stage 1 CKD patient means a CKD patient whose eGRF is 90 mL / min / 1.73 m ² or more, and a stage 2 CKD patient means an eGRF of 60 mL / min / 1.73 m ² or more, Means CKD patients with 89 mL / min / 1.73 m ² or less. Stage 3 CKD patients mean CKD patients with eGRF of 30 mL / min / 1.73 m ² or more and 59 mL / min / 1.73 m ² or less. Stage 4 CKD patients mean CKD patients with an eGRF of 15 mL / min / 1.73 m ² or more and 29 mL / min / 1.73 m ² or less, and stage 5 CKD patients have an eGRF of 15 mL / min. Mean CKD patients less than /min/1.73 m ² .

In the present invention, the sample collected from the CKD patient is not particularly limited as long as it is a sample collected from the CKD patient and can measure FGF-23, such as whole blood, serum, plasma, Examples include urine.

In the present invention, a method for testing the ease of onset of hyperphosphatemia in CKD patients can be performed, for example, by a method comprising the following steps.
(1) collecting a specimen from a CKD patient;
(2) a step of measuring FGF-23 in the sample collected in step (1) and obtaining a measurement value;
(3) A step of creating a calibration curve showing the relationship between the FGF-23 concentration and the measured value by the same method as in step (2) using FGF-23 having a known concentration as a specimen;
(4) From the measurement value obtained in step (2) and the calibration curve showing the relationship between the FGF-23 concentration and the measurement value prepared in step (3), the FGF-23 concentration in the sample is determined. Determining step;
(5) If the FGF-23 concentration in the sample determined in step (4) is higher than the reference value, the CKD patient develops hyperphosphatemia. Easy, if compared to a reference value, the CKD patient is less likely to develop hyperphosphatemia;
(6) As a result of the comparison in step (5), if the FGF-23 concentration in the sample determined in step (4) is above the reference value, the CKD patient is likely to develop hyperphosphatemia. If the value is less than the reference value, determining that the CKD patient is less likely to develop hyperphosphatemia.

<Step (1)>
Examples of the sample collected from the CKD patient in the step (1) include the above-described samples.

<Step (2)>
In step (2), measurement of FGF-23 in the sample collected in step (1) can be performed by any method as long as it can measure FGF-23 in the sample collected in step (1). For example, it can be performed using a method such as an immunoassay. Examples of the immunoassay include any known immunoassay, such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), fluorescent immunoassay (FIA), indirect fluorescent antibody method (Indirect Fluorescence assay), Luminescent immunoassay, physicochemical assay [turbidimetric immunoassay (TIA), latex agglutination (LAPIA), microparticle counting immunoagglutination (PCIA)], Western blotting The ELISA method is preferable. In the immunological measurement method, a sandwich method, a competitive method, or the like can be used, and a homogenis method, a heterogeneous method, or the like can also be used. In addition, FGF-23 in the sample collected in step (1) can be measured using a commercially available FGF-23 measurement reagent based on an immunoassay. Examples of commercially available FGF-23 measurement reagents based on immunoassay include Human Intact FGF-23 ELISA Kit (manufactured by Immutopics), Human FGF-23 ELISA Kit (manufactured by Merck Millipore), and FGF-23 measurement reagent. (Manufactured by Kainos).

A specific embodiment of a method for measuring FGF-23 in the specimen collected in step (1) using an immunological measurement method is shown below.
・ Measurement method 1
A method for measuring FGF-23 in a specimen collected in step (1), comprising the following steps.
(I) reacting the sample collected in step (1) with a first antibody that binds to FGF-23 to produce an immune complex of the first antibody and FGF-23;
(II) reacting the immune complex produced in step (I) with a labeled second antibody in which a label is bound to a second antibody that binds to FGF-23, to thereby react the first antibody, FGF-23 and the labeled antibody Generating an immune complex of a second antibody;
(III) A step of measuring the amount of label in the immune complex produced in step (II).

As the antibody (first antibody, second antibody), either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferred. The antibodies (first antibody and second antibody) include Fabs obtained by papain treatment of antibodies, F (ab ′) ₂ obtained by pepsin treatment, and Fc portions such as Fab ′ obtained by pepsin treatment-reduction treatment. An antibody fragment from which is removed can also be used.

The site (epitope) in FGF-23 recognized by the first antibody may be the same as or different from the site (epitope) in FGF-23 recognized by the second antibody. Is preferred. The antibody (first antibody, second antibody) used in the measurement of FGF-23 is FGF-23 itself or a site (epitope) in FGF-23 recognized by the antibody (first antibody, second antibody) Can be obtained by a conventional antibody production method using a peptide corresponding to the above as an antigen, and a commercially available antibody can be used as an antibody that binds to FGF-23. Examples of antibodies that bind to FGF-23 include monoclonal antibodies produced by hybridomas deposited as FERM BP-7838, FERM BP-7839, FERM BP-7840, and FERM BP-8268, respectively.

The reaction temperature in step (I) and step (II) is not particularly limited as long as it is a reaction temperature that enables measurement of FGF-23, and examples thereof include 0 to 50 ° C., preferably 4 to 40 ° C. . The reaction time in step (I) and step (II) is not particularly limited as long as it is a reaction time allowing measurement of FGF-23, and is, for example, 1 minute to 72 hours, preferably 5 minutes to 20 hours. .

Steps (I) to (III) can also be performed in an aqueous medium. The aqueous medium is not particularly limited as long as it is an aqueous medium capable of measuring FGF-23. Examples thereof include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable. The buffer used for preparing the buffer solution is not particularly limited as long as it has a buffering capacity, but has a pH of 1 to 11, for example, lactic acid buffer, citrate buffer, acetate buffer, succinate buffer, phthalic acid Buffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, Examples include carbonate buffer, glycine buffer, Tris buffer, Good buffer, and the like.

Examples of the good buffer include 2-morpholinoethanesulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, and tris (hydroxymethyl) aminomethane (Tris). Buffer, N- (2-acetamido) iminodiacetic acid (ADA) buffer, piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES) buffer, 2- [N- (2-acetamido) Amino] ethanesulfonic acid (ACES) buffer, 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) buffer, 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) buffer Agent, 3-morpholinopropanesulfonic acid (MOPS) buffer, 2- {N- [tris (hydroxy Til) methyl] amino} ethanesulfonic acid (TES) buffer, N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (HEPES) buffer, 3- [N, N-bis (2- Hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N-tris (hydroxymethyl) methyl] amino} propanesulfonic acid (TAPSO) buffer, piperazine-N, N′-bis (2-hydroxypropane-3-sulfonic acid) (POPSO) buffer, N- (2-hydroxyethyl) -N ′-(2-hydroxy-3-sulfopropyl) piperazine (HEPPSO) buffer, N- (2-hydroxyethyl) -N ′-(3-sulfopropyl) piperazine (EPPS) buffer, tricine [N-tri (Hydroxymethyl) methylglycine] buffer, bicine [N, N-bis (2-hydroxyethyl) glycine] buffer, 3- [N-tris (hydroxymethyl) methyl] aminopropanesulfonic acid (TAPS) buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-cyclohexylamino) propanesulfonic acid ( CAPS) buffer and the like.

The concentration of the buffer solution is not particularly limited as long as it is suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L, and particularly preferably 0.01 to 0.1 mol / L.

Process (I) and process (II) can be performed simultaneously. Although a cleaning step may or may not be provided between step (I) and step (II), it is preferable to provide a cleaning step. Further, although a cleaning step may or may not be provided between step (II) and step (III), it is preferable to provide a cleaning step. The first antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized. When the first antibody is immobilized on an insoluble carrier, the insoluble carrier after the step (I) is washed so that the first antibody produced in the step (I) and the immune complex of the measurement target component are unreacted. It can be separated from the components (component derived from the specimen, excess first antibody, etc.). Similarly, by washing the insoluble carrier after step (II), the immune complex of the first antibody, FGF-23 and labeled second antibody produced in step (II) is allowed to react with unreacted components (excess labeling). Second antibody etc.). As the washing solution, phosphate buffered saline [10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2 (hereinafter referred to as PBS)], PBS containing a surfactant, The above-mentioned aqueous medium etc. can be mention | raise | lifted. Examples of the surfactant include nonionic surfactants such as Tween 20.

The insoluble carrier is not particularly limited as long as it is an insoluble carrier capable of measuring FGF-23. Preferred materials for the insoluble carrier include polymer materials, glass, ceramics, magnetic particles and metals. Examples of the polymer material include polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, nitrocellulose, cellulose acetate, cellulose acetate, and polyethylene terephthalate. Preferable shapes of the insoluble carrier include tubes, beads, plates, fine particles such as latex, sticks, and the like, and a 96-well / sheet polystyrene microtiter plate is preferable.

As a method for immobilizing the first antibody on the insoluble carrier, a known method such as a method using physical bonding, a method using chemical bonding, or a combination thereof is used. Examples of physical bonds include electrostatic bonds, hydrogen bonds, and hydrophobic bonds. Examples of the chemical bond include a covalent bond and a coordination bond.

The first antibody may be directly immobilized on an insoluble carrier or indirectly immobilized on an insoluble carrier. As an indirect immobilization method, for example, a solution of a biotinylated first antibody is added to an insoluble carrier on which avidin is immobilized, and the first antibody is made into an insoluble carrier via specific binding between biotin and avidin. Examples of the immobilization method include a method in which a solution of the first antibody is added to an insoluble carrier on which Fc is immobilized, and the first antibody is immobilized on the insoluble carrier by the interaction between Fc and the first antibody. Furthermore, the first antibody may be immobilized on an insoluble carrier by a covalent bond via a linker. Examples of the linker include a molecule that can be covalently bonded to both the functional group of the first antibody and the functional group held on the surface of the insoluble carrier, and the like, which can react with the functional group of the first antibody. A molecule having one reactive group and the second reactive group capable of reacting with the functional group held on the surface of the insoluble carrier in the same molecule is preferable. Among them, the first reactive activity Molecules in which the group and the second reactive group are different are particularly preferred. The functional group of the first antibody and the functional group held on the surface of the insoluble carrier include carboxyl group, amino group, glycidyl group, sulfhydryl group, hydroxyl group, amide group, imino group, N-hydroxysuccinyl group, maleimide group Etc. The reactive groups in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, psoralen, pyridyl disulfide, vinyl And groups such as sulfone.

・ Measurement method 2
A method for measuring FGF-23 in a specimen collected in step (1), comprising the following steps.
(I) The sample collected in step (1) is reacted with a labeled competitor having a label bound to the competitor and an antibody that binds to both FGF-23 and the labeled competitor, and the labeled Generating an immune complex of the competitor and the antibody;
(II) A step of measuring the amount of label in the immune complex produced in step (I).

Although a cleaning step may or may not be provided between step (I) and step (II), it is preferable to provide a cleaning step. Examples of the cleaning liquid used in the cleaning step include the above-described cleaning liquid. The antibody that binds to both FGF-23 and the labeled competitive substance may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier. Examples of the method for immobilizing the antibody on an insoluble carrier include a method for immobilizing the above-mentioned first antibody on an insoluble carrier. The reaction temperature in step (I) is not particularly limited as long as it is a reaction temperature that enables measurement of FGF-23, and examples thereof include 0 to 50 ° C., preferably 4 to 40 ° C. The reaction time in the step (I) is not particularly limited as long as it is a reaction time allowing measurement of FGF-23, and is, for example, 1 minute to 72 hours, preferably 5 minutes to 20 hours.

Steps (I) and (II) can also be performed in an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium etc. are mentioned, for example. Here, the competitive substance means a substance that is capable of binding to “an antibody that binds to FGF-23” and whose binding is competitive with FGF-23, and FGF-23 itself Is also included. The labeled competitor can be prepared by the same method as the method for preparing the labeled second antibody described later using the competitor and the labeling substance described later.

・ Measurement method 3
A method for measuring FGF-23 in a specimen collected in step (1), comprising the following steps.
(I) The sample collected in step (1) is reacted with a competitive substance and a labeled antibody in which a label is bound to an antibody that binds to both FGF-23 and the competitive substance. Generating an immune complex of the labeled antibody;
(II) A step of measuring the amount of label in the immune complex produced in step (I).

Although a cleaning step may or may not be provided between step (I) and step (II), it is preferable to provide a cleaning step. Examples of the cleaning liquid used in the cleaning step include the above-described cleaning liquid. The competing substance may or may not be immobilized on the insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier. Examples of the method for immobilizing the competitive substance on the insoluble carrier include the same method as the method for immobilizing the first antibody on the insoluble carrier. The reaction temperature in step (I) is not particularly limited as long as it is a reaction temperature that enables measurement of FGF-23, and examples thereof include 0 to 50 ° C., preferably 4 to 40 ° C. The reaction time in the step (I) is not particularly limited as long as it is a reaction time allowing measurement of FGF-23, and is, for example, 1 minute to 72 hours, preferably 5 minutes to 20 hours.

Steps (I) and (II) can also be performed in an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium etc. are mentioned, for example. A labeled antibody can be prepared by using the antibody and a labeling substance described below by the same method as the method for preparing a labeled second antibody described below.

Examples of the labeling substance for labeling the (second) antibody in the measuring method 1 and the measuring method 3 and the labeling substance for labeling the competitive substance in the measuring method 2 include, for example, enzymes, fluorescent substances, luminescent substances, radioisotopes, avidin, biotin, Examples include digoxigenin, a polypeptide containing a tag sequence, metal colloid particles, and colored latex particles. Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like. Examples of the fluorescent substance include FITC (fluorescein isothiocyanate), RITC (rhodamine B-isothiocyanate) and the like. Other fluorescent substances include, for example, quantumdot (Science, 281, 2016-2018, 1998), phycobiliproteins such as phycoerythrin, GFP (Green fluorescent Protein), RFP (Red fluorescent Protein), YFP (Yellow fluorescent Protein), BFP Examples thereof include fluorescent proteins such as (Blue fluorescent Protein). Examples of the light-emitting substance include acridinium and its derivatives, ruthenium complex compounds, and lophine. Further, as the ruthenium complex compound, those shown in Clin. Chem. 37, 9, 1534-1539, 1991, which emits light electrochemically together with an electron donor, are preferable. Examples of the radioisotope include ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ I, and ¹³¹ I. Polypeptides containing tag sequences include FLAG peptides (FLAG tag, Asp Tyr Lys Asp Asp Asp Asp Lys (SEQ ID NO: 1)), polyhistidine (His tag, His His His His His (SEQ ID NO: 2)) , Myc epitope peptide (myc tag, Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu (SEQ ID NO: 3)), hemagglutinin epitope peptide (HA tag, Tyr Pro Tyr Asp Val Pro Asp Tyr Ala (SEQ ID NO: 4)), etc. Is mentioned.

The labeled (second) antibody can be prepared by labeling the (second) antibody with the aforementioned labeling substance. The (second) antibody can be labeled by a reaction that causes a covalent bond between the functional group of the (second) antibody and the functional group of the labeling substance via or without a linker. Examples of the functional group include a carboxyl group, amino group, glycidyl group, sulfhydryl group, hydroxyl group, amide group, imino group, hydroxysuccinyl ester group, maleimide group, and isothiocyanate group. It is possible to cause a condensation reaction between these functional groups.

Examples of the bonding method without using a linker include a method using a carbodiimide compound such as EDC. In this case, it is also possible to use an active ester such as NHS or a derivative thereof. Further, the condensation reaction between the isothiocyanate group and the amino group is preferable because it does not require other reagents and proceeds only by mixing under neutral to weakly alkaline conditions.

Examples of the linker include those having in the molecule both functional groups that react with the functional group of the (second) antibody and functional groups that react with the functional group of the labeling substance. Molecules having a first functional group capable of reacting with an amino acid residue of an antibody and a second functional group capable of reacting with a functional group of a labeling substance in the same molecule are preferred. A molecule in which the functional group is different from the second functional group is particularly preferable. As a functional group of a linker, the above-mentioned functional group is mentioned, for example.

Examples of the method of chemically binding the radioisotope to the (second) antibody include the methods described in the literature (Antibody Immunoconj. Radiopharm., 3, 60, 1990).

When the labeling substance is a polypeptide such as an enzyme, avidin, a fluorescent protein, a phycobiliprotein, or a polypeptide containing a tag sequence, a known genetic recombination technique (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring According to Harbor Laboratory Press (2001), an expression vector containing a DNA encoding a fusion protein of a labeling substance and an antibody is prepared, the expression vector is introduced into a suitable host, and the host is cultured. DNA encoding the fusion protein can be obtained by cloning the DNA encoding the antibody and the labeling substance by PCR or the like, and ligating each DNA by ligase reaction.

Measure the amount of labeling by selecting an appropriate method according to the labeling substance. When the labeling substance is a coloring substance, that is, a substance that absorbs light of a certain wavelength, a spectrophotometer, a multiwell plate reader, or the like can be used. When the labeling substance is a fluorescent substance, a fluorometer, a fluorescent multiwell plate reader, or the like can be used. When the labeling substance is a luminescent substance, a luminescence photometer, a luminescent multiwell plate reader, or the like can be used. When the labeling substance is a radioisotope, the amount of radioisotope can be measured with a scintillation counter, a γ-well counter or the like for the radioactivity.

When the label is an enzyme, measuring the amount of label means measuring the enzyme activity. The amount of labeling can be measured by reacting an enzyme substrate with the enzyme and measuring the produced substance. When the enzyme is a peroxidase, the peroxidase activity can be measured by, for example, an absorbance method, a fluorescence method, a luminescence method, or the like. As a method for measuring the peroxidase activity by the absorbance method, for example, the peroxidase is reacted with a combination of its substrate hydrogen peroxide and an oxidative coloring chromogen, and the absorbance of the reaction solution is measured by a spectrophotometer, a multiwell plate reader, etc. The method etc. which measure by are mentioned. Examples of the oxidative coloring chromogen include a leuco chromogen and an oxidative coupling coloring chromogen.

The leuco chromogen is a substance that is converted into a pigment by itself in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Specifically, tetramethylbenzidine, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7- Bis (dimethylamino) -10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt (DA-64), 10-N-carboxymethylcarbamoyl-3 , 7-bis (dimethylamino) -10H-phenothiazine sodium salt (DA-67), 4,4′-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] An amine (BCMA) etc. are mentioned.

The oxidative coupling chromogen is a substance that forms a dye by oxidative coupling of two compounds in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and an aniline (Trinder reagent), a combination of a coupler and a phenol. Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine. Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N-ethyl-N- (2-hydroxy -3-Sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N , N-di (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl- -(3-sulfopropyl) aniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline, N- (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N -(3-sulfopropyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3- Sulfopropyl) aniline, N-ethyl-N- (3-methylphenyl) -N′-succinylethylenediamine (EMSE), N-ethyl-N- (3-methylphenyl) -N′-acetylethylenediamine, N-ethyl- And N- (2-hydroxy-3-sulfopropyl) -4-fluoro-3,5-dimethoxyaniline (F-DAOS). Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.

As a method for measuring peroxidase activity by a fluorescence method, for example, reacting peroxidase with a combination of its substrate hydrogen peroxide and a fluorescent substance, and measuring the intensity of fluorescence generated by a fluorometer, a fluorescent multiwell plate reader, etc. And the like. Examples of the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, and coumarin.

As a method for measuring peroxidase activity by the luminescence method, for example, the peroxidase is reacted with a combination of hydrogen peroxide and a luminescent substance as a substrate, and the intensity of luminescence generated by a luminescence intensity meter or a luminescence multiwell plate reader is measured. And the like. Examples of the luminescent substance include a luminol compound and a lucigenin compound.

When the enzyme is alkaline phosphatase, alkaline phosphatase activity can be measured by, for example, a luminescence method. Examples of the method for measuring alkaline phosphatase activity by a luminescence method include a method in which alkaline phosphatase and its substrate are reacted and the luminescence intensity of the produced luminescence is measured with a luminescence intensity meter, a luminescence multiwell plate reader, or the like. Examples of alkaline phosphatase substrates include 3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), 2-chloro- 5- {4-methoxyspiro [1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decan] -4-yl} phenyl phosphate disodium Salt (CDP-Star ^™ ), 3- {4-methoxyspiro [1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decane] -4 - yl} phenyl phosphate disodium salt ^(CSPD TM), 9 - [(phenyloxy) (phosphoryloxy) methylidene] -10-methyl-acridan-disodium salt, 9 [(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methyl-acridan-disodium salt (Lumigen ^TM APS-5), and the like.

When the enzyme is β-D-galactosidase, β-D-galactosidase activity can be measured by, for example, an absorbance method (colorimetric method), a luminescence method, or a fluorescence method. As a method for measuring β-D-galactosidase activity by the absorbance method (colorimetric method), for example, β-D-galactosidase and its substrate are reacted, and the absorbance of the reaction solution is measured with a spectrophotometer, a multiwell plate reader or the like. The measuring method etc. are mentioned. Examples of the substrate for β-D-galactosidase include o-nitrophenyl-β-D-galactopyranoside. As a method for measuring β-D-galactosidase activity by a luminescence method, for example, a method in which β-D-galactosidase and its substrate are reacted and the luminescence intensity of the reaction solution is measured with a luminescence intensity meter, a luminescence multiwell plate reader or the like Etc. Examples of the substrate of β-D-galactosidase include galacton-plus (Galacton-Plus, manufactured by Applied Biosystems) or an analogous compound thereof. As a method of measuring β-D-galactosidase activity by a fluorescence method, for example, a method of reacting β-D-galactosidase and its substrate and measuring the fluorescence of the reaction solution with a fluorometer, a fluorescent multiwell plate reader or the like Etc. Examples of the substrate of β-D-galactosidase include 4-methylumbelliferyl-β-D-galactopyranoside.

When the enzyme is luciferase, the luciferase activity can be measured by, for example, a luminescence method. Examples of the method for measuring luciferase activity by the luminescence method include a method of reacting luciferase with its substrate and measuring the luminescence intensity of the reaction solution with a luminescence intensity meter, a luminescence multiwell plate reader, or the like. Examples of the luciferase substrate include luciferin and coelenterazine.

In the measurement method of FGF-23, the antigen-antibody reaction, that is, steps (I) and (II) in measurement method 1, measurement method 2 and step (I) in measurement method 3, are performed using metal ions, salts, sugars, antiseptics. Can be carried out in the presence of an agent, protein, protein stabilizer and the like. Examples of the metal ion include magnesium ion, manganese ion, zinc ion and the like. Examples of the salts include sodium chloride and potassium chloride. Examples of the saccharide include mannitol and sorbitol. Examples of preservatives include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), Bioace, Procrine 300, Proxel GXL, and the like. Examples of the protein include bovine serum albumin (BSA), fetal bovine serum (FBS), casein, block ace (Dainippon Pharmaceutical Co., Ltd.) and the like. Examples of the protein stabilizer include peroxidase stabilization buffer (Peroxidase Stabilizing Buffer, manufactured by DakoCytomation).

<Step (3)>
In the step (3), a calibration curve showing the relationship between the FGF-23 concentration and the measured value is obtained by using the standard product having a known FGF-23 concentration as a specimen and using the above FGF-23 measurement method. A calibration curve can be created from the measured values obtained and the FGF-23 concentration.

<Process (4)>
In the step (4), the FGF-23 concentration in the sample collected in the step (1) is measured by the above-described FGF-23 measurement method using the sample, and the obtained measured value and the above-mentioned It can be determined from the calibration curve.

<Step (5)>
In step (5), if the concentration of FGF-23 in the sample collected in step (1) obtained as described above is not less than the reference value, the CKD patient Compared with the criterion that hyperphosphatemia is likely to develop and, if below the reference value, the CKD patient is less likely to develop hyperphosphatemia.

The reference value in the present invention is not particularly limited as long as it is a reference value that can determine whether or not a CKD patient is likely to develop hyperphosphatemia. For example, eGFR is 60 mL / min / 1.73 m ² or more. Examples include the upper limit value of the 95% confidence interval of the FGF-23 concentration in the subject. Examples of the subject whose eGFR is 60 mL / min / 1.73 m ² or more include healthy persons, stage 1-2 CKD patients, and the like. The upper limit of the 95% confidence interval for FGF-23 concentrations in subjects with an eGFR of 60 mL / min / 1.73 m ² or more is usually 50 to 150 pg / mL, preferably 55 to 100 pg / mL 60-75 pg / mL is particularly preferred.

<Step (6)>
In the step (6), as a result of the comparison in the step (5), the FGF-23 concentration in the sample determined in the step (4) was compared with the above standard. As a result, the FGF-23 concentration in the sample was determined as the standard. If the value is greater than or equal to the value, the CKD patient is likely to develop hyperphosphatemia, and if the value is less than the reference value, it is determined that the CKD patient is less likely to develop hyperphosphatemia.

In the present invention, hyperphosphatemia refers to hyperphosphatemia that develops in CKD patients, that is, a condition in which phosphorus accumulates in the body without being excreted outside the body due to a decrease in renal function. In the present invention, the determination as to whether or not a CKD patient has developed hyperphosphatemia may be any method that enables determination of the development of hyperphosphatemia, for example, collected from a CKD patient. If the serum phosphorus concentration is less than or equal to the upper limit of the serum phosphorus concentration of a healthy person, it is determined that the CKD patient does not develop hyperphosphatemia, and the serum phosphorus collected from the CKD patient When the concentration exceeds the upper limit of the phosphorus concentration in the serum of a healthy person, it can be determined that the CKD patient has developed hyperphosphatemia. Examples of the upper limit of the phosphorus concentration in the serum of the healthy person include 4.1 to 4.6 mg / dL.

In the present invention, the stage of the CKD patient may be any stage, and may be, for example, stage 1-2 or stage 3-5. Even if it is a stage 1 or 2 CKD patient, if the FGF-23 concentration in the sample collected from the CKD patient is equal to or higher than a reference value, it can be determined that the CKD patient is likely to develop hyperphosphatemia. it can. In addition, even for CKD patients at stage 3 to 5, if the FGF-23 concentration in the sample collected from the CKD patient is less than the reference value, it is determined that the CKD patient is unlikely to develop hyperphosphatemia. be able to.

2. Reagent for Testing Ease of Development of Hyperphosphatemia in CKD Patient The present invention also provides a reagent for measuring FGF-23, and FGF-23 concentration in a sample collected from a CKD patient is higher than a reference value. A CKD patient including a reference table in which the CKD patient is likely to develop hyperphosphatemia and the CKD patient is less likely to develop hyperphosphatemia when the CKD patient is less than the reference value, The present invention relates to a reagent for testing the ease of onset of hyperphosphatemia. The reagent of the present invention can be used in the method of the present invention.

The FGF-23 measurement reagent in the reagent of the present invention is a reagent used in the above-described FGF-23 measurement method. The FGF-23 measurement reagent is not particularly limited as long as it is a reagent that can measure FGF-23 in a sample, and examples thereof include a reagent based on an immunological measurement method. Examples of the immunological measurement reagent include reagents based on the above-described immunological measurement method. Further, as the FGF-23 measurement reagent, a commercially available FGF-23 measurement reagent based on an immunoassay can also be used. Examples of commercially available FGF-23 measurement reagents based on immunoassays include Human Intact FGF-23 ELISA KIT (Immutopics), Human FGF-23 ELISA KIT (Merck Millipore), and FGF-23 measurement reagent. (Manufactured by Kainos).

Specific embodiments of the FGF-23 measurement reagent based on the immunoassay are shown below.
・ Measurement reagent 1
A reagent for measuring FGF-23 comprising a first antibody that binds to FGF-23 and a labeled second antibody in which a label is bound to a second antibody that binds to FGF-23.
・ Measurement reagent 2
A reagent for measuring FGF-23, comprising a labeled competitor having a label bound to the competitor, and an antibody that binds to both FGF-23 and the labeled competitor.
・ Measurement reagent 3
A reagent for measuring FGF-23 comprising a competitive substance and a labeled antibody in which a label is bound to an antibody that binds to both FGF-23 and the competitive substance.

First antibody, second antibody, labeled second antibody, competitor, labeled competitor, antibody that binds to both FGF-23 and labeled competitor, and antibody that binds to both FGF-23 and competitor Examples of the labeled antibody to which the label is bound include the first antibody, the second antibody, the labeled second antibody, the competitor, the labeled competitor, the antibody that binds to both FGF-23 and the labeled competitor, Examples thereof include labeled antibodies in which a label is bound to an antibody that binds to both FGF-23 and a competitor.

In measurement reagent 1, the first antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier. In measurement reagent 2, the antibody that binds to both FGF-23 and the labeled competitor may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier. In the measurement reagent 3, the competitive substance may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

The FGF-23 measurement reagent may contain the aforementioned metal ions, salts, saccharides, preservatives, proteins, protein stabilizers, and the like.

The reference table in the reagent of the present invention shows that when the FGF-23 concentration in a sample collected from a CKD patient is higher than the reference value, the CKD patient is likely to develop hyperphosphatemia, and when the concentration is lower than the reference value, It is the reference | standard table | surface in which the reference | standard that the said CKD patient was hard to develop hyperphosphatemia was described.

In the present invention, examples of a method for testing the susceptibility of hyperphosphatemia in CKD patients using a FGF-23 measurement reagent include a method comprising the following steps.
(1) collecting a specimen from a CKD patient;
(2) A step of measuring FGF-23 in the sample collected in step (1) using an FGF-23 measuring reagent and obtaining a measurement value;
(3) A step of creating a calibration curve showing the relationship between the FGF-23 concentration and the measured value by the same method as in step (2) using FGF-23 having a known concentration as a specimen;
(4) From the measurement value obtained in step (2) and the calibration curve showing the relationship between the FGF-23 concentration and the measurement value prepared in step (3), the FGF-23 concentration in the sample is determined. Determining step;
(5) If the FGF-23 concentration in the sample determined in step (4) is higher than the reference value, the CKD patient is likely to develop hyperphosphatemia. Comparing the criteria described in the criteria table that if the CKD patient is less likely to develop hyperphosphatemia if below the reference value;
(6) As a result of the comparison in step (5), if the FGF-23 concentration in the sample determined in step (4) is above the reference value, the CKD patient is likely to develop hyperphosphatemia. If the value is less than the reference value, determining that the CKD patient is less likely to develop hyperphosphatemia.

Examples of CKD patients include the aforementioned CKD patients. Examples of the specimen include the aforementioned specimens. Measurement of FGF-23 in the sample collected in step (1) is performed using an FGF-23 measurement reagent. Examples of the FGF-23 measurement reagent include the aforementioned FGF-23 measurement reagent. A calibration curve showing the relationship between the FGF-23 concentration and the measured value can be created by the method described above. The FGF-23 concentration in the specimen collected in step (1) can be determined by the method described above. Examples of the standard to be compared with the determined FGF-23 concentration include the above-mentioned standard. Examples of the reference value for determining the likelihood of developing hyperphosphatemia in CKD patients by comparing the determined FGF-23 concentration with the reference include the reference values described above.

Hereinafter, although an example explains the present invention, the present invention is not limited to this example.

Determination of FGF23 concentration in serum samples collected from CKD patients (1) FGF-23 measurement kit The following magnetic particle suspension, biotin-conjugated anti-FGF-23 antibody solution, and alkaline phosphatase-labeled anti-FGF-23 antibody An FGF-23 measurement kit containing a fragment solution was prepared.

<Suspension of magnetic particles>
A magnetic particle suspension having the following composition was prepared using commercially available magnetic particles (Dynabeads MyOne Streptavidin T1; manufactured by Dynal) bound with streptavidin as the magnetic particles.
MES (pH 6.5) 50 mmol / L
Streptavidin-coupled magnetic particles 0.75mg / mL
BSA 0.1%
Sodium chloride 0.1 mol / L

<Biotin-conjugated anti-FGF-23 antibody and biotin-conjugated anti-FGF-23 antibody solution>
Using the anti-FGF-23 monoclonal antibody 2C3B produced by the hybridoma deposited as FERM BP-7838 as the first antibody, the antibody and NHS-biotin are mixed and reacted at 37 ° C. for 1 hour. The mixture was applied to a Sephadex G-25 column (manufactured by GE Health Science Japan) to remove unreacted NHS-biotin, and a biotin-conjugated anti-FGF-23 monoclonal antibody was prepared. Using the obtained biotin-conjugated anti-FGF-23 monoclonal antibody, a biotin-conjugated anti-FGF-23 antibody solution having the following composition was prepared.
MES (pH 6.5) 50 mmol / L
Biotin-conjugated anti-FGF-23 monoclonal antibody 2C3B
5 μg / mL
BSA 0.1%
Sodium chloride 0.1 mol / L

<Alkaline phosphatase-labeled anti-FGF-23 antibody fragment and alkaline phosphatase-labeled anti-FGF-23 antibody fragment solution>
As the second antibody fragment, the anti-FGF-23 monoclonal antibody 3C1E produced by the hybridoma deposited as FERM BP-7839 was digested with pepsin, and then G3000SW column (manufactured by Tosoh Corporation; aperture: 21.5 mm; length: 60 cm) F (ab ′) ₂ was separated using an HPLC system (manufactured by Hitachi, Ltd.). The obtained F (ab ′) ₂ was reduced with 2-mercaptoethylamine hydrochloride (manufactured by Nacalai Tesque) and then a HPLC system using a G3000SW column (manufactured by Tosoh Corporation; aperture: 21.5 mm; length: 60 cm) Fab 'was separated by (manufactured by Hitachi, Ltd.). The obtained Fab ′ and alkaline phosphatase were bound by the maleimide method according to the following procedure.

Using maleimide reagent Sulfo-HMCS (manufactured by Dojindo Laboratories), alkaline phosphatase was maleimidized, and the reaction mixture was subjected to Sephadex G-25 gel filtration column (GE Health Science Japan) to make unreacted. Sulfo-HMCS was removed to obtain maleimidated alkaline phosphatase.

The prepared maleimidated alkaline phosphatase and Fab ′ obtained above were mixed to prepare an alkaline phosphatase labeled Fab ′ antibody. Using the obtained alkaline phosphatase labeled Fab ′ antibody, an alkaline phosphatase labeled anti-FGF-23 antibody fragment solution having the following composition was prepared.
MES (pH 6.5) 50 mmol / L
Alkaline phosphatase labeled anti-FGF-23 antibody fragment 5 μg / mL
BSA 0.1%
Sodium chloride 0.1 mol / L

(2) Preparation of calibration curve FGF-23 produced by the method described in the pamphlet of WO2003 / 57733 was added to phosphate buffered saline containing 0.2% BSA (10 mmol / L containing 0.15 mol / L sodium chloride). L Phosphate buffer, pH 7.2) FGF-23 concentrations are 10,000 pg / mL, 3,000 pg / mL, 1,000 pg / mL, 300 pg / mL, 100 pg / mL, 50 pg / mL, 5 pg / A solution diluted to mL, 0 pg / mL was used as a standard solution.

30 μL each of the magnetic particle suspension prepared in (1), the biotin-conjugated anti-FGF-23 antibody solution, and the alkaline phosphatase-labeled anti-FGF-23 antibody fragment solution were added to 10 μL of each standard solution and stirred. The reaction was allowed to proceed at 20 ° C. for 20 minutes. The magnetic particles were collected by magnetic force to remove the reaction solution other than the magnetic particles, and the magnetic particles were washed 5 times with a washing solution [50 mmol / L MOPS buffer (pH 7.35) containing 0.1% Tween 20]. Thereafter, 100 μL of a luminescent substrate solution mainly composed of 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium salt (Lumigen ^™ APS-5) was added and stirred. The amount of luminescence generated (RLU) was measured, and a calibration curve showing the relationship between the FGF-23 concentration and the amount of luminescence (RLU) was created.

(3) Determination of FGF-23 concentration in serum samples collected from CKD patients Samples were collected from 29 CKD patients at stage 3-5 possessed by the nonprofit organization Tsukuba Clinical Laboratory Education and Research Center Except for using each serum sample of 29 serum samples, measurement was performed in the same manner as in (2) above, and after obtaining the luminescence amount for each serum sample, the obtained luminescence amount and the calibration prepared in (2) above From the line, the FGF-23 concentration in each serum sample was determined.

(4) Confirmation of the occurrence of hyperphosphatemia by follow-up of CKD patients In (3) above, the day on which the FGF-23 concentration in the serum collected from each patient was determined for each of the 29 CKD patients The course was observed for 12 to 826 days. During the observation period, serum is collected from each patient at any time, and the phosphorus concentration in the collected serum is determined. If the determined phosphorus concentration is less than 4.5 mg / dL, the CKD patient is hyperphosphatemic. When the determined phosphorus concentration was 4.5 mg / dL or more, it was determined that the CKD patient had developed hyperphosphatemia. The results are shown in Tables 1 and 2.

As is clear from Tables 1 and 2, no hyperphosphatemia was observed in all 5 patients among CKD patients whose FGF-23 concentration was less than 60 pg / mL. On the other hand, in CKD patients whose FGF-23 concentration was 60 pg / mL or more, onset of hyperphosphatemia was observed in 20 of 24 patients. Thus, by comparing the FGF-23 concentration in the serum collected from CKD patients with the reference value of 60 μpg / mL, when the FGF-23 concentration is less than the reference value, the CKD patient It has been found that when the FGF-23 concentration is less than the reference value, it is possible to determine that the CKD patient is likely to develop hyperphosphatemia.

Both the group consisting of 5 CKD patients whose FGF-23 concentration is less than 60 pg / mL and the group consisting of 24 CKD patients whose FGF-23 concentration is 60 pg / mL or more in Example 1 The number of days from the day when the FGF-23 concentration was determined (reference day) to the day when the onset of hyperphosphatemia was confirmed was examined. The result is shown in FIG. FIG. 1 is a hyperphosphatemia non-incidence rate curve according to the Kaplan-Meier method. Since the P value was less than 0.05 in the log rank test, the CKD patient group with an FGF-23 concentration of 60 pg / mL or more was compared with the CKD patient group with an FGF-23 concentration of less than 60 pg / mL. Thus, it was shown that hyperphosphatemia is statistically significantly more likely to develop.

Thus, the FGF-23 concentration in the sample collected from the CKD patient is determined, and if the concentration is less than the reference value, the CKD patient is unlikely to develop hyperphosphatemia, and the concentration is higher than the reference value. It has been found that the CKD patient can be tested for the likelihood of developing hyperphosphatemia by comparing it to a criterion that the patient is likely to develop hyperphosphatemia.

The method for testing the likelihood of developing hyperphosphatemia in CKD patients and the reagent for testing the ease of developing hyperphosphatemia in CKD patients are useful in clinical diagnosis. This contributes to improving the quality of life of CKD patients.

Claims

When fibroblast growth factor-23 (hereinafter referred to as FGF-23) in a sample collected from a patient with chronic kidney disease (hereinafter referred to as CKD) is measured, and the FGF-23 concentration in the sample is greater than or equal to a reference value Compared with the criteria that the CKD patient is likely to develop hyperphosphatemia and the CKD patient is less likely to develop hyperphosphatemia when the CKD patient is below the reference value, the hyperphosphatemia in the CKD patient To test the ease of onset of symptom.
The method of Claim 1 containing the following processes.
(1) collecting a specimen from a CKD patient;
(2) a step of measuring FGF-23 in the sample collected in step (1) and obtaining a measurement value;
(3) A step of creating a calibration curve showing the relationship between the FGF-23 concentration and the measured value by the same method as in step (2) using FGF-23 having a known concentration as a specimen;
(4) From the measurement value obtained in step (2) and the calibration curve showing the relationship between the FGF-23 concentration and the measurement value prepared in step (3), the FGF-23 concentration in the sample is determined. Determining step;
(5) If the FGF-23 concentration in the sample determined in step (4) is higher than the reference value, the CKD patient is likely to develop hyperphosphatemia. Comparing with a criterion that if the CKD patient is less likely to develop hyperphosphatemia if less than a reference value;
(6) As a result of the comparison in step (5), if the FGF-23 concentration in the sample determined in step (4) is above the reference value, the CKD patient is likely to develop hyperphosphatemia. If the value is less than the reference value, determining that the CKD patient is less likely to develop hyperphosphatemia.
The reference value is an upper limit value of a 95% confidence interval of FGF-23 concentration in a subject whose estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m 2 or more. Method.
The upper limit of the 95% confidence interval of the FGF-23 concentration in a subject whose estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m 2 or more is 50 to 150 pg / mL. the method of.
The method according to any of claims 1 to 4, wherein the CKD patient is a stage 3-5 CKD patient.
When the FGF-23 concentration in a specimen collected from a fibroblast growth factor-23 (hereinafter referred to as FGF-23) measurement reagent and a chronic kidney disease (hereinafter referred to as CKD) is a reference value or more, Hyperphosphatemia in CKD patients, including a criteria table that describes the criteria that CKD patients are more likely to develop hyperphosphatemia, and that CKD patients are less likely to develop hyperphosphatemia if below the reference value A reagent for testing the ease of onset.
The reagent according to claim 6, wherein the likelihood of developing hyperphosphatemia in a CKD patient is tested by a method comprising the following steps.
(1) collecting a specimen from a CKD patient;
(2) A step of measuring FGF-23 in the sample collected in step (1) using an FGF-23 measuring reagent and obtaining a measurement value;
(3) A step of creating a calibration curve showing the relationship between the FGF-23 concentration and the measured value by the same method as in step (2) using FGF-23 having a known concentration as a specimen;
(4) From the measurement value obtained in step (2) and the calibration curve showing the relationship between the FGF-23 concentration and the measurement value prepared in step (3), the FGF-23 concentration in the sample is determined. Determining step;
(5) If the FGF-23 concentration in the sample determined in step (4) is higher than the reference value, the CKD patient is likely to develop hyperphosphatemia. Comparing the criteria described in the criteria table that if the CKD patient is less likely to develop hyperphosphatemia if below the reference value;
(6) As a result of the comparison in step (5), if the FGF-23 concentration in the sample determined in step (4) is above the reference value, the CKD patient is likely to develop hyperphosphatemia. If the value is less than the reference value, determining that the CKD patient is less likely to develop hyperphosphatemia.
The reference value is an upper limit value of a 95% confidence interval of FGF-23 concentration in a subject whose estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m 2 or more. reagent.
9. The upper limit of a 95% confidence interval of FGF-23 concentration in a subject whose estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m 2 or more is 50 to 150 pg / mL. Reagent.
The reagent according to any one of claims 6 to 9, wherein the CKD patient is a stage 3 to 5 CKD patient.