Method for measuring fibroblast growth factor-23 and reagent therefor
A method for measuring fibroblast growth factor-23 (FGF-23) in a sample, which comprise the following steps: (1) reacting, in an aqueous medium, FGF-23 in a sample with magnetic particles, a first antibody or a fragment thereof which binds to FGF-23, and a second antibody or a fragment thereof which binds to FGF-23, to form on the magnetic particles an immunocomplex comprising the first antibody or a fragment thereof which binds to FGF-23, FGF-23, and the second antibody or a fragment thereof which binds to FGF-23; (2) collecting the magnetic particles in the reaction mixture after step (1) by magnetic force, and separating the magnetic particles collected by magnetic force from the other components; and (3) measuring the immunocomplex on the magnetic particles separated in step (2). The present invention provides a method for measuring FGF-23 in a sample, which have a high sensitivity and have a wide measurement range.
1. A method for measuring fibroblast growth factor-23 (FGF23) in a serum or plasma sample, comprising:
(1) simultaneously reacting, in an aqueous medium, FGF23 in the sample with (i) a magnetic particle, (ii) a first antibody which binds to FGF23, and (iii) a second antibody which binds to FGF23, to form on the magnetic particle an immunocomplex comprising the first antibody, FGF23, and the second antibody in a reaction mixture,
wherein the first antibody is an anti-FGF-23 monoclonal antibody produced by hybridoma deposited as FERM BP-7838, and the second antibody is an anti-FGF-23 monoclonal antibody Fab or Fab′ fragment produced by digesting anti-FGF23 monoclonal antibody produced by the hybridoma deposited as FERM BP-7839,
the magnetic particle does not directly immobilize the first antibody; and
one of two substances with affinity to each other is fixed to the magnetic particle and the other of the two substances with affinity to each other is bound to the first antibody,
(2) collecting the magnetic particle after step (1) by magnetic force, and separating the magnetic particle collected by magnetic force from the other components; and
(3) measuring the immunocomplex on the magnetic particle separated in step (2).
2. The method of claim 1 , wherein the second antibody is a labeled antibody.
3. The method of claim 2 , wherein the labeled antibody is an alkaline phosphatase-labeled antibody.
4. The method of claim 1 , wherein the measuring of the immunocomplex on the magnetic particle in step (3) is carried out by measurement of chemiluminescence.
5. The method of claim 1 , wherein the two substances with affinity to each other are avidins and biotin.
6. The method of claim 1 , wherein the sample is derived from a dialysis patient under anticoagulant treatment.
7. The method of claim 6 , wherein the sample comprises fibrin.
8. The method of claim 1 , wherein the method detects FGF23 concentrations of 5 pg/mL to 10,000 pg/m L.
9. The method of claim 1 , wherein the concentration of the FGF23 in the sample is from 5 ng/ml to 173.7 pg/ml.
10. The method of claim 1 , wherein a concentration of the magnetic particle in the reaction mixture is 0.1 mg/mL to 10 mg/mL.
11. The method of claim 1 , wherein the magnetic particle has a particle diameter from 1 μm to 6 μm.
12. The method of claim 1 , wherein the reacting step is performed at a temperature from 0° C. to 50° C.
13. The method of claim 1 , wherein the reacting step is performed at a temperature from 20° C. to 40° C.
14. The method of claim 1 , wherein the reacting step is performed for a duration from 5 minutes to 20 minutes.
15. The method of claim 1 , wherein a salt concentration in the reaction mixture is from 40 mmol/L to 400 mmol/L.
16. The method of claim 1 , wherein the reaction mixture comprises at least one selected from the group consisting of a metal ion, a sugar, an antiseptic agent, a protein, a surfactant, and a protein stabilizer.
17. The method of claim 1 , wherein the reaction mixture comprises a metal ion selected from the group consisting of magnesium ion, manganese ion, zinc ion, and a mixture thereof.
18. The method of claim 1 , wherein the reaction mixture comprises a sugar selected from the group consisting of mannitol, sorbitol, and a mixture thereof.
19. The method of claim 1 , wherein the second antibody is labeled with a fluorescence substance, and the measuring of the immunocomplex on the magnetic particle in step (3) is carried out by measurement of fluorescence.
20. The method of claim 1 , wherein the second antibody is labeled with an enzyme, and the measuring of the immunocomplex on the magnetic particle in step (3) is carried out by measurement of chemiluminescence.
21. The method of claim 20 , wherein the enzyme is selected from the group consisting of alkaline phosphatase, peroxidase, β-D-galactosidase, and luciferase.