Method for measuring parathyroid hormone

Abstract

To improve sensitivity in detecting a PTH (parathyroid hormone).SOLUTION: A parathyroid hormone measurement method is the method for measuring a parathyroid hormone (PTH) by a sandwich method. In the method, an antibody for specifically recognizing the PTH as a primary antibody and also obtained by immobilizing two or more kinds of antibodies different in an epitope, or an antibody obtained by immobilizing an antibody to be bound to epitopes of two or more points of the PTH, and an antibody obtained by labelling an antibody for specifically recognizing the PTH so as to recognize an epitope different from that of the primary antibody, are allowed to react with the PTH in a specimen, so as to detect the label of a primary antibody-PTH-secondary antibody being a generated immunoreactive conjugate.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for measuring parathyroid hormone. [Background technology]

[0002] Parathyroid hormone (PTH) is a hormone secreted from the parathyroid gland. PTH is a type of peptide hormone consisting of 84 amino acid residues, and it regulates calcium concentration in the blood. However, when a patient suffers from a parathyroid tumor, renal failure, or functional parathyroid cyst, the blood concentration of this hormone becomes abnormal (Non-Patent Document 1, Non-Patent Document 2). Therefore, in order to diagnose these diseases, it is necessary to examine the blood concentration of this hormone.

[0003] PTH is secreted from the parathyroid gland into the bloodstream, where there are various decomposing enzymes. This causes the hormone to be broken down into several fragments in the bloodstream. Among these decomposition products, the one in which the first six amino acid residues from the N-terminus have been cut off is called intact PTH (iPTH) (Figure 1). The one that has not been broken down at all by enzymes is called whole PTH (wPTH) (Figure 1). The blood concentrations of these two types of PTH are primarily used in diagnosing diseases.

[0004] The measurement of blood levels of PTH usually involves the sandwich method, which uses two types of anti-PTH antibodies (primary and secondary antibodies) with different epitopes (Figure 2). In this method, the sample is first added to the solid phase of the primary antibody and allowed to react. This is then washed, and a secondary antibody (labeled secondary antibody) labeled with an enzyme such as alkaline phosphatase (ALP) is added to this. This is then washed again, and the signal emitted by the labeled secondary antibody bound to the solid phase is measured to determine the blood concentration.

[0005] Although the blood concentration of PTH is very important information clinically, it is not easy to construct a measurement system for it. In particular, since the concentration of PTH in the blood is very low, it is necessary to construct a measurement system with high sensitivity to detect it. When constructing a PTH measurement system, various factors such as reaction time, sample volume, and reaction conditions affect the detection sensitivity. However, it is not any of these factors that have the greatest effect on the detection sensitivity. The detection sensitivity of PTH is most strongly affected by the performance of the antibody used. Basically, the higher the affinity of an antibody with PTH is used, the higher the detection sensitivity will be. However, antibodies with high affinity for PTH are not easily obtained. Therefore, it is very difficult to detect PTH with high sensitivity, and this has been a major challenge until now. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] DB Endres, R Villaueva, CF Sharp Jr, FR Singer. (1991) Immunochemiluminometric and immunoradiometric determinations of intact and total immunoreactive parathyrin:performance in the differential diagnosis of hypercalcemia and hypoparathyroidism. Clin.Chem. 37,162-168. [Non-Patent Document 2] R Lepage, S Whittom, S Bertrand, G Bahsali, PD'Amour. (1992)Superiority of dynamic over static reference intervals for intact, midmolecule, and C-terminal parathyrin in evaluating calcemic disorders. Clin.Chem. 38,2129-2135. Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to improve the detection sensitivity of PTH. [Means for solving the problem]

[0008] As a result of extensive research into the above-mentioned problems, the present inventors have arrived at the present invention. (1) A method for measuring parathyroid hormone (PTH) by the sandwich method, - As the primary antibody, an antibody that specifically recognizes PTH and has two or more types of antibodies with different epitopes immobilized; and - A secondary antibody that specifically recognizes PTH and is labeled with an antibody that recognizes an epitope different from that of the primary antibody. is reacted with PTH in the sample, The method is characterized by detecting the label of the resulting immune reaction complex, which is the primary antibody-PTH-secondary antibody. (2) The method according to (1), wherein an antibody that specifically recognizes any one of amino acids 37 to 53 of whole PTH (wPTH) is used as the primary antibody. (3) The method according to (1) or (2), wherein an antibody that specifically recognizes any one of amino acids 53 to 67 of wPTH is used as the primary antibody. (4) In a method for measuring PTH by the sandwich method, - As a primary antibody, an antibody that specifically recognizes PTH and is capable of binding to two or more epitopes of PTH is immobilized; and - A secondary antibody that specifically recognizes PTH and is labeled with an antibody that recognizes an epitope different from that of the primary antibody. is reacted with PTH in the sample, The method is characterized by detecting the label of the resulting immune reaction complex, which is the primary antibody-PTH-secondary antibody. (5) The method according to (4), wherein an antibody that specifically recognizes any one of amino acids 37 to 53 of wPTH is used as the primary antibody. (6) The method according to (4) or (5), wherein an antibody that specifically recognizes any one of amino acids 53 to 67 of wPTH is used as the primary antibody.

[0009] The present invention will be described in detail below. First, the first invention will be described.

[0010] The first invention relates to a method for improving the detection sensitivity of PTH, characterized in that two or more types of antibodies with different epitopes are immobilized and used as primary antibodies (Figure 3). The type of antibody used is not particularly limited as long as each antibody has a different epitope, and examples that can be used include IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgA1, IgGA2, IgD, IgE, IgY, and mixtures of these.

[0011] The origin of the antibody is not particularly limited, and antibodies derived from mouse, rat, rabbit, camel, shark, alpaca, chicken, ostrich, goat, sheep, etc., or mixtures thereof can be used.

[0012] Furthermore, the antibody used herein may be a monoclonal antibody, a polyclonal antibody, or a mixture of monoclonal antibodies. Furthermore, the antibody used herein is not particularly limited in format as long as it can bind to PTH, and may be an intact antibody, F(ab), F(ab'), F(ab')2, single-chain antibody, scFv, sdAb, or a mixture thereof. Furthermore, the antibody used herein is not particularly limited in type as long as it has the property of binding to PTH, and may include an affinity molecule such as an aptamer.

[0013] The solid phase used for the primary antibody is not particularly limited, and examples thereof include magnetic particles, gold colloid, polystyrene, and the like.

[0014] The label used for the secondary antibody is not particularly limited, and examples thereof include enzymes such as alkaline phosphatase, horseradish peroxidase, and luciferase, and fluorescent proteins and compounds.

[0015] The specimen is not particularly limited, and examples thereof include blood, plasma, serum, and urine.

[0016] Next, the second invention will be described. The second invention is characterized in that an immobilized antibody that specifically recognizes PTH and can bind to two or more epitopes of PTH is used as the primary antibody. Examples of such antibodies include bi-specific antibodies, Tandem scFv (BiTEs), diabodies, and Tri-Fab (Figure 4). In other respects, the second invention is similar to the first invention. Effect of the Invention

[0017] According to the present invention, the detection sensitivity of PTH can be improved. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a diagram relating to fragments of PTH. [Diagram 2] FIG. 1 is a diagram showing the principle of detecting PTH by the sandwich method. [Diagram 3] FIG. 1 is a diagram showing the principle of detecting PTH using two or more types of antibodies with different epitopes as primary antibodies. [Figure 4] FIG. 1 shows the principle of detecting PTH using a bi-specific antibody as a primary antibody. [Diagram 5] FIG. 1 shows a calibration curve prepared using a wPTH monoclonal antibody as a secondary antibody. [Figure 6] FIG. 1 shows a calibration curve prepared using an iPTH monoclonal antibody as a secondary antibody. [Figure 7]This figure shows the effect on the S / N ratio when the mixing ratio is changed when a wPTH monoclonal antibody is used as a secondary antibody and two types of antibodies with different epitopes are used as primary antibodies. [Figure 8] This figure shows the effect on the S / N ratio when changing the mixing ratio when an iPTH monoclonal antibody was used as the secondary antibody and two types of antibodies with different epitopes were used as the primary antibodies. EXAMPLES

[0019] The present invention will be specifically illustrated by the following examples, but the present invention is not limited to these examples. The compositions and pH values ​​of the chemicals used in the examples are as follows. PBS (pH 7.5) 8.1mM disodium hydrogen phosphate dodecahydrate 1.5mM Potassium hydrogen phosphate 137mM Sodium Chloride 2.7mM Potassium Chloride Carbonate buffer (pH 9.6) 12mM Sodium Carbonate 38mM Sodium Bicarbonate Wash buffer (pH 7.4) 1mM Tris(hydroxymethyl)-aminomethane 7.5mM Sodium Chloride 0.05% tween20 Blocking buffer (pH 7.5) 8.1mM disodium hydrogen phosphate dodecahydrate 1.5mM Potassium hydrogen phosphate 137mM Sodium Chloride 2.7mM Potassium Chloride 1% skim milk Incubation buffer (pH 7.5) 8.1mM disodium hydrogen phosphate dodecahydrate 1.5mM Potassium hydrogen phosphate 137mM Sodium Chloride 2.7mM Potassium Chloride 0.1% skim milk ALP buffer (pH9.8) 1M Diethanolamine 0.5mM Magnesium chloride 10mM 4-Methylumbelliferyl phosphate Beads washing solution (pH 7.5) 1mM Tris(hydroxymethyl)-aminomethane 7.5mM Sodium Chloride 0.05% tween20.

[0020] [Example 1] (1) Preparation of immunogen First, 20 mg of ovalbumin (OVA) was prepared and dissolved in 2 ml of ultrapure water. Next, 20 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfо-SMCC) was weighed out and dissolved in 4 ml of ultrapure water. These solutions were then mixed and left to stand at room temperature for 1 hour to react. The solution was then passed through a PD-10 column (GE) equilibrated with PBS to desalt and remove unreacted sulfо-SMCC.

[0021] Next, a peptide consisting of the 34th to 84th amino acids of wPTH was prepared with cysteine ​​introduced at the N-terminus (PTH34-84). 20 mg of this peptide was weighed out and dissolved in 5 ml of PBS. This solution was then added to the OVA solution previously desalted with PD-10 and left to stand at room temperature for 2 hours to bind PTH34-84 to OVA (OVA_PTH34-84). PBS was then added to this solution to adjust the volume to 20 ml. The reaction solution was then placed in a dialysis tube and dialyzed against a PBS solution at 4°C to remove unreacted peptide. After dialysis, OVA_PTH34-84 was collected from the dialysis tube and used as an antigen for immunization.

[0022] (2) Immunization of experimental animals Mice (female, ICR, 4 weeks old) were prepared as immunized animals. Next, the previously prepared OVA_PTH34-84 solution was mixed with Freund's complete adjuvant at a 1:1 ratio. This mixture was then sonicated on ice to emulsify. 100 μl of this solution was then injected into the abdominal cavity of the prepared mice to immunize them.

[0023] One week after the first immunization, the mice were immunized again as follows. First, OVA_PTH34-84 recovered from dialysis was mixed with Freund's incomplete adjuvant in a 1:1 ratio. The mixture was then sonicated on ice to emulsify. 100 μl of this solution was then injected into the abdominal cavity of the mice for immunization. From the second immunization onwards, this immunization procedure using Freund's incomplete adjuvant was carried out once a week for 7 weeks.

[0024] (3) Cell fusion and screening After immunization, the mice were euthanized and spleen cells were collected. These cells were then mixed with myeloma cells in a 2:1 ratio and cell fusion was performed by electrofusion. After electrofusion was completed, the cells were collected and suspended in a fusion cell medium (E-RDF + 10% bovine serum + 5% BM conditioned + HAT) until the cell density of the spleen cells reached 6.7 × 10 4 The cell suspension was then plated onto a 384 well plate and cultured for 11 days at 37°C in 5% CO2. After the culture was completed, a small amount of culture medium was collected from each well of the plate. The wells in which antibodies that strongly bind to PTH were produced were examined by ELISA as follows.

[0025] (A) Anti-mouse antibody was dissolved in carbonate buffer to a concentration of 0.5 μg / ml, and 50 μl of this solution was dispensed into each well of an ELISA plate (384 wells). The plate was then left to stand at room temperature for 1 hour, and each well was washed three times with washing buffer. (B) 100 μl of blocking buffer was added to each well and incubated at room temperature for 1 hour, after which each well was washed once with washing buffer. (C) 50 μl of incubation buffer was added to each well. (D) 2 μl of culture supernatant was added to each well and allowed to stand at room temperature for 1 hour, after which each well was washed three times with washing buffer. (E) wPTH was dissolved in the incubation buffer to a concentration of 5 ng / ml. 50 μl of this solution was added to each well and allowed to stand at room temperature for 1 hour. Then, each well was washed three times with the washing buffer. (F) 50 μl of an antibody that recognizes the N-terminal region of wPTH and is labeled with ALP was added to each well and allowed to stand at room temperature for 1 hour. Then, each well was washed three times with the washing buffer. (G) 50 μl of ALP buffer was added to each well and allowed to stand for 30 minutes. (H) Fluorescence intensity was measured (Excitation 360 nm / Emission 465 nm) and wells containing antibodies that strongly bind to wPTH were examined.

[0026] (4) Cloning Several wells containing antibodies that strongly bind to wPTH were selected, and cells were collected from each well. These cells were then suspended in cell culture medium (E-RDF + 10% bovine serum) or cell fusion medium, and cultured at 37°C in a 5% CO2 environment. After confirming cell growth, limiting dilution was performed on each cell as described below to obtain monoclonal cells. (A) The collected cells were suspended in 6.4 ml of cell culture medium or cell fusion medium. (B) This cell suspension was dispensed into 64 wells of a cell culture plate (384 wells) at 50 μl per well. (C) An equal volume of fresh medium was added to the remaining 3.2 ml of cell suspension, and the cells were thoroughly suspended. (D) This cell suspension was dispensed into 64 wells of a cell culture plate (384 wells) at 50 μl per well.

[0027] (E) The above dilution and dispensing procedures were repeated until the expected number of cells in the well was 1 cell / well or less. (F) The cell culture plate was placed in an environment of 37°C and 5% CO2 and cultured. (G) The reactivity of the antibodies secreted into the culture supernatant with PTH was examined by ELISA under the same conditions as those used in the screening ELISA. (H) Among the wells containing antibodies that strongly react with wPTH, only those wells with the expected number of cells in the well of 1 cell / well or less were selected. These wells were then observed under a microscope to confirm that the cells were monoclonal. The cells were then collected from the wells. These cells were then suspended in new cell culture medium or cell fusion medium and cultured at 37°C in 5% CO2.

[0028] (5) Epitope Identification First, 20 mg of ovalbumin (OVA) was prepared and dissolved in 2 ml of ultrapure water. Next, 20 mg of sulfo-SMCC was weighed out and dissolved in 4 ml of ultrapure water. These solutions were then mixed and left to stand at room temperature for 1 hour to react. This solution was desalted by passing it through a PD-10 column (GE) equilibrated with PBS to remove unreacted sulfo-SMCC.

[0029] Next, we prepared a peptide consisting of the 37th to 53rd amino acids of wPTH with cysteine ​​introduced at the N-terminus (PTH37-53), a peptide consisting of the 46th to 60th amino acids of wPTH with cysteine ​​introduced at the N-terminus (PTH46-60), a peptide consisting of the 53rd to 67th amino acids of wPTH with cysteine ​​introduced at the N-terminus (PTH53-67), a peptide consisting of the 60th to 74th amino acids of wPTH with cysteine ​​introduced at the N-terminus (PTH60-74), and a peptide consisting of the 67th to 84th amino acids of wPTH with cysteine ​​introduced at the N-terminus (PTH67-84). Then, we weighed out 4 mg of each of these peptides and dissolved them in 1 ml of PBS. Next, the OVA solution desalted with PD-10 was divided into 5 equal parts, and each peptide solution dissolved in PBS was added to this OVA solution. The solution was then left to stand at room temperature for 2 hours to allow OVA to bind to each peptide (referred to as OVA_PTH37-53, OVA_PTH46-60, OVA_PTH53-67, OVA_PTH60-74, and OVA_PTH67-84, respectively). After the reaction, PBS was added to each solution to adjust the volume to 4 ml. The reaction solution was then placed in a dialysis tube and dialyzed against a PBS solution at 4°C to remove unreacted peptides. After dialysis, each sample was collected from the dialysis tube and used in the following experiment to identify the epitope of the monoclonal antibody.

[0030] (A) OVA_PTH37-53, OVA-PTH46-60, OVA-PTH53-67, OVA-PTH60-74, or OVA-PTH67-84 was dissolved in carbonate buffer to a concentration of 0.5 μg / ml, and 100 μl of each solution was dispensed into each well of a separate ELISA plate (96 wells). The plate was then left to stand at room temperature for 1 hour, and each well was washed three times with washing buffer. (B) 200 μl of blocking buffer was added to each well of the prepared ELISA plate and left to stand at room temperature for 1 hour, after which each well was washed once with washing buffer. (C) 70 μl of incubation buffer was added to all wells of the prepared ELISA plate. Then, 30 μl of culture supernatant of the monoclonal cells was added to each well of each plate. After leaving it at room temperature for 1 hour, each well was washed three times with washing buffer.

[0031] (D) ALP-labeled anti-mouse antibody was diluted 10,000-fold with incubation buffer. 100 μl of this solution was then added to all wells of the prepared ELISA plate and allowed to stand at room temperature for 1 hour. Each well was then washed three times with washing buffer. (E) 100 μl of ALP buffer was added to all wells of the prepared ELISA plate, and the plate was allowed to stand for 30 minutes. (F) Fluorescence intensity was measured (Excitation 360nm / Emission 465nm) to determine which fragment of PTH each antibody reacted with. As a result of the experiment, it was confirmed that a monoclonal antibody (PTH115) that recognizes the amino acids 37 to 53 of wPTH and a monoclonal antibody (PTH113) that recognizes the amino acids 53 to 67 of wPTH were isolated.

[0032] (6) Purification of antibodies and fluorescein (FITC) labeling The PTH115-producing cells and the PTH113-producing cells were each collected from the culture medium. These cells were then suspended in PBS and washed. Next, these cells were diluted to 1×10 6 The cells were suspended in serum-free medium for hybridomas (Cyteva) at a concentration of 100 cells / ml and cultured at 37° C. in an environment of 5% CO 2 .

[0033] After cell culture, the culture supernatants containing the antibodies were collected and filtered. These solutions were then passed through a Protein A column (Tosoh Corporation) equilibrated with PBS. PBS was then passed through the column to wash each column. 100 mM Gly-Cl (pH 2.5) was then passed through the column to elute each antibody from the column. The purified antibodies were then concentrated and placed in dialysis tubes and dialyzed against PBS solution at 4°C. After dialysis, the monoclonal antibodies PTH115 and PTH113 were collected from the dialysis tubes, and the protein concentrations were measured by the BCA method.

[0034] The concentration and volume of the purified monoclonal antibodies PTH115 and PTH113 were adjusted with PBS to 1 mg / ml and 1 ml, respectively. Then, each of these antibody solutions was placed in a dialysis tube and dialyzed against 50 mM sodium borate solution (pH 9) at 4°C. After the dialysis was completed, each antibody was recovered from the dialysis tube. Next, 1.9 mg of 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester was weighed out and dissolved in 1 ml of DMSO. Then, 10 μl of this solution was added to 1 ml of each antibody solution dialyzed against boric acid solution. Then, these solutions were mixed by inversion overnight at 4°C, and each antibody was labeled with FITC. After the reaction, 100 μl of glycine solution dissolved in ultrapure water to a concentration of 12.5 mg / ml was added to each of the reaction solutions to stop the reaction. Then, each of these solutions was placed in a dialysis tube and dialyzed against PBS solution at 4°C to remove unreacted FITC. After dialysis, the FITC-labeled monoclonal antibodies PTH115 and PTH113 were collected from the dialysis tube.

[0035] (7) Evaluation of antibody performance The performance of FITC-labeled monoclonal antibodies PTH115 and PTH113 was evaluated as follows.

[0036] First, the FITC-labeled monoclonal antibody PTH115 or PTH113 was diluted with the incubation buffer to adjust the concentration to 0.1 mg / ml. Next, a certain amount of this antibody solution or a mixture of these solutions was added to the particles on which the anti-FITC antibody was immobilized, and reacted at room temperature for one hour. After that, the particles were washed three times with bead washing solution, and a certain amount of the secondary antibody labeled with ALP was added. Here, either an antibody that recognizes the first to sixth amino acid residues counting from the N-terminus of wPTH (referred to as the wPTH antibody) or an antibody that recognizes the seventh to 34th amino acid residues counting from the N-terminus of PTH (referred to as the iPTH antibody) was used as the secondary antibody. Then, a certain amount of a calibrator solution (six types, Cal1-Cal6), with a known wPTH concentration, was added to this and reacted for a certain time.

[0037] After that, B / F separation was performed, and the ALP substrate was added, and the signal emitted from the ALP-labeled secondary antibody immobilized on the beads was measured. The signal (N) when a calibrator solution (Cal1) containing no PTH was added was set to 1, and the signal (S) when each calibrator solution (Cal2-6) was added was calculated as a relative value (S / N ratio). The PTH concentration was plotted on the horizontal axis and the S / N on the vertical axis, and the measurement results when each calibrator solution was used were plotted to create a calibration curve. This series of operations was performed fully automatically at 37°C using a Tosoh AIA-600II.

[0038] As a result of the experiment, it was found that the S / N ratio of the standard curve was improved when the monoclonal antibodies PTH115 and PTH113 were mixed in a 1:1 ratio (PTH113+PTH115) compared to when they were used alone (Fig. 5, Fig. 6). In addition, when the mixture ratio of the monoclonal antibodies PTH115 and PTH113 was changed (wPTH concentration 16.1pg / ml or 1.56ng / ml, and the secondary antibody was wPTH antibody or iPTH antibody), it was found that the S / N ratio of the standard curve was improved when the PTH113:PTH115 ratio was in the range of 2:8 to 8:2 compared to when each antibody was used alone (Fig. 7, Fig. 8). In addition, this improvement in the S / N ratio was confirmed when either the wPTH antibody or the iPTH antibody was used as the secondary antibody (Fig. 5, Fig. 6, Fig. 7, Fig. 8). These results demonstrated that the detection sensitivity of PTH can be improved by using two or more antibodies with different epitopes as primary antibodies.

Claims

1. In a method for measuring parathyroid hormone (PTH) by a sandwich method, As a primary antibody, the following (i) and (ii) were immobilized: (i) A monoclonal antibody that specifically recognizes any of the amino acids from 37 to 53 of whole PTH (wPTH). (ii) a monoclonal antibody that specifically recognizes any of the amino acids 53 to 67 of wPTH; and The secondary antibody is a labeled antibody that specifically recognizes PTH and recognizes an epitope different from that of the primary antibody. is reacted with PTH in the sample, The method comprises detecting the label of the resulting immune reaction complex, which is primary antibody-PTH-secondary antibody.

2. The method described in claim 1, wherein the mixing ratio of the primary antibodies (i) and (ii) is in the range of 2:8 to 8:2.

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