JP2727477B2 - PTHrP immunoassay by sandwich method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention is directed to the use of Humoral hypercalcemia which is most frequently associated with malignant tumors.
parathyroid hormone-related protein (hereinafter referred to as “PT”), which is the main causative substance of “malignancy” (hereinafter abbreviated as “HHM”).
HrP ”). More specifically, a method for measuring PTHrP, which can measure PTHrP in a simple and extremely sensitive manner by applying the technique of immunological measurement and can be used immediately for differential diagnosis of HHM, and a test method that can be used for this method About the kit.

[0002]

2. Description of the Related Art Conventionally, PTH which is a main cause substance of HHM is used.
Since rP had not been identified, blood calcium levels increased in cancer patients, and as a result, hypercalcemia was suspected at the stage when various clinical symptoms (disturbed consciousness, dry mouth, etc.) advanced, and various types of diagnosis were made. Laboratory tests were being performed.
As one of such clinical tests, measurement of serum calcium concentration is performed. As a result, when the serum calcium concentration is high, hypercalcemia due to tumor bone metastasis (Local osteolitic hypercalcemia) or, rarely, tumor ectopic production of parathyroid hormone (hereinafter abbreviated as “PTH”) Needs to be identified. If bone metastasis was strongly suspected, bone scintigraphy had to be performed to confirm the presence of bone metastases. For differential diagnosis of PTH ectopic production, it was necessary to measure the blood PTH concentration and confirm it by increasing the concentration.

On the other hand, there is no differential diagnosis method for HHM which is the most frequent. If the differential diagnosis of HHM is performed, PTHrP produced from the tumor
Is known to act on the PTH receptor in the kidney, resulting in an increase in urinary cAMP (primary nephrogenic cAMP) concentration. It is considered as. However, there are some problems in the measurement of the renal primordial cAMP concentration, and even if the renal primordial cAMP concentration is increased, the increase in the concentration due to other factors cannot be completely denied. For example, when measuring serum calcium concentration, nearly half of serum calcium is bound to protein, especially albumin, and in cancer patients, serum protein concentration may be reduced due to so-called cachexia, The result is that the total serum calcium concentration is lower with decreasing albumin. Therefore, it is necessary to accurately measure serum calcium and serum albumin, and to correct the serum calcium concentration with the serum albumin concentration. The renal progenitor cAMP concentration also decreases with aging, is affected by circadian fluctuations and various drugs, and reaches a high level in various diseases, hyperparathyroidism, manic depression, myocardial infarction, uremia, etc. It is known. For these reasons, it is not easy to make a differential diagnosis of HHM based on serum calcium concentration and renal primordial cAMP concentration because various factors must be considered.

[0004] In order to make a simple and accurate differential diagnosis of HHM, it is theoretically sufficient to measure the concentration of PTHrP, which is produced by a malignant tumor and is a main causative substance causing hypercalcemia. Recently, Suva
(Science 1987: 237: 893-6) elucidated the entire primary structure of PTHrP by cloning cDNA extracted from a lung cancer cell line. According to this, PTHrP is composed of 141 amino acid residues represented by the following formula.

Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu -His-His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile-Arg-Ala-Thr-Ser-Glu-Val-Ser-Pro-Asn-Ser-Lys-Pro-Ser -Pro-Asn-Thr-Lys-Asn-His-Pro-Val-Arg-Phe-Gly-Ser-Asp-Asp-Glu-Gly-Arg-Tyr-Leu-Thr-Gln-Glu-Thr-Asn-Lys -Val-Glu-Thr-Tyr-Lys-Glu-Gln Pro-Leu-Lys-Thr-Pro-Gly-Lys-Lys-Lys-Lys-Gly-Lys-Pro-Gly-Lys-Arg-Lys-Glu-Gln-Glu-Lys-Lys-Lys-Arg-Arg- Thr-Arg-Ser-Ala-Trp-Leu-Asp-Ser-Gly-Val-Thr-Gly-Ser-Gly-Leu-Glu-Gly-Asp-His-Leu-Ser-Asp-Thr-Ser-Thr- Thr-Ser-Leu-Glu-Leu-Asp-Ser-Arg-Arg-His The N-terminal amino acid sequence having the physiological activity shows high homology with that of the N-terminal of PTH, and PTHr
This is consistent with the findings so far which are considered to have physiological activities very similar to P.

Based on this report, the present inventors have directly
The possibility of a method for measuring HrP was examined.
First, from the findings that PTHrP and PTH have substantially the same physiological activity, their blood concentrations are:
It was expected to be as low as several pmol / l.
Therefore, since a method capable of measuring an extremely low concentration must be used as the measurement method, the present inventor has studied the measurement by the radioimmunoassay method, which is the most sensitive measurement method for peptides at present. However, in spite of having sufficient measurement sensitivity, blood PTHrP
The increase in concentration was not sufficiently confirmed.

The reason for this is that the antibody binds to the antigen, that is, the antibody recognition site is a site that is difficult to bind when viewed from the whole PTHrP molecule, and the antibody recognition site is embedded in a so-called binding protein. It is conceivable that the antigen secreted from the tumor is extremely rapidly degraded in the blood, so that the measured value cannot be obtained sufficiently high. However, in any case, PTHrP could not be measured directly. Therefore, the fact is that there is no simple differential diagnosis method for HHM.

[0008] As a result of the research conducted by the present inventors, it has been found that PTHrP present in blood more frequently exists in a fragmented state in a truncated state than in the complete molecule. . Also, unexpectedly, it was found that those fragments, particularly the C-terminal part, stably exist in a relatively long state.

[0009] The form (mode of existence) of PTHrP in blood is considered to be an important factor, that is, the sensitivity of the measurement system itself is high when measuring low concentrations of substances in a radioimmunoassay system. And the substance recognized by the measurement system, that is, the antigen, requires extremely high identity.

Therefore, the present inventors first focused on the C-terminal fragment of the PTHrP fragment, and since it is relatively stable in the bloodstream, PTHrP can be easily converted by the RIA method using an antibody that recognizes this. He found that measurement was possible and filed a patent application.
No. 0).

[0011]

SUMMARY OF THE INVENTION
Although clinical utility is high as a method for measuring P,
Since this measured value reflects the amount of the C-terminal portion having no direct bioactivity, it is desired to directly measure the N-terminal portion having bioactivity for more accurate HHM diagnosis. Therefore, development of such a measuring method was required.

[0012]

The present inventors further provide a PT
Research was conducted to develop a method for directly measuring the N-end of HrP. However, the N-terminal fragment was more easily decomposed than the C-terminal fragment, and only a trace amount was present in blood. Therefore, it became clear that an extremely sensitive measurement system was required. Therefore, the present inventors have further studied and found that PTHrP is N-end-middle and C-throat in the bloodstream.
It was recognized that it was disassembled into edges. Further, it has been found that the above object can be achieved by performing a so-called 2-site immunometric assay using an antibody that recognizes an N-terminal portion and an antibody that recognizes an intermediate portion based on this recognition.

That is, the present invention is characterized in that an antibody specifically recognizing the N-terminal portion of PTHrP and an antibody specifically recognizing an intermediate portion thereof are allowed to act on a specimen, and the resulting antibody-antigen-antibody reaction is detected. This is an immunoassay for PTHrP.

In order to carry out the method of the present invention, an antibody that recognizes basically two different epitopes is required. As this antibody, an antibody that specifically recognizes the N-terminal part of PTHrP (hereinafter, referred to as “N-terminal antibody”) and an antibody that specifically recognizes the intermediate part (hereinafter, “intermediate part antibody”) are used. You.

[0015] The N-terminal fragment of PTHrP used as an antigen for preparing the N-terminal antibody may be of any length as long as it contains the N-terminal. It is desirable to use those having about amino acid chain length.

The intermediate part recognized by the intermediate antibody is
It refers to a structural portion on the N-terminal side (N-terminal side from the 87th position) of a specific structural site in which the base amino acids of PTHrP are continuous, and does not include the N-terminal portion. In order to prepare an intermediate antibody, the intermediate fragment used as an antigen generally preferably has a chain length of about 20 to 35 amino acids.

It is preferable that the N-terminal fragment and the intermediate fragment do not overlap with each other in the structure of PTHrP.

As the N-terminal and intermediate fragments, not only peptides having the same amino acid sequence as PTHrP but also peptides having homology thereto can be used. In the present specification, a peptide having homology to a certain peptide means that some amino acids in the sequence are changed, deleted, etc., and are not the same as the original peptide. Means almost the same peptide as

Preferred examples of the peptide which can be used as the N-terminal fragment include the following formula (I): Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly -Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala (I) [PTHrP (1-34)] and a peptide having homology thereto.

On the other hand, preferred specific examples of the peptide which can be used as the intermediate fragment include the following formula (II): Glu-Ile-Arg-Ala-Thr-Ser-Glu-Val-Ser-Pro-Asn-Ser PTHrP represented by -Lys-Pro-Ser-Pro-Asn-Thr-Lys-Asn-His-Pro-Val-Arg-Phe-Gly-Ser-Asp-Asp-Glu-Gly-Arg-Tyr (II) [PTHrP (35-67)] and a peptide having homology thereto and the following formula (III): Tyr-Leu-Thr-Gln-Glu-Thr-Asn-Lys- Val-Glu-Thr-Tyr-Lys-Glu-Gln-Pro-Leu-Lys-Th -Pro-NH ₂ (III) peptide [PTHrP (35-67)] having the sequence No. 67-86 of PTHrP represented by and peptides having this homologous thereof.

In order to prepare the N-terminal antibody and the intermediate antibody using each of the above-mentioned fragments of PTHrP, known methods may be used. For example, the antigen selected as described above may be prepared by a conventional method. Animals, such as sheep, goats,
Sensitize animals such as rabbits and mice.

In order to measure PTHrP in a sample using the N-terminal antibody and the intermediate antibody obtained in this way, a fixed amount of the sample and one of these antibodies are labeled, and then, simultaneously Alternatively, an N-terminal antibody-antigen-intermediate antibody labeled complex may be generated by sequentially reacting, and the amount of the labeled complex may be measured.

The method of the present invention will be specifically described below, taking as an example the case where the intermediate antibody is used as a labeled antibody and the N-terminal antibody is used as an immobilized antibody. First, the intermediate antibody is labeled with a radioactive substance such as radioactive iodine, an enzyme, a luminescent substance, a fluorescent substance, or the like according to a conventional method (labeled antibody). On the other hand, the N-terminal antibody binds to biotin, proteins, insoluble substances, etc. (immobilized antibody). Next, the immobilized antibody, a fixed amount of the labeled antibody and the specimen are mixed to obtain an immobilized antibody-antigen-labeled antibody complex. The unreacted antibody is removed from the complex by a known separation means such as filtration, and the amount of label is measured by measuring the radioactivity, enzyme activity, or fluorescent activity of the complex in the complex. Finally, the amount of PTHrP is determined from the amount of label measured, and this is used as the measured value.

In the above method, instead of immobilizing the N-terminal antibody, a secondary antibody of the N-terminal antibody or the intermediate antibody may be used, and after forming the complex, this may be separated from the unreacted antibody. . In the above description, the intermediate antibody was used as the labeled antibody, and the N-terminal antibody was used as the solid-phased antibody.

Samples used in the method of the present invention include blood,
Serum, plasma, urine, ascites, pleural effusion, milk, amniotic fluid, spinal fluid, secretory fluid, exudate or leaked fluid are used.

In order to easily carry out the present invention, PTH
It is preferable to use an rP standard and a reagent kit comprising an N-terminal antibody and an intermediate antibody, one of which is labeled. The other unlabeled antibody may be immobilized.

[0027]

The development of N-terminal RIA of PTHrP has been promoted in the past. However, the effect of serum and the N-terminal fragment were low in the blood, and some of the healthy subjects could not measure enough. There was a problem. Some C-end measurement systems are also being developed, but all have the drawback that they do not directly reflect the physiological activity of PTHrP.

On the other hand, the reason that the method of the present invention solved the above-mentioned drawbacks and measured PTHrP was that the present invention is based on the fact that the site generated by decomposition in the bloodstream is from the N-end to the middle part. This is considered to be due to utilizing the antibody provided by the above and a 2-site immunometric assay having high measurement sensitivity.

[0029]

According to the method of the present invention, healthy subjects and HH
The PTHrP concentration in the M sample can be measured, and H
Since the measured value of HM can be measured specifically higher than the value of a healthy person, the differential diagnosis of HHM can be easily performed.
Further, the measurement system of the present invention can be applied to P
Since the THrP concentration can be easily measured, it is extremely advantageous also in this respect.

[0030]

The present invention will be described in more detail with reference to the following examples. Example 1 Preparation of reagent: (1) Preparation of antibody 1. Preparation of PTHrP (35-67) antibody From PTHrP (35-67) 2 mg and bovine thyroglobulin (Sigma) 8 mg, using a known technique. Then, a complex was prepared, and 200 μg thereof was mixed with Freund's complete adjuvant (manufactured by Calbiochem) to prepare an emulsion, which was then administered to the sheep skin. A further 100 μg each was boosted at 4-week intervals. Two weeks after each immunization, blood was collected from the jugular vein, the serum was separated, and PTHrP (3
5-67) The antiserum was collected. The antiserum was selected by using the labeled antigen described later and examining the titer and sensitivity. The obtained antiserum was used as a γG fraction by the salting-out method, and the buffer solution (0.0) was used.
It was dialyzed against 2M phosphate buffer (pH 7.4). This was added to a PTHrP (35-67) affinity column described below, and the column was washed with the same buffer, and the antibody was eluted from the column with an eluate (0.01 M Gly-HCl (pH 2.2)). And affinity purified.

2. Preparation of PTHrP (67-86) Antibody A complex was prepared from 2 mg of PTHrP (67-86) amide and 8 mg of bovine albumin (manufactured by Miles) using a known technique. It was administered intradermally to obtain antiserum. This was subjected to affinity purification in the same manner using a PTHrP (67-86) affinity column.

3. Preparation of PTHrP (109-141) Antibody A complex was prepared from 2 mg of PTHrP (109-141) and 8 mg of bovine thyroglobulin (Sigma) using a known technique, and sheep skin was similarly prepared. Was administered intracellularly to obtain antiserum. This was affinity-purified similarly using a PTHrP (109-141) affinity column.

4. Preparation of PTHrP (1-34) Antibody A complex was prepared from 2 mg of PTHrP (1-34) amide and 8 mg of bovine albumin (manufactured by Miles) using a known technique, and 100 μg of Freund was completely used. An emulsion was prepared by mixing with adjuvant (manufactured by Calbiochem), and administered subcutaneously to mice. Further, 50 μg each was boosted three times at 4-week intervals. Four weeks later, 50 μg of the complex was intraperitoneally administered, and three days later, mouse lymphocytes and mouse myeloma were fused with cells using a known technique to prepare a monoclonal antibody, and the purified antibody was purified using an anion exchange column. Produced.

(2) Preparation of labeled antigen 1. Preparation of labeled antigen of PTHrP (35-67) 2 μg of PTHrP (35-67) was added to 70 MBq of Na.
Labeled with ¹²⁵ I using chloramine T according to the standard method of Greenwood and Hunter. Then 2
By adding to a high-performance chromatography (manufactured by Tosoh Corporation) using a reversed-phase column equilibrated with a 0.1% trifluoroacetic acid solution containing 0% acetonitrile and eluting with a 0.1% trifluoroacetic acid solution containing 40% acetonitrile. Unreacted ¹²⁵ I and PTHrP (35-67) labeled antigen were separated. The separated labeled antigen was buffered (0.01 M phosphate buffer containing 0.5% bovine serum albumin (pH
It was diluted to about 7.4 kB / ml in 7.4)) to obtain a labeled antigen of PTHrP (35-67).

2. Preparation of PTHrP (67-86) Labeled Antigen 2 μg of PTHrP (67-86) amide was labeled with 70 MBq of Na ¹²⁵ I using chloramine T according to the standard method of Greenwood and Hunter. afterwards,
In the same manner as described above, PTHrP (67-86) -labeled antigen was separated by high-performance chromatography using a reversed-phase column. The separated labeled antigen was buffered (0.01 M phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin).
Diluted to about 7.4 kB / ml with
PTHrP (67-86) labeled antigen was used.

3. Tyr ¹⁰⁸ -PTHrP (108-1)
41) Preparation of labeled antigen 2 μg of Tyr ¹⁰⁸ -PTHrP (108-141) was added to 7
Labeled with 0 MBq Na ¹²⁵ I using chloramine T according to the standard method of Greenwood and Hunter. Thereafter, Tyr ¹⁰⁸ -PTHrP (108-14) was similarly subjected to high-performance chromatography using a reversed-phase column.
1) The labeled antigen was separated. The separated labeled antigen was diluted to about 7.4 kB / ml with a buffer (0.1 M Tris buffer (pH 8.5) containing 0.5% bovine serum albumin) to give Tyr ¹⁰⁸ -PTHrP. (108-
141) A labeled antibody was used.

4. Preparation of Tyr ⁰ -PTHrP (1-34) Labeled Antigen 2 μg of Tyr ⁰ -PTHrP (1-34) was added to 70 MBq
By the Na ¹²⁵ I, it was labeled with chloramine T according to standard methods of Greenwood and Hunter. Thereafter, a Sephadex G-25 Superfine (Pharmacia) column (1 ×) equilibrated with a 0.01 M phosphate buffer containing 0.5% bovine serum albumin and 0.1% Tween 20 was used. 30I) and eluted with the same buffer to separate unreacted ¹²⁵ I from PTHrP (1-34) labeled antigen. The separated labeled antigen was added to a buffer (0.01 M phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin and 0.1% Tween 20) at about 7.4 kB / ml.
, And Tyr ⁰ -PTHrP (1-3
4) Used as a labeled antigen.

(3) Preparation of Labeled Antibody 1. Preparation of PTHrP (35-67) Labeled Antibody 50 μg of PTHrP (35-67) antibody was treated with 23 MBq of Na ¹²⁵ I using chloramine T according to the standard method of Greenwood and Hunter. Labeled. afterwards,
0.5% bovine serum albumin and 0.1% Tween 20
Was added to a Sephacryl S-300 HR (manufactured by Pharmacia) column (1 × 30 cm) equilibrated with a 0.01 M phosphate buffer solution containing, and eluted with the same buffer solution to remove unreacted ¹²⁵ I. PTHrP (35-67)
Separated from the labeled antibody. The separated labeled antibody was diluted with a buffer (0.01 M phosphorus buffer containing 0.5% bovine serum albumin, pH 7.4) to a concentration of about 37 kB / ml and labeled with PTHrP (35-67). An antibody was used.

2. Preparation of PTHrP (67-86) Labeled Antibody 50 μg of the PTHrP (67-86) antibody was labeled with 23 MBq of Na ¹²⁵ I using chloramine T according to the standard method of Greenwood and Hunter. afterwards,
0.5% bovine serum albumin and 0.1% Tween 20
S-300 HR (Pharmacia) column (1 × 3) equilibrated with 0.01M phosphate buffer containing
0 cm) and eluted with the same buffer to separate unreacted ¹²⁵ I from PTHrP (67-86) labeled antibody. The labeled antibody separated was a buffer (0.01 M phosphoric acid buffer containing 0.5% bovine serum albumin, pH 7.5).
4)), the mixture was diluted to about 37 kB / ml to obtain a PTHrP (67-86) labeled antibody.

3. Preparation of PTHrP (109-141) Labeled Antibody 50 μg of PTHrP (109-141) antibody was added to 23 MB.
q was labeled with chloramine T according to the standard method of Greenwood and Hunter with Na ¹²⁵ I. afterwards,
0.5% bovine serum albumin and 0.1% Tween 20
S-300 HR (Pharmacia) column (1 × 3) equilibrated with 0.01M phosphate buffer containing
0 cm) and eluted with the same buffer to separate unreacted ¹²⁵ I from PTHrP (109-141) labeled antibody. The separated labeled antibody was added to a buffer (0.
It was diluted to about 37 kB / ml with 0.01 M phosphorus buffer (pH 7.4) containing 5% bovine serum albumin to obtain a PTHrP (109-141) labeled antibody.

4. Preparation of PTHrP (1-34) Labeled Antibody 50 μg of PTHrP (1-34) antibody was added to 23 MBq of N
a ¹²⁵ I was labeled with chloramine T according to the standard method of Greenwood and Hunter. afterwards,
0.5% bovine serum albumin and 0.1% Tween 20
S-300 HR (Pharmacia) column (1 × 3) equilibrated with 0.01M phosphate buffer containing
0 cm) and eluted with the same buffer to separate unreacted ¹²⁵ I from PTHrP (1-34) labeled antibody. The labeled antibody separated was a buffer (0.01 M phosphoric acid buffer containing 0.5% bovine serum albumin, pH 7.5).
4)), the mixture was diluted to about 37 kB / ml to obtain a PTHrP (1-34) labeled antibody.

(4) Preparation of Primary Antibody Beads 1. Preparation of PTHrP (1-34) Antibody Beads 100 polystyrene beads (4 mm in diameter) were 5%
After washing with Scat (Daiichi Kogyo), PTHrP (1-34) antibody 1 prepared in Example (1) -4 above
00 μg was added to 20 ml of 0.01 M phosphate buffer (pH 6.0).
The solution diluted in 4) was reacted at 25 ° C. for 18 hours.
Thereafter, the cells were washed with a 0.01 M phosphate buffer (pH 6.4) and stored in a 0.01 M phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin. The binding ability of the antibody beads was confirmed by examining the binding ability of the PTHrP (1-34) -labeled antigen to the beads described above.

(5) Preparation of Affinity Column 1. Preparation of PTHrP (35-67) Affinity Column 1 g of CNBr-activated Sepharose 4B gel (Pharmacia) was diluted with diluted hydrochloric acid (1 mM HCl) 2
After washing on a glass filter with 00 ml, the reaction solution was washed with 10 ml of a 0.1 M sodium carbonate buffer (pH 8.3 containing 0.5 M sodium chloride), and immediately
The suspension was suspended in a reaction solution containing 0.5 mg of THrP (35-67) and mixed by inversion at room temperature for 20 hours. Thereafter, the gel was thoroughly washed alternately with the above-mentioned diluted hydrochloric acid and the reaction solution on a glass filter, and the buffer solution (0.02 M phosphate buffer solution (pH 7.0)) was used.
4) Saved during).

2. Preparation of PTHrP (67-86) affinity column 1 g of CNBr-activated Sepharose 4B gel (manufactured by Pharmacia) was washed on a glass filter with 200 ml of dilute hydrochloric acid (1 mM HCl), and then the reaction solution (0.5 M chloride) was added. 0.1 M sodium carbonate buffer containing sodium (pH 8.
3)) Wash with 10 ml, and immediately wash PTHrP (67-
86) The suspension was suspended in a reaction solution containing 0.5 mg of amide and mixed by inversion at room temperature for 20 hours. Thereafter, the gel was thoroughly washed alternately with the diluted hydrochloric acid and the reaction solution on a glass filter, and stored in a buffer solution (0.02 M phosphate buffer (pH 7.4)).

3. Preparation of PTHrP (109-141) Affinity Column 1 g of CNBr-activated Sepharose 4B gel (Pharmacia) was washed with 200 ml of dilute hydrochloric acid (1 mM HCl) using a glass filter, and the reaction solution (0.5 M sodium chloride) was used. 0.1M sodium carbonate buffer (pH 8.
3)) Wash with 10 ml, and immediately wash PTHrP (109
-141) The suspension was suspended in a reaction solution containing 0.5 mg, and mixed by inversion at room temperature for 20 hours. Thereafter, the gel was thoroughly washed alternately with the diluted hydrochloric acid and the reaction solution on a glass filter, and stored in a buffer solution (0.02 M phosphate buffer (pH 7.4)).

(6) Preparation of Standard Solution 500 ml of commercially available unprepared milk was added at 4 ° C. and 3000 rpm.
Centrifuge at 30 ° C. for 30 minutes to remove the upper fat layer and sediment. An S-Sepharose (Pharmacia) column (2 × 1) in which 400 ml of this was equilibrated with a 0.01 M phosphate buffer pH 7.4 containing 0.1 M sodium chloride.
2 cm), washed with the buffer used for equilibration, and eluted with 0.01 M phosphate buffer (pH 7.4) containing 1.0 M sodium chloride. The eluted fraction was diluted with a buffer (0.01 M phosphate buffer (pH 7.4) containing 0.5% bovine albumin and 0.01 M sodium chloride) as a standard solution, and PTHrP (1 −
34) The concentration was assayed by RIA, and the molar concentration of PTHrP in the standard solution was calculated.

(7) Preparation of Reagent for Second Antibody Sheep gamma globulin was sensitized to horses using a known technique to obtain an antiserum. This antiserum was diluted approximately 40-fold with a buffer (0.01 M phosphate buffer (pH 7.4) containing 0.01 M sodium chloride) to obtain a second antibody reagent.

Example 2 PTHrP (1-34) RIA PTHrP (1-3) at a concentration of 318.6 pmol / l
4) from buffer (0.5% bovine serum albumin, 0.01
0.01M phosphate buffer containing sodium chloride (p
H7.4)) was used to prepare a series of 2-fold dilutions at 19.9 pmol.
/ L, which was used as a standard reagent. A buffer (0.01 M phosphoric acid buffer (pH 7.4) containing 0.5% bovine serum albumin and 0.01 M sodium chloride) was used as a standard reagent having a concentration of 0 pmol / l.

A test tube was prepared, and 100 μl of each of these standard reagents, a standard solution roughly purified from milk, and a sample was placed in each of 2 tubes.
Collected in test tubes. 100 μl of PTHrP (1-34) antiserum (manufactured by Peninsula) was added to each test tube, and the mixture was gently stirred and reacted at 25 ° C. for 22 hours. Then, P
100 μl of THrP (1-34) standard antigen was added to each test tube, and the mixture was gently stirred and reacted at 25 ° C. for 22 hours.
Then 100 μl of normal rabbit serum reagent (Peninsula)
And rabbit second antibody reagent (Peninsula) 100 μl
Was added, and the mixture was stirred well and reacted at 25 ° C. for 30 minutes. The resulting antigen-antibody-second antibody conjugate or labeled antigen-antibody-second antibody conjugate was treated for 30 minutes at 4 ° C, 300 ° C.
The precipitate was precipitated by centrifugation at 0 rpm, and the supernatant containing unreacted antigen or labeled antigen was aspirated using an aspirator. The test tube of each standard reagent was measured with a well-type scintillation counter, and the average amount of radioactivity was calculated. In addition, the radioactivity of the standard reagent of 0 pmol / l was obtained in the same manner, and this was defined as B ₀ , and the above B was divided by B ₀ to calculate the percentage, which was defined as B / B ₀ (%). Using a semilogarithmic graph, the molar concentration of the standard reagent was plotted on the logarithmic side, and B / B ₀ (%) was plotted on the other side to prepare a standard curve.
The average amount of radioactivity B was similarly calculated for the sample, and
B ₀ (%) was determined. The corresponding concentration was determined from the standard curve created from this.

Example 3 PTHrP IRMA (1) PTHrP (1-34) antibody beads (primary antibody)
-PTHrP (35-67) labeled antibody was converted from a standard solution having a concentration of 609 pmol / l of IRMA to a buffer solution (0.5
% Bovine serum albumin, 0.01 M phosphoric acid buffer (pH 7.4) containing 0.01 M sodium chloride).
A two-fold dilution series was prepared up to a concentration of 2.38 pmol / l, and this was used as a standard reagent. A buffer (0.01 M phosphoric acid buffer (pH 7.4) containing 0.5% bovine serum albumin and 0.01 M sodium chloride) was used as a standard reagent having a concentration of 0 pmol / l.

A tray for beads was prepared, and 100 μl of these standard reagents and samples were collected in each of two wells. One PTHrP (1-34) antibody bead was added to each well and reacted at room temperature for 6 hours. After that, the beads in each well were washed twice with 2 ml of purified water.
200 μl of a THrP (35-67) -labeled antibody was added to each well and reacted at room temperature for 18 hours. 2 ml of purified water
The beads in each well were washed twice with and the beads were transferred to a separately prepared test tube.

The test tubes of each standard reagent were measured with a well-type scintillation counter, and the average radioactivity was calculated.
This was designated B. In addition, the radioactivity of the standard reagent of 0 pmol / l was obtained in the same manner, and this was set as B _0, and B ₀ was subtracted from the above B to obtain B-B ₀ (cpm). The molar concentration of the labeled reagent in the horizontal axis side using log-log graph, by plotting the B-B ₀ (cpm) to the other, to generate a standard curve. In the same manner for the sample to calculate the average amount of radioactivity B, B-B ₀ seek (cpm), fit to a standard curve generated with this value to determine the corresponding concentration.

(2) PTHrP (1-34) antibody beads (primary antibody) / I of PTHrP (67-86) labeled antibody
RMA 609 pmol / l standard to buffer (0.5
% Bovine serum albumin, 0.01 M phosphoric acid buffer (pH 7.4) containing 0.01 M sodium chloride) to prepare a series of 4-fold dilutions up to a concentration of 2.38 pmol / l, which was used as a standard reagent. . As a standard reagent having a concentration of 0 pmol / l, a buffer solution (0.5% bovine serum albumin,
A buffer of 0.01 M phosphoric acid (pH 7.4) containing 0.01 M sodium chloride was used.

A tray for beads was prepared, and 100 μl of each of these standard reagents and samples was collected in each of two wells. One PTHrP (1-34) antibody bead was added to each well and reacted at room temperature for 6 hours. After that, the beads in each well were washed twice with 2 ml of purified water.
200 μl of a THrP (67-86) -labeled antibody was added to each well and reacted at room temperature for 18 hours. 2 ml of purified water
The beads in each well were washed twice with and the beads were transferred to a separately prepared test tube.

A test tube for each standard reagent was measured using a well-type scintillation counter, and the average radioactivity was calculated.
This was designated B. In addition, the radioactivity of the standard reagent of 0 pmol / l was determined in the same manner, and this was set as B _0, and B ₀ to B ₀
The subtracted This was B-B ₀ and (cpm). Using log-log graph, the molar concentration of the labeled reagent in the horizontal axis side, a standard curve was generated by plotting the B-B ₀ to the other (cpm). In the same manner for the sample to calculate the average radioactivity B, it was determined B-B ₀ to (cpm). This value was fitted to the prepared standard curve to determine the corresponding concentration.

(3) PTHrP (1-34) Antibody Beads (Primary Antibody) A standard solution of PTHrP (109-141) labeled antibody having a concentration of 609 pmol / l IRMA was added to a buffer (0.5%).
Bovine serum albumin, 0.01 M phosphoric acid buffer containing 0.01 M sodium chloride (pH 7.4)
A two-fold dilution series was prepared up to a concentration of 2.38 pmol / l and used as a standard reagent. A buffer (0.5% bovine serum albumin, 0.5%) was used as a standard reagent at a concentration of 0 pmol / l.
A buffer of 0.01M phosphoric acid (pH 7.4) containing 01M sodium chloride was used.

A tray for beads was prepared, and 100 μl of each of these standard reagents and samples was collected in each of two wells. One PTHrP (1-34) antibody bead was added to each well and reacted at room temperature for 6 hours. After that, the beads in each well were washed twice with 2 ml of purified water.
200 μl of a THrP (109-141) -labeled antibody was added to each well, and reacted at room temperature for 18 hours. Purified water 2
The beads in each well were washed twice with ml, and the beads were transferred to a separately prepared test tube.

The test tubes of each standard reagent were measured with a well-type scintillation counter, and the average radioactivity was calculated.
This was designated B. Further, the amount of radioactivity of the standard reagent of 0 pmol / l was determined in the same manner, and this was defined as B0.
The subtracted This was B-B ₀ and (cpm). Using log-log graph, the molar concentration of the labeled reagent in the horizontal axis side, a standard curve was generated by plotting the B-B ₀ to the other (cpm). It was determined and the calculated B-B ₀ (cpm) Average amount of radioactivity B in the same manner for the sample. This value was fitted to the prepared standard curve to determine the corresponding concentration.

(4) PTHrP (35-67) antibody (1
Secondary antibody) .PTHrP (1-34) labeled antibody IRMA at a concentration of 609 pmol / l from standard solution to buffer (0.5
% Bovine serum albumin, 0.01 M phosphoric acid buffer (pH 7.4) containing 0.01 M sodium chloride) to prepare a series of 4-fold dilutions up to a concentration of 2.38 pmol / l, which was used as a standard reagent. . A buffer (0.5% bovine serum albumin, 0.5%) was used as a standard reagent at a concentration of 0 pmol / l.
A buffer of 0.01M phosphoric acid (pH 7.4) containing 01M sodium chloride was used.

Test tubes were prepared, and 100 μl of each of these standard reagents and samples was collected in each of two test tubes.
PTHrP (35-67) antiserum (100-fold dilution) 1
00 μl and PTHrP (1-34) standard antigen 100
μl was added to each test tube, and the mixture was gently stirred and reacted at 25 ° C. for 22 hours. Thereafter, 500 μl of the second antibody reagent was added, and the mixture was stirred well and further reacted at 25 ° C. for 30 minutes. The resulting complex was precipitated by centrifugation at 3000 rpm at 4 ° C. for 30 minutes, and the supernatant was aspirated using an aspirator. The test tube of each standard reagent was measured with a well-type scintillation counter, and the average amount of radioactivity was calculated. In addition, the radioactivity of the standard reagent of 0 pmol / l was obtained in the same manner, and this was set to B _0, and B ₀ was subtracted from the above B to obtain B-B ₀ (cpm). Using a log-logarithmic graph, the molar concentration of the labeling reagent is plotted on the horizontal axis and B
-B ₀ was to create a standard curve by plotting the (cpm).
The average amount of radioactivity B was calculated in the same manner for the sample, and B-
B ₀ (cpm) was determined. This value was fitted to the prepared standard curve to determine the corresponding concentration.

[Brief description of the drawings]

FIG. 1. PTHrP of PTHrP (35-67) antibody
(35-67) Drawing showing generation on affinity column

FIG. 2 is a drawing showing the production of unregulated milk on an S-Sepharose column after centrifugation.

FIG. 3 is a drawing showing an example of a standard curve of IRMA of PTHrP (1-34) antibody beads (primary antibody) and PTHrP (35-67) purified antibody.

FIG. 4 is a drawing showing an example of a standard curve of IRMA of PTHrP (1-34) antibody beads (primary antibody) and PTHrP (67-86) purified antibody.

FIG. 5: PTHrP (35-67) antibody (primary antibody)
Drawing showing a standard curve example of IRMA of PTHrP (1-34) purified antibody

FIG. 6 shows the relationship between healthy subjects and H performed by the measurement method of the present invention.
Drawing showing PTHrP measurement results of HM

Claims (15)

(57) [Claims] 1. A PTHrP characterized in that an antibody specifically recognizing the N-terminus of PTHrP and an antibody specifically recognizing an intermediate portion thereof are allowed to act on a specimen, and the resulting antibody-antigen-antibody reaction is detected. Immunoassay. 2. The PT according to claim 1, wherein one of an antibody specifically recognizing the N-terminal portion of PTHrP and an antibody specifically recognizing the intermediate portion is labeled.
HrP immunoassay. 3. The method for immunologically measuring PTHrP according to claim 2, wherein the label is an antibody labeled with a label selected from a radioactive substance, an enzyme, a substance exhibiting luminescence, or a fluorescent substance. 4. An unlabeled antibody among the antibodies specifically recognizing the N-terminal portion and the intermediate portion of PTHrP, wherein the unlabeled antibody is immobilized.
The method for immunological measurement of PTHrP according to the above item. 5. The PTHrP (1-34) wherein the N end of PTHrP is represented by the formula (I).
The immunoassay for PTHrP according to any one of the above items. Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His- His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala (I) 6. An antibody obtained by immunizing an animal with PTHrP (1-34) represented by the formula (I) or a peptide having homology thereto, wherein the antibody specifically recognizing the N-terminal portion of PTHrP is: The method for immunologically measuring PTHrP according to any one of claims 1 to 5. Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His- His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala (I) 7. The immunoassay for PTHrP according to claim 1, wherein the intermediate part of PTHrP is PTHrP (35-67) represented by the formula (II). Glu-Ile-Arg-Ala-Thr-Ser-Glu-Val-Ser-Pro-Asn-Ser-Lys-Pro-Ser-Pro-Asn-Thr-Lys-Asn-His-Pro-Val-Arg-Phe- Gly-Ser-Asp-Asp-Glu-Gly-Arg-Tyr (II) 8. An antibody which specifically recognizes an intermediate part of PTHrP, wherein PTHrP (35-67) represented by the formula (II):
8. The PTH according to claim 1, which is an antibody obtained by immunizing an animal with a peptide having homology thereto.
Immunological assay for rP. Glu-Ile-Arg-Ala-Thr-Ser-Glu-Val-Ser-Pro-Asn-Ser-Lys-Pro-Ser-Pro-Asn-Thr-Lys-Asn-His-Pro-Val-Arg-Phe- Gly-Ser-Asp-Asp-Glu-Gly-Arg-Tyr (II) 9. The method for immunologically measuring PTHrP according to claim 1, wherein the intermediate part of PTHrP is PTHrP (67-86) represented by the formula (III). Tyr-Leu-Thr-Gln- Glu-Thr-Asn-Lys- Val-Glu-Thr-Tyr-Lys-Glu-Gln-Pro- Leu-Lys-Thr-Pro-NH 2 (III) 10. An antibody which specifically recognizes an intermediate part of PTHrP is a PTHrP (67-8) represented by the formula (III):
6) or an antibody obtained by immunizing an animal with a peptide having homology thereto.
The immunoassay for PTHrP according to any one of the above items. Tyr-Leu-Thr-Gln- Glu-Thr-Asn-Lys- Val-Glu-Thr-Tyr-Lys-Glu-Gln-Pro- Leu-Lys-Thr-Pro-NH 2 (III) 11. The PT, wherein the label is represented by the formula (I):
The antibody according to claim 2, 3 or 4, wherein the antibody is an antibody that specifically recognizes HrP (1-34) or a peptide having homology thereto, and the label is radioactive iodine introduced into the antibody. Immunological assay for PTHrP. Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His- His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala (I) 12. The PT, wherein the label is represented by the formula (II):
The antibody according to claim 2, 3 or 4, wherein the antibody is an antibody that specifically recognizes HrP (35-67) or a peptide having homology thereto, and the label is radioactive iodine introduced into the antibody. Immunological assay for PTHrP. Glu-Ile-Arg-Ala-Thr-Ser-Glu-Val-Ser-Pro-Asn-Ser-Lys-Pro-Ser-Pro-Asn-Thr-Lys-Asn-His-Pro-Val-Arg-Phe- Gly-Ser-Asp-Asp-Glu-Gly-Arg-Tyr (II) 13. The method according to claim 11, wherein the label is represented by the formula (III):
The antibody according to claim 2, 3, or 4, which is an antibody that specifically recognizes THrP (67-86) or a peptide having homology thereto, and wherein the label is radioactive iodine introduced therein. Immunological assay for PTHrP. Tyr-Leu-Thr-Gln- Glu-Thr-Asn-Lys- Val-Glu-Thr-Tyr-Lys-Glu-Gln-Pro- Leu-Lys-Thr-Pro-NH 2 (III) 14. An antibody which specifically recognizes the N-terminal part of labeled PTHrP, an antibody which may be solid-phased which specifically recognizes an intermediate part of PTHrP, and a standard product for measuring PTHrP. Inspection kit. 15. An optionally solid-phased antibody which specifically recognizes the N-terminal of PTHrP, labeled PTHrP
A test kit comprising an antibody that specifically recognizes an intermediate part of the above and a standard product for PTHrP measurement.