JPH0424554A - Method and kit for detecting calpastatin abnormality disease - Google Patents

Abstract

PURPOSE:To make it possible to detect the disease with abnormality in calpastatin production by comparing the quantity of the calpastatin in serum, plasma or urine with a normal value, and measuring the quantity. CONSTITUTION:Monoclonal-antibody bonded beads and oxygen-labeled monoclonal antibodies are formed. A sample is inputted into a tube. The solid- state antibody beads are inputted into the tube one by one. Incubation is repeatedly performed at 37 deg.C for 20 minutes. Absorbance is measured. The organ-type calpastatin is measured based on the increase in coloring absorbance with the increase in concentration of the organ-type calpastatin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for detecting disorders of calpastatin production such as human myocardial infarction, human cerebral thrombosis, and human cancer, and a kit for quantifying the same.

[Conventional technology]

Calpastatin is calpain (BC3,4,22゜17
) is an endogenous inhibitor protein that specifically inhibits
G, I, et al [Journal of Biology
Chemistry (J, Biol, Chem, ) No. 24
1, p. 3097 (1968)) as an inhibitor of the activator of phosphorylase b kinase, Nijimura 1, Rashi Journal of Biochemistry (J, Biochem).
), Vol. 84, p. 1657 (1978)] reported that it is a specific inhibitor protein of calpain. To date, its cDNA sequence has also been reported [Asada K, LA, Bnzyme Inhib,
Volume 3, page 49 (1989)].

The physiological significance of calpastatin is that by specifically inhibiting calpain, it inhibits various enzymes (protein kinase C, transglutaminase, etc.), cytoskeletal proteins (MAP2, neurofilament, keratin, etc.)
It was understood as one of the stimulus response systems of cells by regulating the activity of receptor proteins (EGF receptor, steroid hormone receptor, etc.).

On the other hand, the content of calpastatin in various rat tissues was investigated using the calpain inhibitory activity of calpastatin as an index.
It has been reported that it exists in large amounts in the liver, lungs, myocardium, and hepatocytes per unit tissue weight [Murac
hi) T. et al., Biochemistry International (8io-chetrlInt.), Vol. 2, p. 661, (1981)]. Furthermore, a rabbit anti-calpastatin polyclonal antibody was produced using calpastatin purified from human red blood cells as an antigen, and a method for measuring the amount of calpastatin in red blood cells by enzyme immunoassay (BIA) has been reported.
B. et al., Journal of Applied Biochemistry (J, Appl. Biochem,),
Vol. 2, p. 117 (1984)]. Furthermore, Pontremo Ii S, et al. [Biochemical and Biophysical Research Communications (Biochem, Biophy,
Res, Comm, ), Volume 157, Page 867 (
[1988)] used calpastatin derived from red blood cells as an antigen, produced a mouse anti-calpastatin monoclonal antibody, and measured the calpastatin content in red blood cells by Western blotting. It has been reported that this is significantly lower than that of

In addition, HTLV, the virus that causes adult T-cell leukemia,
Intracellular calpastatin content increases in cells infected with -I compared to uninfected cells [Adana
h i) Y, et al., Biological Chemistry B io ], Chem.

Hoppe-8ayler), Volume 369, No. 223
Page (1988)] has been reported.

[Problem to be solved by the invention]

However, these reports merely describe changes in calpastatin content within red blood cells, cells, and tissues. Furthermore, the relationship between human diseases and changes in the amount of calpastatin in tissues and cells has only been reported for essential hypertension; other reports are based solely on in vitro or animal experiments. . Furthermore, there has been no report to date of the presence of calpastatin in human serum, plasma, or urine, or of the relationship between the amount of calpastatin in human serum, plasma, or urine and a specific disease. Furthermore, regarding the method for measuring calpastatin, the activity assay method [Murakami T., et al., Journal
of Biochemistry, Volume 90, Page 1809 (
1981)], EIA using polyclonal antibodies, Western protting method using monoclonal antibodies,
EIA using monoclonal antibodies (Patent application No. 1-211
258 specification), but these methods only describe methods for measuring calpastatin extracted from cells/tissues or purified; There is no mention of reactivity, the presence of calpastatin in serum, plasma or urine, or the relationship between the amount of calpastatin in serum, plasma or urine and disease.

An object of the present invention is to provide a novel measuring method and a diagnostic kit for detecting diseases accompanied by abnormal production of calpastatin using the amount of calpastatin in serum, plasma, or urine as an indicator.

[Means to solve the problem]

To summarize the present invention, the first aspect of the present invention relates to a method for detecting a disease accompanied by abnormal production of calpastatin, and is characterized by measuring the amount of calpastatin in serum, plasma, or urine in comparison with a normal value.

Moreover, the second invention relates to a detection kit for a disease accompanied by abnormal production of calpastatin, and is characterized by containing an anti-calpastatin antibody.

In view of the above-mentioned current situation, the present inventors have conducted extensive research,
By measuring the amount of calpastatin in serum, plasma, or urine, diseases accompanied by abnormal production of calpastatin can be detected, and we have created a kit that can easily quantify calpastatin ■ in serum, plasma, or urine with high sensitivity. , completed the present invention.

The present invention will be explained in detail below.

Diseases associated with abnormal calpastatin production are as follows (1), (
These are diseases such as 2) and (3).

(1) Diseases caused by destruction of cells and organs themselves that contain relatively high amounts of calpastatin.

(2) Diseases caused by quantitative abnormalities in cells or organs themselves that contain relatively high amounts of calpastatin.

(3) Diseases caused by qualitative abnormalities in cells or organs themselves that contain relatively high amounts of calpastatin.

To specifically explain (1), (2), and (3) above, (
Examples of 1) include myocardial infarction and cerebral infarction; examples of (2) include cancer; and examples of (3) include arteriosclerosis and thrombosis.

The present inventors confirmed that greater amounts of calpastatin are present in serum, plasma, or urine derived from cerebral thrombosis, cerebral infarction, myocardial infarction, or cancer than in healthy individuals, and found that calpastatin in serum, plasma, or urine We found that this can be used as an indicator of diseases associated with abnormal calpastatin production.

In the present invention, the method for measuring the amount of calpastatin in serum, plasma, or urine is not particularly limited, and examples thereof include activity measurement methods and immunological methods, but immunological methods are preferred in terms of sensitivity and simplicity. preferable. Immunological methods include methods using antibodies, and in this case, immunoassay methods using one or more types of antibodies can be used. Monoclonal antibodies and polyclonal antibodies can be used as antibodies, and monoclonal antibodies are preferably used in terms of their specificity and affinity. On the other hand, immunoassay methods include enzyme immunoassay, radioimmunoassay, latex agglutination method, immunoturbidimetry, fluorescence/luminescence immunoassay, and Western blotting.
Enzyme immunoassay is desirable because it is simple and non-radioactive.

By preparing the anti-calpastatin antibody used for measuring the amount of calpastatin as a kit, the amount of calpastatin in the sample can be easily measured. The reagents used in the kit may be in solution form or may be in lyophilized form.

〔Example〕

The present invention will be explained below with reference to examples, but the present invention is not limited to the scope of the following examples.

Example 1 Sandwich EIA using two types of monoclonal antibodies
Measurement of organotypic calpastatin amount by method (1) Preparation of monoclonal antibody-bound beads) 1ybr
idoma CS L 4-7
No. 14 (PBRM P-10914)] monoclonal antibody C3L4-7 produced by Patent Application No. 1-21125
0, 1M phosphate buffer (pH 8,0) containing 1 mg prepared according to the method described in Specification No. 8, 50
-Polystyrene balls (manufactured by Tanezu Kagaku Co., Ltd., particle size 6.35)
mm) were added and reacted at 5°C for 16 hours to immobilize the antibody on the beads. After washing the beads with physiological saline, they were immersed in phosphate buffered saline containing 1% bovine serum albumin (BS^) and left overnight at 5°C to obtain anti-calpastatin monoclonal antibody-conjugated beads.

(2) Production of enzyme-labeled monoclonal antibody Hybrid
oma CS L 1-3
No. 3 (FBRM P-10913)] monoclonal antibody C3LI-3 produced by Patent Application No. 1-211258.
The antibody was prepared according to the method described in the specification, and peroxidase (manufactured by Boehringer Mannoim) was added to the antibody (
[Journal of Histochemistry and Cytochemistry - (
J,) Listochem, Cytochem,)
Vol. 22, p. 1084 (1987)]1 to obtain a labeled antibody. That is, dissolve 10 mg of peroxidase in 2- purified water, and add 0.2 ml of 0.1M potassium periodate! Add. After reacting at room temperature for 20 minutes, the mixture was dialyzed against 1 mM acetate buffer (pH 9,0) at 4°C overnight. On the other hand, C3L1-3 antibody 2 m
g was dissolved in 1.5-phosphate buffered saline (pH 7.4) and dialyzed against 10 mM carbonate buffer (pH 9.5) overnight at 4°C, which was then treated with periodic acid as described above. After mixing with peroxidase and reacting at room temperature for 2 hours, add sodium borohydride (4 mg/ml to 0.0
．． After adding 11nl of Ultrogel A and reacting at 4°C for 2 hours, it was equilibrated with phosphate buffered saline (p17.4).
Fractionated by gel filtration using c^22 (manufactured by LKB),
Both portions with matching peroxidase activity and antibody activity were collected, sodium merthiolate was added to a final concentration of 0.01%, and the mixture was stored at 4°C.

(3) Measurement of calpastatin The EIA method was performed as follows. Put 300 μl of the sample into a tube, and add 1 immobilized antibody bead into the tube.
The first incubation was performed at 37° C. for 20 minutes. Next, the beads are washed three times with physiological saline, 300 μl of labeled antibody solution (300-fold dilution) is placed in the tube containing the beads, and a second incubation is performed at 37° C. for 20 minutes.

The beads were then washed three times with 31 nl of saline and the beads were transferred to another tube containing 0.1% of the coloring reagent (0-) unilene diamine l mg/-1■, 0.1M. Add citric acid (pH 5,0),
The reaction was allowed to proceed at room temperature for 15 minutes, and 1 ml of IN H,SO was added to stop the reaction. Measure the absorbance at a wavelength of 492 nm.

The absorbance of the color development increased as the organotypic calpastatin concentration increased, and it became clear that organotypic calpastatin could be measured by this measurement method.

Example 2 Sandwich EIA using two types of monoclonal antibodies
Measurement of total amount of organ type and erythroid type calpastatin by method (1) Preparation of monoclonal antibody-conjugated beads) 1ybr
idoma CS L 5-12 (Microtechnical Laboratory 1st
No. 0915 (FBRM P-10915)) has filed a patent application for the monoclonal antibody C3L5-12 produced by
The antibody was prepared according to the method described in No. 1258, and monoclonal antibody-bound beads were produced in the same manner as in Example 1-(1) using the antibody.

(2) Preparation of enzyme-labeled monoclonal antibody It was prepared in the same manner as in Example 1-(2) above.

(3) Measurement of organ type and erythroid type calpastatin total amount Using the antibody-bound beads prepared in Example 2-(1) and the enzyme-labeled antibody prepared in Example 2-(2), Example 1-
Measurement was performed in the same manner as in (3). The absorbance of the color development increased with the increase in the amount of calpastatin in organ type and erythroid type, and it became clear that the total amount of calpastatin could be measured by this measurement method.

Example 3 Measurement of organ-type calpastatin Production of an EIA kit and measurement of the amount of organ-type calpastatin in the serum of various patients (1) Production of an EIA kit The composition of the EIA kit (1 batch) is shown below.

1. Antibody-immobilized beads 1 (beads on which the monoclonal antibody C3L4-7 mentioned above is immobilized) 2. Peroxidase-labeled antibody 0.3- (antibody labeled with the monoclonal antibody C3LI-3 mentioned above with peroxidase) 3 , coloring reagent 0.3-(0-
Citric acid buffer pH 5,0 containing 10 mg/-1 of phenylenediamine HCI and 0.01% hydrogen peroxide) 4. Reaction stop solution (IN sulfuric acid) 1. Oif
5. Standard product (gi device pear-shaped calpastatin 30.90.2
70.81 Q 1% BSA/TBS including pmol
) (2)) Measurement of organ-type calpastatin in patient plasma E above
The amount of organotypic calpastatin was measured based on Example 1 using the IΔ kit using serum from 23 healthy subjects, 7 patients with cerebral infarction, 7 patients with myocardial infarction, 5 patients with cerebral thrombosis, and 3 patients with gastric cancer. .

As a result of determining a calibration curve using a standard product, a calibration curve as shown in FIG. 1 was obtained. In the graph in Figure 1, the horizontal axis is calpastatin concentration (pmol/β), and the vertical axis is absorbance (A
4s2nm). The amount of organ-type calpastatin was determined from the absorbance obtained by measuring the sample using this calibration curve.

The measurement results of organotypic calpastatin in serum are shown in FIG. That is, FIG. 2 is a graph in which the amount of calpastatin in serum measured according to the present invention is plotted for each case.

As shown in FIG. 2, differences were observed in the distribution of organotypic calpastatin levels in serum between healthy subjects and patients with cerebral infarction, myocardial infarction, cerebral thrombosis, and gastric cancer. That is, it was found that the amount of organotypic calpastatin in the serum of patients with the above disease was higher than that of healthy individuals.

Example 4 Production of EIA kit for measurement of total amount of calpastatin in organ type and red blood cell type and measurement of total amount of calpastatin in serum of various patients (1) Production of EIA kit The composition of the EIA kit (one batch) is shown below.

1. 1 antibody-immobilized bead (bead on which the monoclonal antibody C3L5-12 mentioned above is immobilized) 2. Peroxidase-labeled antibody 0.3 mj' (
3. Coloring reagent 0.3 (0-
10■/-1 hydrogen peroxide of phenylenediamine HCI 0
．． 4. Reaction stop solution (IN sulfuric acid > i, o -
5. Standard product (red blood cell type calpastatin 30.90.2
(1% O3^/TBS containing 70.810 pmol) (2)) Measurement of total amount of calpastatin in patient serum EI above
The total amount of calpastatin was measured based on Example 2 using the A kit and using the same sample as in Example 3-(2). As a result, measurement results almost consistent with those shown in FIG. 2 were obtained.

Example 5 Change over time in total amount of calpastatin in serum after onset of myocardial infarction Using the kit of Example 4, changes over time in total amount of calpastatin in serum after onset of myocardial infarction were investigated in three patients with myocardial infarction. The results are shown in FIG. In other words, Figure 3 shows the number of days after the attack (days,
It is a graph showing the relationship between calpastatin concentration (pmol/i, vertical axis) and calpastatin concentration (pmol/i, vertical axis). As shown in Figure 3, the total amount of calpastatin in the serum increased significantly one day after the attack, and remained high for 2 to 13 days. As a result, creatinine kinase, which has traditionally been used as a biochemical diagnostic method for myocardial infarction, normalizes within 3 to 4 days after myocardial infarction, even though myocardial necrosis persists for several weeks. Considering that this was insufficient, this shows that a new detection method and kit have been provided that can diagnose myocardial infarction even in patients hospitalized several days after the attack.

[Effects of the Invention] As described in detail above, the present invention can reduce the production of calpastatin in cases where the amount of calpastatin in serum, plasma, or urine is typified by myocardial infarction, cerebral infarction, cerebral thrombosis, cancer, etc. It was discovered that it serves as a marker for diseases accompanied by abnormalities, and new detection methods and quantitative kits for the above-mentioned diseases were developed.

[Brief explanation of the drawing]

Figure 1 is a graph showing the calibration curve used to measure the amount of calpastatin, Figure 2 is a graph showing the measurement results of calpastatin in serum, and Figure 3 is the change over time in the total amount of calpastatin in serum after the onset of myocardial infarction. This is a graph showing.

Claims (1)

[Scope of Claims] 1. A method for detecting a disease accompanied by abnormal production of calpastatin, which comprises measuring the amount of calpastatin in serum, plasma, or urine in comparison with a normal value. 2. A kit for detecting a disease accompanied by abnormal calpastatin production, the kit comprising an anti-calpastatin antibody.