JPS59111062A - Measurement of beta-enolase - Google Patents

Abstract

PURPOSE:To improve measuring sensitivity, by a method wherein beta-enolase to be examined and enzyme labeled anti-betabeta-enolase antibody are immobilized by a solid phase through anti-betabeta-enolase antibody and, thereafter, the activity of the label enzyme bonded to the solid phase is measured. CONSTITUTION:An anti-betabeta-enolase antibody is obtained by a method wherein a mammal such as a rabbit or a goat is immunized with betabeta-enolase isozyme and the serum thereof is subjected to salting out. In addition, as label enzyme, beta- D-galactosidase or alkali phosphatase is used while, as the bonding method of the label enzyme and the betabeta-enolase antibody, a method not deactivating the respective activities of the label enzyme and the anti-betabeta-enolase antibody may by employed and a polyfunctional reagent such as glutaraldehyde or N,N-o- phenylmaleimido can be used. As a solid phase for insolublizing an anti-betabeta- enolase antibody, polysaccharides such as agarose, synthetic resin such as polystyrene, glass or polyacrylamide are used. By this method, measurement with high sensitivity and preciseness is enabled without receiving the influence of other components in live body fluids in an object to be examined.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for measuring β-type enolase.

Enolase is an enzyme that catalyzes the reaction of 2-phosphoglycerol, Mg, and phosphoenolpyruvate, and has a molecular weight of approximately 40,000 to 50,000
It has a dimeric structure with three types of subunits (α, β, and T) as its constituent units, and there are various isozymes. There are three types of β-type enolase isozymes: homodimeric ββ, and hybrid types αβ and βγ. The α subunit is a protein that exists non-specifically in living tissues, whereas the β subunit is specific to muscle tissues such as skeletal muscle and cardiac muscle, and the γ subunit is a protein specific to nerve tissues. be. As described above, enolase is an enzyme that exists in living tissues, but it is expected that it will leak into body fluids such as blood due to tissue lesions.

Conventionally, enolase has been measured by measuring its enzyme activity or by electrophoresis, but the former method has low sensitivity and is therefore inappropriate for measuring trace amounts of enolase in biological body fluids. Furthermore, it was not possible to specifically measure only β-type enolase. Moreover, the latter method requires complicated operations and is not far from being used as a daily measurement method.

The present invention relates to a method for measuring β-type enolase with excellent specificity and sensitivity, which improves these drawbacks. , enzyme-labeled anti-ββ
The enolase antibody is reacted with the enolase antibody, or the reaction product obtained by reacting the enzyme-labeled anti-ββ enolase antibody with β-enolase in the sample is allowed to act on the anti-ββ enolase antibody-bound solid phase. After immobilizing the sample β-enolase and the enzyme-labeled anti-ββ enolase antibody on a solid phase, the activity of the labeled enzyme bound to the solid phase is measured, and this measured value and a previously known amount of ββ enolase are used as a test solution. This is a measurement method characterized by determining the β-type enolase in a sample by comparing it with a calibration curve prepared in the same manner as above.

To obtain the anti-ββ enolase antibody used in the method of the present invention, first, mammals such as rabbits, goats, and rats are immunized with ββ enolase isozyme, and the serum is isolated by known methods such as salting out, chromatography, and dialysis. Obtain antibodies.

The antibody can be used as it is, but it can also be used with only the antigen-binding site isolated. For example, Fab parts obtained by treatment with proteases such as papain and pepsin, Fa
b' portions, F(a b')2 portions, etc. may also be used.

The labeling enzyme used in the method of the present invention only needs to be able to measure the activity as highly sensitively as possible. For example, β-D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase, malate dehydrogenase, etc. are used, but in particular β-D-galactosidase is preferred because it has high measurement sensitivity.

The method of binding the labeled enzyme and ββ enolase antibody used in the preparation of the enzyme-labeled anti-ββ enolase antibody is such that the respective activities (catalytic activity, antibody binding ability) of the labeled enzyme and the anti-ββ enolase antibody are not lost. Any method is fine. Specifically, glutaraldehyde, N, N
Known polyfunctional reagents such as '-0-phenylene dimaleimide and m-maleimidobenzoyl-N-hydroxysuccinimide ester can be used.

As a solid phase (insoluble carrier) for insolubilizing the anti-ββ enolase antibody, polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, glass, and polyacrylamide are used. Various forms of the solid phase can be used, such as spherical, tubular, columnar, or fibrous. Binding of the anti-ββ enolase antibody to a solid phase is usually carried out by physical adsorption, but methods used to insolubilize proteins or enzymes can also be used. For example, when using an insoluble polysaccharide, use bromine cyanide, sodium periodate, epichlorohydrin, 1''-carponylimitazole,
The binding reaction is performed by activation with P-)luenesulfonyl chloride or the like. Alternatively, an appropriate spacer may be introduced into the solid phase, and then an anti-ββ enolase antibody may be bound to the solid phase via the spacer. As the spacer, for example, 6-aminocaproic acid, hexamethylene diamine, or albumin is used. Furthermore, if the bond between the anti-ββ enolase antibody and the solid phase is a reversible bond, for example, an SS bond, the immunoreactant bound to the surface phase can be cleaved and removed from the solid phase after measurement.
(bonds are cleaved by reducing agents), the solid phase can be used repeatedly.

A spherical or fibrous solid phase can also be used by filling a column, but in this case, the appropriate amount of antibody immobilized on the solid phase is 0.1 to 20 mg per insoluble carrier, but even larger amounts can be used. Measurement sensitivity and accuracy can be improved by immobilizing anti-ββ enolase antibodies.

Hydrophobic proteins such as gelatin or salts such as sodium chloride are used to suppress or eliminate the interference caused by various biological fluid components coexisting in the specimen.

According to the method of the present invention, it is possible to measure β-enolase with high sensitivity and accuracy without being affected by other biological fluid components in the sample.

When β-type enolase in serum was measured using the method of the present invention and its relationship with pathological conditions was examined, a correlation with muscle diseases such as muscular dystrophy and myocardial infarction was observed. Taleatine kinase is conventionally known as a marker for muscle diseases, and a correlation was also observed between the measured value of β-type enolase and creatine kinase activity using the method of the present invention.

Example 1 +1) Antiserum (1 lil) of skeletal muscle was mixed with 10 times the amount of 50 mM phosphate buffer (pH 7,0
Homogenize with 5mM MgSO4 and centrifuge (4°C, 107,000 xg-, 1 hour).
After that, the supernatant was heat-treated at 55°C for 5 minutes, and then heated for 15 minutes.
mM phosphoric acid tikilj solution (pH 7, 0, 5mM Mg
0M Sephatex equilibrated with SO4), 15
mMIJ acid buffer (pH 18, 0° 4mM MgS
DEAE Sephadex equilibrated with O4), 1
0mM Tris-HCl buffer (pH 8, 5, 4mM MgS
QAE Sephadex equilibrated with O4), 5m
Column chromatography on CM Sephadex equilibrated with M phosphate buffer (pH 6, 0, containing 5 mM MgSO4) was sequentially performed to obtain a purified ββ enolase preparation. By the above method, approximately 10 g of skeletal muscle is
mg of purified sample was obtained. Anti-ββ enolase serum was prepared by immunizing rabbits with this preparation.

(2) ββ enolase-specific antibody and its F (a b
') Preparation of 2 fragments Antiserum 1 obtained from 3 rabbits
Immunoglobulin G (abbreviated as IgG) fraction was precipitated by adding ammonium sulfate to 50% saturation under cooling.

Centrifuge the precipitate (10,0OOX g, 15 minutes, 4℃)
The solution was collected in 0.01M phosphate buffer (pH 7.0) containing 0.1% NaN3, and dialyzed against the same buffer at room temperature.

The dialysate fluid was transferred to antigen-binding Sepharose 4B (Sepharose 4B).
ose4B, Pharmacia 1M) column (1,0X
20cm) to adsorb the antibody, then IM onto the column.
0.1 M glycine-hydrochloric acid buffer containing NaCl (pH
2, 5) was used for elution. The obtained antibody fractions were collected, neutralized, and then concentrated in a collodion bag (SM 13,200, Zatorius) under reduced pressure to obtain 15 mg of antibody 1gG. Add 1.6 mg of pepsin (derived from pig intestinal mucosa, manufactured by Sigma) to 1 g of antibody, and incubate at 37°C for 16 days.
F(ab')2 fragments were obtained after time treatment.

(3) Preparation of antibody Fab′-β-D-galactosidase complex Antibody F(ab”)2 fragment was reduced with 2-mercaptoethylamine to obtain a Fab′ fragment, and then an excess of N-N′-0-phenylene dimaleimide was added. (manufactured by Aldrisohi) Maleimide-Fab added to the solution and reacted.
' was obtained, and this was reacted and combined with β-D-galactosidase (manufactured by Boehringer, hereinafter abbreviated as Gal). Fa
The amount of b'-Gal complex was expressed as Gal activity. The activity is
When 4-methylumbelliferyl-β-D-galactoside is reacted as a substrate and the fluorescence of the produced 4-methylumbelliferone is measured with a fluorophotometer, 1μ7' of product is produced per minute. The amount was defined as 1 unit.

As described above, the enzyme-labeled antibody prepared from 10 mg of F(ab'') can be used to measure about 70,000 to 80,000 samples.

(4) Preparation of antibody-bound solid phase
(a b') 2 fragments were physically adsorbed.

That is, diluted F (a b')2 solution (pH 7,
0.

Soak a piece of silicone rubber in A28 (1=0.5),
It was left at 4°C overnight. After removing the antibody solution, the solid phase was
tt+phosphate buffer wave buffer+7.0.0. IM Na
Cl, 1mM MgCl2, 0.1% bovine serum albumin, and 0.1% NaN3 (hereinafter referred to as solution A), and stored in solution A at 4° C. for 2 days or more before use for measurement.

The used antibody F (ab')2 solution could be used repeatedly, and the antibody-bound solid phase was stable for at least one month.

(5) Measurement operation 0.01, 0.03, 0.1.0.3.1.
0,3.0.10. Using Ong's ββ enolase, this 0.01mE and O.01M phosphate buffer (pH 7.0
: 0.3M NaCl, 1mM MgCl2.0.
1% bovine serum albumin, 0.5% gelatin, 0.1% N
Contains aN3 (hereinafter referred to as B solution) 0.49mF! and one of the above antibody-bound solid phases were mixed and shaken at 30°C for 5 hours. After that, the reaction solution was removed by suction, and the solid phase was mixed with solution A 1-2.
Enzyme-labeled antibody (3 mu/
0.2ml! ) and stored at 4°C overnight. The next day, the reaction solution was removed by suction, the solid phase was washed with solution A, and the Gal activity bound on the solid phase was measured.

The ββ enolase specimen used as a specimen was made of steel using the method described in (1) above. This specimen is SD
S gel electrophoresis showed a single band, 5mM MgS
It was stable for at least 6 months when stored at -30°C in a 50% glycerin solution containing O4. For comparison, αα enolase and γγ enolase preparations were cleaved from the brain by a method similar to the above (11), and the same measurement procedure was performed using these as specimens.

A calibration curve showing the relationship between the amount of ββ enolase and the fluorescence intensity is shown in FIG. 0.01 to 1 per measurement using the method of the present invention
Ong ββ enolase could be measured, and no crossover between αα and Tγ enolases was observed.

Example 2 Using four serum samples as specimens, the accuracy of β-enolase measurement by the method of the present invention, that is, intra-day variation and day-to-day variation, was investigated. The operation was performed in the same manner as in Example 1, and the concentration of β-type enolase in the sample was determined by comparing the measured value of fluorescence intensity in each reaction with the above-mentioned calibration curve (ββ
(expressed as enolase disozyme equivalent), its mean value and standard deviation (expressed as SD) were calculated. The results are shown in Table 1.

Table 1 Example 3 β-type enolase was measured in 59 normal human serum samples and 10 patient serum samples. The measurement operation was performed in the same manner as in Example 1, and the relationship between the measured values and the pathological condition was observed, and the results were shown in FIG. 2. In other words, serum β-type enolase was on average 55-6n/mR in normal people, but
It was found that all values were higher than normal values in patients with enne) type dystrophy.

Example 4 β-type enolase was measured in 100 serum samples. The measurement procedure was performed in the same manner as in Example 1, and for comparison, creatine kinase, which is known as a marker for muscle diseases, was measured at the same time. Creatine kinase was measured using the known measurement reagent CPK-Pestwako (bisubstrate creatine phosphate, adenosine diphosphate, hexose kinase, manufactured by Wako Pure Chemical Industries, Ltd.).
(including glucose 6-phosphate dehydrogenase, diaphorase, etc.).

The results are shown in Figure 3. A significant correlation was observed between β-type enolase and creatine kinase by the method of the present invention,
The correlation coefficient was 0.901.

Example 5 Serum β-type enolase in patients with myocardial infarction was measured over time. The measurement operation was performed in the same manner as in Example 1, and creatine kinase was measured at the same time for comparison.

As shown in FIG. 4, a good correlation was observed between β-type enolase and creatine kinase obtained by the method of the present invention.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a calibration curve for ββ enolase isoenzyme measurement according to the method of the present invention. FIG. 2 is a diagram showing the relationship between the serum concentration of β-enolase and pathological conditions, and FIG. 3 is a diagram showing the relationship with serum creatine kinase. FIG. 4 is a diagram showing changes in β-type enolase in patient serum in comparison with creatine kinase. Patent applicant Amano Pharmaceutical Co., Ltd. Figure 1 OQfl 0.1
1 10 Enolase (ng) Fig. 2 iE Ordinary person Decocieci type Cis I- Lofi Fig. 3 0 200
40OfioO creatine kinase (ml/ml) Figure 4 0612211 48 hours 7 days elapsed (hours or days)

Claims (1)

[Claims] An enzyme-labeled anti-ββ enolase antibody is reacted with the reaction product obtained by reacting β-type enolase in the specimen with the anti-ββ enolase antibody-bound solid phase, or the enzyme-labeled anti-ββ enolase antibody is reacted with the β-type enolase in the specimen. By allowing the resulting reaction product to act on the anti-ββ enolase antibody-bound solid phase, the analyte β is released through the anti-ββ enolase antibody.
1. A method for measuring β-type enolase, which comprises immobilizing a β-type enolase and an enzyme-labeled anti-ββ enolase antibody on a solid phase, and then measuring the activity of the labeled enzyme bound to the solid phase.