JP3299787B2 - Radioimmunoassay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a radioimmunoassay.

[0002]

2. Description of the Related Art As a conventional immunoassay, an antigenic substance is bound to a first antibody immobilized on an insoluble solid, and a second antibody labeled with radioactivity or an enzyme is reacted with the antigen. A method for measuring the amount (so-called sandwich method) is known.

[0003] Among the sandwich methods, in the enzyme immunoassay using an enzyme-labeled second antibody, the amount of labeled second antibody used is not limited, so that a highly sensitive second antibody cannot be obtained. However, the sensitivity of the labeled second antibody can be compensated for by using a high concentration. In this case, high sensitivity cannot be obtained because the enzyme activity is smaller than the radioactivity of the radioactive substance. Therefore, a sensitization method in which many labeling enzymes are bound to one molecule of the second antibody bound to the antigenic substance has been adopted. [For example, avidin-biotin complex method ("Enzyme Immunoassay" by P. Tissen)) and the like. However, even if these methods are used, the measurement sensitivity in the enzyme immunoassay is not improved as much as the method of labeling with radioactivity, and it is difficult to obtain a highly sensitive measurement system (JP-A-63-118656). Reference).

On the other hand, a radioimmunoassay using a radioactively labeled second antibody is a method that can easily obtain high measurement sensitivity because radioactivity can be detected even at a concentration lower than the enzyme activity. However, since the amount of radioactivity that can be added to the measurement system is limited, when the concentration of the second antibody is low and only a low-sensitivity second antibody is obtained, the second antibody cannot sufficiently bind to the antigenic substance. And the sensitivity of the measurement system is not sufficiently improved.

More specifically, in the reaction solution, the ratio of the concentration of the antigenic substance bound to the second antibody to the concentration of the antigenic substance not bound to the antibody ([AgAb2] / [A
g]) is determined as follows by the concentration of the second antibody ([Ab2]) and the dissociation constant (Kd).

[0006]

(Equation 1)

That is, the higher the concentration of the second antibody and the lower the dissociation constant, the more the second antibody binds to the antigenic substance, and the higher the measurement sensitivity.

However, the amount of radioactivity that can be used in the measurement system is limited, and the concentration of the second antibody is limited. Therefore, when the dissociation constant of the second antibody is large, a measurement system with sufficient sensitivity can be obtained. Not possible [Kobata Kodo et al. “Nuclear medicine” 2
6 No. 3 (1989)].

[0009] As described above, conventionally, it has been considered particularly important in immunoassays to obtain a highly sensitive second antibody in order to obtain a highly sensitive assay system. However, it is difficult to obtain a highly sensitive antibody, and a great deal of labor has been spent on it.

In the prior art, since the second antibody is directly labeled, if two or more types of measurement systems are to be prepared for measuring two or more types of antigenic substances, the respective antigenic substances must be prepared. Since two or more types of second antibodies recognizing, must be separately labeled, this leads to an increase in cost.

[0011]

SUMMARY OF THE INVENTION The present invention provides a method for radioimmunoassay to obtain a highly sensitive assay even when the second antibody has low sensitivity. It is intended to provide a completely new concept of measurement that can be used. More specifically, the present invention relates to a parathyroid hormone-related protein (PTHrP), which was conventionally difficult to obtain a highly sensitive antibody.
The purpose of the present invention is to provide a radioimmunoassay.

[0012]

SUMMARY OF THE INVENTION The present invention provides a first antibody (A) on an insoluble solid that recognizes a parathyroid hormone-related protein (B), a parathyroid hormone-related protein (B), and the first antibody. Reacting a second antibody (C) recognizing the parathyroid hormone-related protein from a different animal species at a concentration where the concentration of the second antibody is higher than the dissociation constant between the second antibody and the parathyroid hormone-related protein. To obtain an immune complex (D), and then reacting the immune complex with a radiolabeled probe (E) obtained by radiolabeling a probe that specifically reacts with the second antibody.
A radioimmunoassay, wherein the parathyroid hormone-related protein (B) is measured by measuring the radioactivity of a solid or a reaction solution.

The measuring method of the present invention requires a highly sensitive measuring method because the concentration in a test sample (eg, in blood) is low.
In addition, antigenicity has a problem that it is difficult to produce a highly sensitive antibody for one or all of two or more antigenic determination sites on a molecule, which is required for measurement by the sandwich method. This is particularly effective when measuring a substance.

The antigenic substances having the above-mentioned problems include, for example, parathyroid hormone-related protein (PT
HrP), atrial natriuretic hormone (ANP),
Endothelin (ET), corticotropin (ACT)
H) and other peptide hormones.
Among them, PTHrP is particularly suitable as an antigenic substance in the measurement method of the present invention because PTHrP has a particularly low blood concentration and makes it difficult to obtain a highly sensitive antibody.

Parathyroid hormone-related protein (PTHrP) used as an antigenic substance in the present invention
(B) may be any as long as it has antigenicity in the test sample to be subjected to the measurement. The protein can be obtained according to a conventionally known method and used as an antigen for preparing an antibody or as a standard solution for preparing a calibration curve. As a method for obtaining the protein, for example, P
A method of incorporating the THrP gene into an arbitrary vector, introducing the THrP gene into a host such as Escherichia coli suitable for the vector used, expressing the resultant, and extracting and purifying this, and the like can be mentioned.

As the first antibody (A), a monoclonal antibody obtained by immunizing vertebrates such as rabbits, goats, sheep and guinea pigs with the above parathyroid hormone-related protein by a conventionally known method, or Toyama Sakuji,
Tamoe Ando, "Monoclonal Antibody Experiment Manual" (198
7) A hybridoma cell (preferably a cell obtained by cell fusion of an antibody-producing cell (preferably a mouse cell) and a myeloma cell (preferably a cell of the same type as the antibody-producing cell) by the method described in Kodansha, etc. (Derived from mouse). These antibodies were obtained by ammonium sulfate fractionation, DEAE cellulose chromatography, affinity chromatography (Nao Matsui, Hideo Naruchi, Mitsuko Usui, “Introduction to Immunological Experiments” (19)
81) Purify and use by methods such as the Society Press Center.

Examples of the insoluble solid include inorganic carriers such as glass (porous glass, frosted glass, etc.), silica gel, siliceous carriers such as bentonite, and magnetic materials.
And organic carriers such as plastics, polymer gels (polysaccharides such as dextran and agarose or polyacrylamide gels), filter paper and the like. Of these, preferred are glass (glass beads and glass test tubes) and plastic (plastic beads, plastic tubes, plastic trays), which can easily and stably bind antibodies, and are easy to handle.

As a method for binding the first antibody to the insoluble solid, a method for chemically binding the antibody to glass, for example, a method using a silane coupling agent and, if necessary, a cross-linking agent (JP-A-54-108696). ) Or physical adsorption (U.S. Pat. Nos. 4,280,992 and 3,682,761), a method of physically adsorbing an antibody to plastic ("Enzyme Immunoassay" by P. Tississen), or avidin immobilized on a solid; There is a method of immobilizing efficiently by reacting an antibody with biotin chemically bonded thereto.

As the second antibody (C), a cloned antibody derived from an animal species different from that of the first antibody and recognizing the above-mentioned protein (for example, when the first antibody is derived from rabbit, goat, sheep, guinea pig, etc.) Antibody: When the first antibody is derived from sheep, an antibody of rabbit, goat, guinea pig, etc.): or when the first antibody is a monoclonal antibody recognizing the above protein, a polyclonal antibody against the above protein may be mentioned. Examples of the second antibody include antiserum, hybridoma culture solution, ascites, and immunoglobulin obtained by purifying these. When using avidin or streptavidin as a probe, biotin is chemically bound after purifying the second antibody by a known method.

In order to improve the measurement sensitivity by the present method as compared with the conventional radioimmunoassay, particularly when the sensitivity of the second antibody is low, that is, the dissociation between the second antibody and the parathyroid hormone-related protein. The case where the constant is large is effective. The dissociation constant between the second antibody and the protein is 10 ^-6
９9 × 10 ^-12 mol / l, more preferably 10 ^-8 to 10
^In the case of ^-11 mol / l, particularly improvement of the measurement sensitivity can be achieved as compared with the conventional radioimmunoassay, but it is not limited to this value depending on the concentration of the protein to be measured.

The concentration of the second antibody (concentration as an immunoglobulin) is determined mainly by the dissociation constant (Kd) of the reaction between the second antibody and the protein, and is 0.1 Kd to 100 Kd.
Preferably, 1 Kd to 10 Kd (for example, Kd is 1 × 10 ⁻⁹ mole)
/ l, the concentration of the second antibody is 1 × 10 ^-10 to 1
× 10 ^-7 mole / l, preferably 1 × 10 ^-9 to 1 × 10 ^-8 mo
le / l) is desirable, but is not necessarily limited to this value by the concentration of the protein in the test sample and the labeling rate or concentration of the radiolabeled probe (D).

Here, the dissociation constant is determined by a general method [for example, P. Tijssen, “Practice and Theory of Enzyme Immunoassay” (P. Tijssen,
“Practice and theory of enzyme immunoassays” (19
85) Scatchard plot analysis method described in Elsevier Science Publishers)].

That is, the dissociation constant (Kd) is determined by the concentration of the antigenic substance bound to the antibody [AbAg], the concentration of the antigenic substance not bound to the antibody [Ag], and the concentration of the antibody not bound to the antigenic substance. It is represented by the following equation by the concentration [Ab].

[0024]

(Equation 2)

Here, assuming that [Ag] is F, [AbAg] is B, and the total concentration of the antibody in the reaction solution is [Ab] _TOTAL , [Ab] = [Ab] _TOTAL- B. Can be transformed as follows.

[0026]

(Equation 3)

In this equation, the ratio of the concentration of the antigenic substance bound to the antibody to the concentration of the total antigenic substance in the solution (B /
T), and B / F can be measured by a tracer experiment. B is the amount of the total antigenic substance added to the reaction solution and B
Since it can be calculated from / T, B and B / T can be measured while changing the amount added to the reaction solution in various ways. When this result is plotted on a graph in which B is the horizontal axis and B / F is the vertical axis, the reciprocal of the slope of the approximate straight line is the dissociation constant.

The immune complex (D) can be obtained by reacting the first antibody (A), the parathyroid hormone-related protein (B) and the second antibody (C) at the above concentration simultaneously or sequentially. For example, a buffer solution containing an insoluble solid to which a first antibody is bound or an insoluble solid to which avidin is immobilized and a biotinylated first antibody, a test sample containing a parathyroid hormone-related protein, and a buffer solution containing a second antibody are simultaneously used. Alternatively, an immune complex can be obtained by sequentially keeping the temperature. After the incubation, the reaction solution is removed from the insoluble solid. Next, after adding a washing solution (for example, distilled water, physiological saline, phosphate buffer, a diluted solution of a surfactant, etc.),
Several operations to wash insoluble solids (eg 1 to 3 times)
By repeating, the immune complex can be isolated from unreacted substances.

The radiolabeled probe (E) is obtained by radiolabeling a probe that specifically reacts with the second antibody.
Examples of such a probe include an antibody that recognizes an immunoglobulin derived from an animal of the same species as the second antibody (for example, an anti-rabbit immunoglobulin / goat antibody when the second antibody is derived from a rabbit). When biotin is bound to the second antibody, avidin and streptavidin can be used as the probe. The probe is a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C,
Preferably, it is used by radiolabeling with ¹²⁵ I) which has a long half-life and a high specific activity and is easy to measure. Examples of the radioactive labeling method include the method described in “Radio Immunoassay” (1974), published by Kodansha, edited by Minoru Irie.

The reaction between the immune complex (D) and the radiolabeled probe (E) can be carried out by keeping the immune complex and the buffer solution containing the probe warm. Insulation is performed under normal conditions (for example, 2 to 50 ° C., preferably 20 to 3 ° C.).
(0 ° C, 5 minutes to 2 days, preferably 5 hours to 1 day). After incubation, the reaction product of the immune complex and the radiolabeled probe was washed with a washing solution (distilled water, physiological saline, phosphate buffer, etc.), and a well-type scintillation gamma counter (ARC-1000, manufactured by Aloka). Radioactivity).

The concentration of the parathyroid hormone-related protein in the test sample is determined by previously performing a measurement using a standard solution or the like of the protein to prepare a calibration curve, and comparing this with the radioactivity of the test sample. You can ask.

[0032]

The present invention will be described in more detail with reference to the following Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 Measurement of Parathyroid Hormone-Related Protein (PTHrP) (a) Preparation of PTHrP Standard Solution Bjorn Nilsson, Lars Abrahamsen, Method in Enzymology (Biorn Nilsson, Lars Abraha)
msen, Method in Enzymol.) 185 (1990) 144, the PTHrP gene (cDNA) obtained from Yale University in the United States [Proceedings of National Academy of Science, such as Arthur E. Brodas, et al. (Arthur E. Broadus, et al., Proc. Na
Acad. Sci. USA) 85 (1988) 597]
It was incorporated into a fusion protein expression vector (pRIT2T, manufactured by Pharmacia) and introduced into Escherichia coli (N4830-1 strain).

After the Escherichia coli was grown, a fusion protein of PTHrP and protein A was produced. The fusion protein was extracted, and PTHrP and protein A were chemically cleaved by BrCN treatment to obtain crude PTHrP. This crude PTHrP is subjected to affinity chromatography,
Purified by ion exchange chromatography and gel filtration. After the obtained PTHrP was hydrolyzed, its concentration was quantified by an amino acid analysis method, and then 0.32 in phosphate-buffered saline containing 5% bovine serum albumin, 0.8% bovine gamma globulin and 0.05% Tween 20. ~ 1
It was adjusted to 000 pmole / l.

(B) Preparation of Anti-PTHrP Monoclonal Antibody (Primary Antibody) -Binding Plastic Beads A mouse (Balb / c) was treated with PTH bound to thyroglobulin.
From the spleen cells obtained by immunization with rP (1-34) (manufactured by Peninsula) and mouse myeloma cells, "Monoclonal Antibody Experiment Manual" (1)
987) Monoclonal hybridoma was obtained by the method described in Kodansha. Next, the obtained monoclonal hybridoma was ascites-purified, and the ascites was purified to obtain anti-PTHrP.
Monoclonal immunoglobulins were produced. Anti-PT made
HrP monoclonal immunoglobulin was coated on plastic beads by the method described in the above-mentioned document “Practice and Theory of Enzyme Immunoassay”.

(C) Preparation of anti-PTHrP double-cloned antibody (second antibody) PTHrP (50-83) synthesized by an automatic solid phase peptide synthesizer manufactured by Applied Biosystems was bound to thyroglobulin, and the rabbit was lysed. Immunization and anti-P
THrP antiserum was obtained. By purifying the antiserum by affinity chromatography, a highly specific anti-P
A THrP monoclonal antibody was obtained and used as a second antibody.

(D) Measurement of dissociation constant of anti-PTHrP double-cloned antibody (second antibody) with PTHrP (1-87) (antigenic substance) (i) Preparation of ¹²⁵ I-PTHrP (1-87) (1-87) was labeled with ¹²⁵ I according to the method described by Minoru Irie, "Radioimmunoassay" (1974) published by Kodansha, purified by reversed-phase high-performance liquid chromatography, and then 2% bovine serum albumin was purified. It was adjusted to 400 Bq / 0.1 ml with a phosphate buffered saline solution.

(Ii) Preparation of B / F separating agent Anti-rabbit immunoglobulin / goat serum (manufactured by Institute of Immunology) 1/256, normal rabbit serum (manufactured by Institute of Immunology)
1/124 and polyethylene glycol 12.5%
Was prepared as a B / F separating agent.

(Iii) Measurement of dissociation constant 0.1 ml of the standard PTHrP solution was added to 0.1 ml of the second antibody (anti-PTHrP double-clone immunoglobulin) solution.
0.1 ml of ¹²⁵ I-PTHrP (1-87) solution was added,
After incubating at 4 ° C. for 48 hours, B / F separating agent 0.5
The supernatant was removed by centrifugation, and the radioactivity of the precipitate was measured using a well-type scintillation gamma counter (ARC-1000, manufactured by Aloka).

The Scatchard analysis was performed by the method described in the above-mentioned document “Practice and Theory of Enzyme Immunoassay”, the binding constant was determined, and the reciprocal number was taken as the dissociation constant.

FIG. 1 is a Scatchard plot according to this embodiment. Further, the dissociation constants obtained from this example were as follows. Dissociation constant 0.3 nmole / l

(E) Preparation of second antibody solution From the dissociation constant determined in the preceding section, the second antibody was 1.5 nmole / l
At a concentration of. The second antibody was diluted with phosphate buffered saline containing 2% bovine serum albumin so that the final concentration in the reaction solution was the same.

(F) Preparation of ¹²⁵ I-labeled anti-rabbit immunoglobulin antibody Anti-rabbit immunoglobulin / goat antibody (Dako)
0.25 to 40 μg was added to 1 according to the method described in “Radio Immunoassay” edited by Minoru Irie (1974) published by Kodansha.
Labeled with 8.5 MBq of ¹²⁵ I, purified by gel filtration, 4 KBq / 0.3 ml in phosphate buffered saline containing 4% bovine serum albumin, 0.53% bovine gamma globulin.
Was prepared.

(G) Measurement of PTHrP (i) Preparation of calibration curve A standard PTHrP solution (0.32 to 1000 pm) was placed in a test tube.
(ole / l) 0.2 ml, 0.1 ml of second antibody solution and anti-PT
One HrP monoclonal antibody (primary antibody) -bound plastic bead was added, and the mixture was kept at room temperature for 20 hours. Thereafter, the reaction solution was removed, and the plate was washed with distilled water. Next, 0.3 ml of an anti-rabbit immunoglobulin / goat antibody solution labeled with ¹²⁵ I was added,
After keeping at room temperature for 20 hours, the reaction solution was removed, washed with distilled water, and the radioactivity of the beads was measured with a well-type scintillation gamma counter (ARC-1000, manufactured by Aloka).

FIG. 2 is a calibration curve according to this embodiment. In addition, the measurement sensitivity, the measurement range and the PTHrP concentration were 8
The measurement accuracy (coefficient of variation) at pmole / l and 200 pmole / l was as follows.

(Ii) Measurement of PTHrP in Humoral Hypercalcemia Patient Sample The measurement was carried out using humoral hypercalcemia patient plasma as a test sample instead of standard PTHrP according to the measurement method described in the preceding section. The PTHrP concentration in the test sample was determined by comparing the calibration curve prepared in the previous section with the radioactivity of the test sample. The PTHrP concentration in the test sample was as follows. Patient sample measurements 5.8 pmole / l and 6.0 pmole / l

Comparative Example 1 PTHrP (1-8) was used at a concentration lower than the dissociation constant between the second antibody and the antigenic substance.
7) Measurement (a) Preparation of PTHrP Standard Solution The procedure was the same as in Example 1 (a). (B) Preparation of Anti-PTHrP Monoclonal Antibody (Primary Antibody) -Binding Plastic Beads According to Example 1 (b). (C) Preparation of anti-PTHrP double-cloned antibody (second antibody) The procedure was the same as in Example 1 (c).

(D) Preparation of Second Antibody Solution According to Example 1 (e). The second antibody was used after being diluted so that the final concentration in the reaction solution was 0.0015 nM. (E) Preparation of ¹²⁵ I-labeled anti-rabbit immunoglobulin antibody The procedure was as in Example 1 (f). (F) Measurement of PTHrP According to Example 1 (g).

FIG. 3 shows a calibration curve according to this comparative example. In addition, the measurement sensitivity, the measurement range and the PTHrP concentration were 4
The measurement accuracy (coefficient of variation) at 0 pmole / l and 200 pmole / l was as follows.

Comparative Example 2 Measurement of PTHrP (1-87) by Conventional Sandwich Method (Method of Directly Labeling a Second Antibody) (a) Preparation of PTHrP Standard Solution The procedure was the same as in Example 1 (a). (B) Preparation of Anti-PTHrP Monoclonal Antibody (Primary Antibody) -Binding Plastic Beads According to Example 1 (b). (C) Preparation of anti-PTHrP double-cloned antibody (second antibody) The procedure was the same as in Example 1 (c).

(D) Preparation of ¹²⁵ I-labeled anti-PTHrP monoclonal antibody (labeled second antibody) An anti-PTHrP monoclonal antibody (second antibody) was prepared in Example 1.
Labeling and purification were carried out according to (f), and the concentration was adjusted to 4 KBq / 0.1 ml with phosphate buffered saline containing 2% bovine serum albumin.

(E) Measurement of PTHrP A standard PTHrP solution (0.32 to 200 pmo
le / l) or 0.2 ml of test sample of humoral hypercalcemia patient plasma, add 0.1 ml of ¹²⁵ I-labeled anti-PTHrP monoclonal antibody solution and one anti-PTHrP monoclonal antibody-bound plastic bead, , And the reaction solution was removed, washed with distilled water, and the radioactivity of the beads was measured with a counter.

FIG. 4 is a calibration curve according to this comparative example. In addition, the measurement sensitivity, the measurement range, and the PTHrP concentration were 40 pmol.
Measurement accuracy (coefficient of variation) at e / l and 200pmole / l
And patient sample measurements were as follows:

Reference Example 1 Adrenocorticotropin (ACT)
Measurement of H) (a) Preparation of ACTH standard solution After hydrolysis of human ACTH obtained from Sigma, based on a value quantified with an amino acid analyzer (manufactured by Hitachi, Ltd.),
2.2-140 pm in phosphate buffered saline containing 5% bovine serum albumin and 0.8% bovine gamma globulin
ole / l.

(B) Preparation of Plastic Beads Conjugated with Anti-ACTH Monoclonal Antibody (Primary Antibody) Mouse spleen cells immunized with ACTH (manufactured by Sigma) and monoclonal hybridomas obtained from mouse myeloma cells were ascitesed. , By purifying ascites, anti-A
CTH monoclonal immunoglobulin was produced. The prepared anti-ACTH monoclonal immunoglobulin was prepared in Example 1 (b).
Coated on plastic beads according to.

(C) Preparation of anti-ACTH double-cloned antibody (second antibody) ACT obtained from Sigma in accordance with Example 1 (c)
Immunize rabbits with H to obtain anti-ACTH antiserum, purify,
A highly specific anti-ACTH monoclonal antibody was obtained.

(D) Measurement of dissociation constant of anti-ACTH double-cloned antibody (second antibody) with ACTH (antigenic substance) The measurement was performed in the same manner as in Example 1 (d). FIG. 5 is a Scatchard plot according to the present reference example. In addition, the dissociation constants obtained from this reference example were as follows. Dissociation constant 0.009 nmole / l

(E) Preparation of second antibody solution From the dissociation constant determined in the preceding section, the second antibody was 0.15 nmole /
l concentration. The second antibody was used after being diluted with phosphate buffered saline containing 2% bovine serum albumin so that the final concentration in the reaction solution was the same.

(F) Preparation of ¹²⁵ I-labeled anti-rabbit immunoglobulin antibody The procedure was the same as in Example 1 (f).

(G) Measurement of ACTH A standard ACTH solution (10-640 pg / ml) was placed in a test tube.
2 ml, 0.1 ml of anti-ACTH double clone antibody solution and anti-AC
After one TH monoclonal antibody-bound plastic bead was kept at room temperature for 20 hours, the reaction solution was removed and washed with distilled water. Next, 0.3 ml of an anti-rabbit immunoglobulin / goat antibody solution labeled with ¹²⁵ I was added, and the mixture was incubated at room temperature for 20 hours. The reaction solution was removed, washed with distilled water, and the radioactivity of the beads was measured with a counter. did.

FIG. 6 shows a calibration curve according to this reference example. In addition, the measurement sensitivity, the measurement range, and the ACTH concentration were 8.
The measurement accuracy (coefficient of variation) at 9 pmole / l and 140 pmole / l was as follows.

Reference Example 2 Measurement of ACTH at a concentration where the second antibody is lower than the dissociation constant between the second antibody and the antigenic substance (a) Preparation of ACTH standard solution The procedure of Reference Example 1 (a) was followed. (B) Preparation of Anti-ACTH Monoclonal Antibody (Primary Antibody) -Binding Plastic Beads The procedure was the same as in Reference Example 1 (b). (C) Preparation of anti-ACTH double-cloned antibody (second antibody) The procedure was the same as in Reference Example 1 (c).

(D) Preparation of Second Antibody Solution According to Reference Example 1 (e). The second antibody was used after being diluted so that the final concentration in the reaction solution was 0.0015 nM. (E) Preparation of ¹²⁵ I-labeled anti-rabbit immunoglobulin antibody The procedure was as in Example 1 (f). (F) Measurement of ACTH According to Reference Example 1 (g).

FIG. 7 is a calibration curve according to this reference example. In addition, the measurement sensitivity, the measurement range and the ACTH concentration were 36
The measurement accuracy (coefficient of variation) at pmole / l and 140 pmole / l was as follows.

Reference Example 3 Measurement of ACTH by Conventional Sandwich Method (Method of Directly Labeling a Second Antibody) (a) Preparation of ACTH Standard Solution The same procedure as in Reference Example 1 (a) was used. (B) Preparation of Anti-ACTH Monoclonal Antibody (Primary Antibody) -Binding Plastic Beads The procedure was the same as in Reference Example 1 (b). (C) Preparation of anti-ACTH double-cloned antibody (second antibody) The procedure was the same as in Reference Example 1 (c).

(D) Preparation of ¹²⁵ I-labeled anti-ACTH polyclonal antibody (labeled second antibody) An anti-ACTH polyclonal antibody (second antibody) was prepared in Example 1.
Labeling and purification were carried out according to (f), and the concentration was adjusted to 4 KBq / 0.1 ml with phosphate buffered saline containing 2% bovine serum albumin.

(E) Measurement of ACTH A test tube was charged with a standard ACTH solution (2.2 to 140 pmole / l).
) Take 0.2 ml, add 0.1 ml of ¹²⁵ I-labeled anti-ACTH double-clonal antibody solution and one anti-ACTH monoclonal antibody-bound plastic bead, incubate at room temperature for 20 hours, remove the reaction solution, and add distilled water. After washing, the radioactivity of the beads was measured with a counter.

FIG. 8 is a calibration curve according to the present reference example. In addition, the measurement sensitivity, the measurement range, and the ACTH concentration were 8.
The measurement accuracy (coefficient of variation) at 9 pmole / l and 140 pmole / l was as follows.

Reference Example 4 Measurement of Endothelin (ET) (a) Preparation of ET Standard Solution Human endothelin (ET) obtained from Peptide Laboratories, Inc. was prepared by using 5% bovine serum albumin and 0.8% bovine gamma globulin. In phosphate buffered saline containing
It was adjusted to 500 pmole / l.

(B) Anti-ET monoclonal antibody (first antibody)
Preparation of conjugated plastic beads Anti-ET monoclonal immunoglobulin prepared according to Example 1 (b) was coated on plastic beads.

(C) Anti-ET monoclonal antibody (second antibody)
The rabbit was immunized with ET to obtain an anti-ET antiserum, which was purified to obtain a highly specific anti-ET monoclonal antibody.

(D) Anti-ET monoclonal antibody (second antibody)
Of dissociation constant of ET with an ET (antigenic substance) According to Example 1 (d). The dissociation constants obtained from this reference example were as follows. Dissociation constant 0.08 nmole / l

(E) Preparation of Second Antibody Solution From the dissociation constant obtained in the previous section, the second antibody was 0.6 nmole / l
At a concentration of. The second antibody was used after being diluted with phosphate buffered saline containing 2% bovine serum albumin so that the final concentration in the reaction solution was the same.

(F) Preparation of ¹²⁵ I-labeled anti-rabbit immunoglobulin antibody The procedure was as in Example 1 (f). (G) Measurement of ET A standard ET solution (5-500 pmole / l) 0.2 m was placed in a test tube.
1, 0.1 ml of anti-ET monoclonal antibody solution and one anti-ET monoclonal antibody-bound plastic bead were kept at room temperature for 20 hours, then the reaction solution was removed and washed with distilled water. next
^After adding 0.3 ml of an anti-rabbit immunoglobulin / goat antibody solution labeled with ¹²⁵ I and keeping the mixture at room temperature for 20 hours, the reaction solution was removed, washed with distilled water, and the radioactivity of the beads was measured with a counter.

FIG. 9 is a calibration curve according to the present reference example, expressed in% of the combined radioactivity with respect to the total radioactivity. The measurement sensitivity, measurement area, and measurement accuracy were as follows. Measurement sensitivity <5 pmole / l Measurement range 5-500 pmole / l

Reference Example 5 Measurement of ET by Conventional Sandwich Method (Method of Directly Labeling Second Antibody) (a) Preparation of ET Standard Solution The procedure was the same as in Example 1 (a). (B) Preparation of Plastic Beads Conjugated with Anti-ET Monoclonal Antibody (Primary Antibody) According to Example 1 (b). (C) Preparation of anti-ET monoclonal antibody (second antibody) The procedure was the same as in Example 1 (c).

(D) Preparation of ¹²⁵ I-labeled anti-ET monoclonal antibody (labeled second antibody) The anti-ET monoclonal antibody (second antibody) was labeled and purified according to Example 1 (f), and 2% bovine It was adjusted to 4 KBq / 0.1 ml with phosphate buffered saline containing serum albumin.

(E) Measurement of ET A standard ET solution (5-500 pmole / l) 0.2 was placed in a test tube.
Take 0.1 ml of ¹²⁵ I-labeled anti-ET monoclonal antibody solution 0.1
After adding 1 ml of anti-ET monoclonal antibody-conjugated plastic beads and keeping the mixture at room temperature for 20 hours, the reaction solution was removed, washed with distilled water, and the radioactivity of the beads was measured with a counter.

FIG. 10 is a calibration curve according to the present reference example, expressed as a percentage of the amount of bound radioactivity relative to the total amount of radioactivity. Also,
The measurement sensitivity, measurement area, and measurement accuracy were as follows. Measurement sensitivity 25 pmole / l Measurement range 25-500 pmole / l

Reference Example 6 Measurement of Atrial Natriuretic Peptide (ANP) In order to examine the relationship between the dissociation constant between the second antibody and the antigenic substance and the measurement sensitivity, the concentration of the second antibody was changed to change the ANP.
Was measured.

(A) Preparation of ANP standard solution Human ANP obtained from Peptide Laboratories, Inc.
It was adjusted to 50 pg / ml with a phosphate buffer containing 0.1% bovine serum albumin and the like.

(B) Preparation of Anti-ANP Monoclonal Antibody (Primary Antibody) -Binding Plastic Beads The anti-ANP monoclonal immunoglobulin prepared according to Example 1 (b) was coated on plastic beads.

(C) Preparation of anti-ANP monoclonal antibody (second antibody) A rabbit was immunized with ANP to obtain an anti-ANP antiserum, which was purified to obtain a highly specific anti-ANP monoclonal antibody.

(D) Measurement of dissociation constant of anti-ANP double-cloned antibody (second antibody) with ANP (antigenic substance) The same as in Example 1 (d). The dissociation constants obtained from this reference example were as follows. Dissociation constant 0.078 nmole / l

(E) Preparation of Second Antibody Solution From the dissociation constant determined in the preceding section, the second antibody
It was decided to use at a concentration of 15 nmole / l. The second antibody is
The solution was diluted with a phosphate buffer containing 0.1% bovine serum albumin or the like so that the final concentration in the reaction solution was the same.

(F) Preparation of ¹²⁵ I-labeled anti-rabbit immunoglobulin antibody The procedure was the same as in Example 1 (f).

(G) Measurement of ANP In a test tube, 0.2 ml of a standard ANP solution (50 pg / ml) and an anti-ANP monoclonal antibody solution (final concentration: 0.0015 to 1)
After 0.1 ml of 5 nmole / l) and one anti-ANP monoclonal antibody-conjugated plastic bead were kept at room temperature for 20 hours, the reaction solution was removed and washed with distilled water. Next, 0.3 ml of an anti-rabbit immunoglobulin / goat antibody solution labeled with ¹²⁵ I was added, and the mixture was incubated at room temperature for 20 hours. The reaction solution was removed, washed with distilled water, and the radioactivity of the beads was measured with a counter.

FIG. 11 is a second antibody dose curve according to this reference example. From the point where the concentration of the second antibody exceeds the dissociation constant between the second antibody and the antigenic substance, the radioactivity of the beads is significantly improved.

[0089]

By comparing Example 1 and Comparative Example 1, in the method of the present invention, the second antibody is reacted with the second antibody at a concentration higher than the dissociation constant of parathyroid hormone-related protein (PTHrP). Thus, it is clear that the sensitivity is significantly higher than when the reaction is performed at a concentration lower than the dissociation constant. Also, as compared with the conventional method (method of directly labeling the second antibody) (Comparative Example 2), a remarkable improvement in the measurement sensitivity was observed, and only a low-sensitivity (large dissociation constant) second antibody was obtained. It was shown that measurement of PTHrP can be performed with high sensitivity. In addition, this method was shown to be useful for other peptide hormones which are difficult to obtain highly sensitive antibodies as shown in Reference Examples, but were relatively sensitive (Kd = 9 × 10 ^−12). (mol / l) Even when the second antibody was obtained (for example, in the case of ACTH), improvement of the measurement sensitivity by the method of the present invention was recognized. That is, in the method of the present invention, the method is particularly effective when the sensitivity of the usable second antibody (dissociation constant between the second antibody and the antigenic substance) is at least greater than 9 × 10 ⁻¹² mole / l. It is thought that there is.

In Reference Example 6, the radioactivity of the beads was significantly increased from the point where the concentration of the second antibody exceeded the dissociation constant between the second antibody and the antigenic substance. That is, it was found that it is important to use the second antibody at a concentration higher than the dissociation constant. From the above results, it is clear that a highly sensitive measurement system can be constructed by the method of the present invention without depending on the sensitivity of the usable second antibody. According to the present invention, a highly sensitive parathyroid hormone-related protein (PT
A method for measuring HrP) is provided.

[Brief description of the drawings]

FIG. 1 is a Scatchard plot of the anti-PTHrP antibody obtained in Example 1 (d).

FIG. 2 is a calibration curve for PTHrP measurement obtained in Example 1.

FIG. 3 is a calibration curve for PTHrP measurement obtained in Comparative Example 1.

FIG. 4 is a calibration curve for PTHrP measurement obtained in Comparative Example 2.

FIG. 5 is a Scatchard plot of the anti-ACTH antibody obtained in Reference Example 1 (d).

FIG. 6 is a calibration curve for ACTH measurement obtained in Reference Example 1.

FIG. 7 is a calibration curve for ACTH measurement obtained in Reference Example 2.

FIG. 8 is a calibration curve for ACTH measurement obtained in Reference Example 3.

FIG. 9 is a calibration curve for ET measurement obtained in Reference Example 4.

FIG. 10 is a calibration curve for ET measurement obtained in Reference Example 5.

FIG. 11 is a second antibody dose curve of the ANP measurement system obtained in Reference Example 6.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Masato Takada 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. Mitsubishi Tsuka Corporation Tsukuba Research Institute (72) Inventor Mayumi Goto Ami-cho, Inashiki-gun, Ibaraki 8-3-1, Mitsubishi Yuka Corporation Tsukuba Research Laboratory (56) References JP-A-2-205774 (JP, A)

Claims (3)

(57) [Claims] 1. Parathyroid hormone-related protein (B)
A first antibody (A) on an insoluble solid recognizing, a parathyroid hormone-related protein (B), and a second antibody (C) recognizing the parathyroid hormone-related protein derived from an animal species different from the first antibody Reacting the second antibody at a concentration higher than the dissociation constant between the second antibody and the parathyroid hormone-related protein to obtain an immune complex (D). After reacting a probe that specifically reacts with the antibody with a radioactively labeled probe (E), the radioactivity of a solid or a reaction solution is measured, whereby the parathyroid hormone-related protein (B) is measured.
A radioimmunoassay characterized by measuring the following. 2. The radioimmunoassay according to claim 1, wherein the concentration of the second antibody is 1 Kd to 10 Kd with respect to the dissociation constant (Kd) between the second antibody and the parathyroid hormone-related protein. Law. 3. The dissociation constant between a second antibody and said parathyroid hormone-related protein is 10 ^{-6 to} 9 × 10 ^-12 mol.
The radioimmunoassay according to claim 1 or 2, which is included in the range of e / l.