JPH038515B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying human-derived neural tissue-specific enolase released from clinically useful neural tissue or neuroendocrine cells.

Enolase is an enzyme that catalyzes the reaction of 2-phosphoglycerate and phosphoenolpyruvate.
It has a molecular structure and consists of three types of subunits (α, β, γ) with a molecular weight of about 40,000 to 50,000 [L. FIetcher et al.: Biochim. Biophys. Acta No.
Volume 452, pages 245-252, 1976].

Among these, γγ type and αγ type are neuron-specific enolase (hereinafter referred to as neuron-specific enolase).
Abbreviated as NSE. ). Immunohistochemically, this NSE is present in neuronal cell bodies, axons, and synapses, but is not present in normal glial cells. Enolase present in glial cells is αα type and non-neuronalenolase (NNE).
It is abbreviated as ).

It is known that NSE accounts for more than 1% of the soluble protein in nerve tissue, increases in amount as nerve cells differentiate, and increases particularly rapidly during the period of nerve cell formation. As described above, NSE is present in large amounts in nerve cells, and the amount increases with the differentiation of cell bodies, suggesting a close relationship with the higher functions of nerve cells. Recently, it has been confirmed that NSE exists in cells originating from neural tissues such as intraneural cysts and tumor cells that develop from them [PJ Marangos et al.: Journal of Neurochemistry (J. Neurochem.) Volume 33,
pp. 319-329, 1979] and [Tapia et al. (P.J.
Tapia et al.): The Lancet 1st
Volume, pages 808-811, 1981]. This suggests that NSE will leak into body fluids (blood, cerebrospinal fluid, etc.) as nerve tissue is damaged or tumor cells disintegrate or divide.

The present invention was completed against this background, and its purpose is to detect cancer and tumors by quantifying minute amounts of NSE in human body fluids with high sensitivity using enzyme immunoassay. The aim is to use it as a means of diagnosis, judgment of therapeutic effects, and judgment of prognosis. However, the quantitative method of the present invention is not limited to the above, and it is also possible to quantify NSE in tissues other than body fluids.

Conventional methods for quantifying enolase include radioimmunoassay (hereinafter referred to as RIA) using antisera for each type of enolase isozyme in order to distinguish and specifically quantify various enolase isozymes using limited tissue materials without separation procedures. ) has been used. For example [Marangos et al. (PJ
Marangos et al.): Journal of Neurochemistry (J.Neurochem.) Volume 33, 319-329
Page, 1979] measured NSE and NNE in rat, monkey, and human brain tissues using the RIA method in units of ng. [KWBrown et al.: CIinica, Chimica,
Acta) Volume 101, pages 257-264, 1980]
NSE in human cerebrospinal fluid and serum by RIA method
, and states that the limit of its sensitivity is 150 pg. Also [AMParma et al.: Journal of Neurochemistry (J.
Neurochem.) Volume 36, pages 1093-1096, 1981
] detected NSE in the range of 108 pg to 2 ng using the more sensitive RIA method.

As described above, the RIA method allows the quantification of enolase at a level of approximately 100 pg. However, although the RIA method is sufficient for quantifying large amounts of enolase such as in brain tissue, it is not suitable for quantifying trace amounts of enolase in cell culture fluids or body fluids (e.g. cerebrospinal fluid, blood, urine, etc.). Not suitable. Furthermore, a disadvantage of the RIA method is that it requires special facilities because radioactive isotopes are used.
Stability is low due to the need for equipment and the half-life of the isotope used.

Therefore, the present inventors attempted to quantify human enolase by enzyme immunoassay method (hereinafter referred to as EIA method) using Escherichia coli β-D-galactosidase as a labeling enzyme, and found that approximately 3 pg of human enolase was detected. The present invention was completed after learning that trace amounts of enolase can be measured. According to the method of the present invention, it is possible to quantify human enolase with a sensitivity approximately 30 times greater than that of the RIA method, and furthermore, no special equipment is required, and the reagents used for the reaction are stable for a long period of time.

That is, the present invention involves reacting an anti-enolase antibody-bound solid phase with human enolase in a specimen and causing an enzyme-labeled anti-enolase antibody to act on the reaction product, or reacting human enolase in the specimen with an enzyme-labeled anti-enolase antibody. Human enolase and enzyme-labeled anti-enolase antibodies are immobilized on the solid phase via the anti-enolase by allowing the reaction product obtained to act on the anti-enolase antibody-bound solid phase. The present invention relates to a method for quantifying human enolase, which is characterized in that an anti-enolase antibody against enolase derived from an animal other than humans is used as an anti-enolase antibody in a method for determining the amount of human enolase in a sample by measuring tissue enzyme activity.

The features of the method of the present invention, ie, how it is possible to use an anti-enolase antibody against animal-derived enolase instead of an anti-human enolase antibody, will be described below.

First, in human enolase quantification, the γ subunit of enolase used to prepare the antibody-bound solid phase and enzyme-labeled antibody has immunochemically common parts with humans depending on the type of animal. There are many substances (e.g. human gamma subunit is found in cows,
They found that some γ subunits have a lot in common with those of pigs, while others do not (for example, the γ subunits of rats and rabbits have few common parts).

Therefore, when using a polyclonal antibody obtained using bovine or porcine enolase as an anti-enolase antibody in the human enolase quantification of the present invention, that is, an anti-enolase antibody obtained using bovine or porcine enolase, this antibody is In many cases, the reactivity against human enolase is almost the same as that of an anti-human enolase antibody, so we decided to select a lot that has the same reactivity against human enolase as an anti-human enolase antibody and use it as a substitute.

On the other hand, the rat and rabbit γ subunit is
It has little in common with the human γ subunit,
When polyclonal antibodies obtained using rat and rabbit enolase, ie anti-enolase antibodies, are used, they do not show the same reactivity with human enolase as anti-human enolase antibodies. Therefore, as is, anti-enolase antibodies derived from rats and rabbits cannot be used as a substitute for anti-human enolase antibodies.

However, the present inventors have demonstrated that by using a method that selectively utilizes the parts common to human gamma subunits derived from rats and rabbits, cows and rabbits can be produced.
We thought that it could be used as a substitute for human enolase antibody, as in the case of pigs. Therefore, the present inventors tried the following measures for this purpose. (1) Purification of anti-enolase antibodies derived from gamma subunits derived from rats and rabbits by affinity chromatography using human enolase. (2) Select a lot that has the same reactivity to human enolase as an anti-human enolase antibody from among several immunized animals derived from the γ subunit of rats and rabbits. (3) Anti-enolase monoclonal antibodies are isolated using rat and rabbit-derived enolases that have the same reactivity with human enolase as anti-human enolase antibodies.

In this way, it became possible to quantify human enolase using animal-derived anti-enolase without using anti-human enolase.

Thus, in the method of the present invention, when quantifying human enolase, instead of using anti-human enolase antibodies that are difficult to obtain, anti-enolase antibodies prepared using enolase obtained from non-human animals can be used instead. A major feature of this method is that it can stably produce reagents used for quantitative determination and can be widely applied to clinical medicine.

The labeled enzyme used in the present invention only needs to be able to measure activity as highly sensitively as possible, for example, β-
Any commonly used enzyme such as D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase, malate dehydrogenase, etc. may be used, but β-D-galactosidase has particularly high measurement sensitivity, so it can be used for high-sensitivity measurement. preferred.

The method of binding the enzyme and enolase antibody used to prepare the enzyme-labeled anti-enolase antibody is based on the respective activities (catalytic activity,
Any method that does not cause loss of antibody binding ability (antibody binding ability) may be used; specifically, glutaraldehyde, carbodiimide, N,N'-O-phenylene dimaleimide, m-maleimidobenzoyl-
N-hydroxysuccinimide ester (m-
Maleimidoben-zoyl-N-
Known bifunctional reagents such as HydroxysuccinimideEster) can be used.

As the solid phase (insoluble carrier), polysaccharides such as agarose, dextran, cellulose, synthetic resins such as polystyrene, glass, polyacrylamide, etc. are used, and the form is bead-like,
It is preferably fibrous. Although physical adsorption may be used to bind the anti-enolase antibody to the solid phase, methods commonly used to insolubilize proteins or enzymes may also be used. For example, methods used for insolubilization may be used. For example, when using an insoluble polysaccharide, the insoluble polysaccharide may be cyanogen bromide, sodium periodate, epichlorohydrin, 1,1'-carbonyldiimidazole, p
- Activate with toluenesulfonyl chloride or the like to carry out a binding reaction.

In addition, after introducing an appropriate spacer into the solid phase,
An anti-enolase antibody may be bound via a spacer. Furthermore, if the bond between the anti-enolase antibody and the solid phase is a reversible bond, for example, an S-S bond, the immunoreactant bound to the solid phase is cleaved and removed from the solid phase after measurement (for example, an S-S bond). cleaved by a reducing agent), the solid phase can also be used repeatedly. When using a column, the appropriate amount of antibody to be insolubilized in the solid phase is 0.1 to 20 mg per ml of insoluble carrier, but if possible, insolubilize a larger amount of anti-enolase antibody to improve measurement sensitivity and accuracy. It is possible to improve.

The anti-enolase antibody protein used here may be used as is, or may be one in which only the antigen-binding site has been isolated. For example, Fab parts obtained by treatment with proteases such as papain and pepsin,
Fab′ portion, F(ab′) ₂ portion, etc. can also be used. There is a report by Ishikawa et al. on the method for preparing the Fab' portion [Scandinavian Journal of the Immunology (Scand. J. Immunol.) No. 8].
Vol. 43, 1978).

Gelatin and the like are used as hydrophobic buffers used to suppress or eliminate the interference caused by biological fluid components, and common salts are used as salts.

According to the present invention, human enolase to be measured can be measured with high sensitivity and accuracy without being affected by biological body fluid components, and furthermore,
It is also easy to apply to automatic measurement systems.

Human enolase in body fluids (e.g. blood, spinal fluid, urine, etc.) can be determined using the method of quantifying human enolase according to the present invention.
NSE can be determined by quantifying NSE and detecting diseases that involve the destruction of neural tissues or cells that are embryologically close to nerve cells, such as tissues containing neuroendocrine cells (anterior pituitary gland, thyroid gland, pancreatic islets of Langerhans, adrenal glands, etc.). It is effective in the early diagnosis of diseases such as tumors that arise from tissues, in determining the efficacy of treatment, and in determining the quality of the prognosis.

Additionally, among certain types of cancer (e.g., esophageal cancer, kidney cancer, rectal cancer, pancreatic cancer, liver cancer, etc.), NSE is present in large amounts in the body fluids of patients with malignant undifferentiated cancers, and in benign cancers, NSE levels are low. The method of the present invention is also useful for diagnosing such diseases.

The method for quantifying human enolase of the present invention is several tens of times more sensitive than conventional assay methods and can quantify trace amounts of human enolase, making it extremely useful for the above purpose.

This will be explained in detail below using examples.

Example 1 (1) Preparation of antiserum Anti-γ serum was obtained from bovine brain using the method of Suzuki et al. [F.
Suzuki, (J.Biochem.) Volume 87, pages 1587-1594,
It was created by immunizing rabbits with γγ enolase purified according to (1980).

(2) Preparation of enolase-specific antibody and its F (ab') ₂ fragment Add 100 ml of antiserum obtained from 3 rabbits under cooling (4
℃) Ammonium sulfate was added to achieve 50% saturation to precipitate a 1 gG (immunoglobulin G) fraction.
The precipitate was collected by centrifugation (10,000 x g, 15 minutes, 4°C) and _diluted with 0.1M phosphate buffer (PH
7.0) and dialyzed against the same buffer at room temperature.
Next, add this to antigen-bound Sepharose 4B.
(Sepharose4B, manufactured by Pharmacia) Column (1.0
×20cm). Elution of the antibody adsorbed to the column was performed with a 0.1M glycine-hydrochloric acid buffer (PH2.5) containing 1M NaCI. The eluted purified antibody fractions were collected, neutralized, and concentrated in a collodion bag (SM13200, Sartorius) under reduced pressure.
Approximately 15 mg of antibody IgG was obtained. This includes 1.6mg of pepsin.
(derived from pig intestinal mucosa, manufactured by Sigma), and heated at 37℃.
After treatment for 16 hours, F(ab') ₂ fragment was obtained.

(3) Preparation of antibody Fab′-Gal complex Antibody F(ab′) ₂ fragment was reduced with 2-mercaptoethylamine to produce Fab′(SH) fragment, and then treated with excess N,N′-o-phenylene dimaleimide. (N,N'-o-Phenylendimaleimide,
(manufactured by Aldrich) and reacted to obtain maleimide-Fab', which was reacted and bound with β-D-galactosidase (hereinafter abbreviated as Gal, manufactured by Boehringer). The amount of Fab'-Gal complex is expressed as Gal activity, 1 unit activity=1 μmole product/min. The enzyme-based antibody prepared from 10 mg of F(ab') ₂ in this manner can be used to measure about 70,000 to 80,000 samples.

(4) Preparation of antibody-bound solid phase Antibody F(ab') ₂ fragment was physically adsorbed onto polystyrene beads (diameter 6.5 mm, manufactured by Sekisui Chemical Co., Ltd.). That is, the above F(ab') ₂ diluted appropriately
Polystyrene bead pieces were immersed in the solution (PH7.0, A280≒0.5), left overnight at 4°C, the antibody solution was collected, and the solid phase was soaked in 0.01M phosphate buffer (PH7.0),
Next, add solution A [0.01M phosphate buffer, pH7.0,
It was thoroughly washed with 0.1M NaCl, 1mMgCI ₂ , 0.1% bovine serum albulin (BSA), 0.1% NaN ₃ ) and stored in solution A at 4° C. for 2 days or more before being used for measurement. Antibody F
The (ab′) ₂ solution could be used repeatedly, and the antibody-conjugated solid phase remained stable for at least a year.

(5) Measurement procedure G solution containing standard enolase (0.01M phosphate buffer, PH7.0, containing 0.3M NaCI, 1mMgCI ₂ , 0.1%
Antibody-conjugated beads were placed one by one in 0.5 ml of BSA, 0.5% gelatin, 0.1% NaN ₃ and shaken at 30°C. 5
After a period of time, the reaction solution was removed by suction, and the solid phase was washed in the test tube (1 ml of solution A x 2). The solid phase was transferred to an enzyme-labeled antibody (3 munits/0.2 ml) diluted with Solution A and allowed to stand at 4°C overnight. The next day, the reaction solution was removed by suction, the solid phase was washed with solution A, and then bound onto the solid phase.
Gal activity was measured. Gal activity is associated with o-nitrophenyl-β-D-galactopyranoside (o-
o-nitrophenyl (o-nitrophenyl-β-D-galactopyranoside) was used as a substrate.
nitrophenyl) was measured using a spectroscopic altimeter.

The four enolase standards used were prepared by the previously described method of Suzuki et al. The final specimen is
No other proteins were detected by SDS gel electrophoresis. The content of purified enolase was determined using the Bio-Rad protein assay method [MM Bradford: Analytical Biochem.
Vol. 72, pp. 248-254, 1976).

These standard enolase products (approximately 1 mg/ml) are
It is immunoreactively stable for at least 3 months at -30°C in 30mM phosphate buffer (PH 6.8) containing 2.5mM _MgCI2 , 50% glycerol.

The calibration curves of four types of enolases are shown in FIG. In the figure, -●- indicates bovine γγ enolase, and -○- indicates human γγ enolase.
Enolase, -▲- means human αγ enolase -◇
- represents human αα enolase, -Δ- represents human ββ enolase. γγ enolase measurement system has a minimum of about 50p
Human γγ enolase was 100% cross-reactive with a measurement system using an antibody prepared by immunizing a rabbit with bovine γγ enolase.
No cross-reactivity between human αα enolase and human ββ enolase was observed.

Hybrid human αγ enolase cross-reacted by approximately 30%.

Example 2 An antibody prepared by immunizing a rabbit with γγ enolase purified from rat brain was purified by affinity chromatography using human enolase, dried in the same manner as in Example 1, and a calibration curve was obtained. I asked for it. In this example, a cylinder made by cutting a piece of silicone (string with a diameter of 3 mm) into a length of 4 mm was used as the antibody-binding solid phase, and a cylinder of 4 mm in length was used to measure the enzyme activity bound on the solid phase.
-methylumbelliferyl-β-D-galactoside (4-methylumbelliferyl-β-D-
4-methylumbelliferon produced using galactoside as a substrate
was measured using a fluorophotometer. The results are shown in Figure 2.
With the method of this example, as in Example 1, trace amounts of human NSE can be quantified.

[Brief explanation of the drawing]

FIG. 1 shows a standard curve for enolase isozyme when an antibody prepared by immunizing a rabbit with bovine brain γγ enolase is used in the reaction system. FIG. 2 shows a standard curve for γγ enolase when an antibody prepared by immunization with rat brain γγ enolase is used in the reaction system. In Figures 1 and 2, -●- indicates bovine γγ enolase, -○- indicates human γγ enolase, -▲-
represents human αγ enolase, −◇− represents human αα enolase, and −Δ− represents human ββ enolase.

Claims (1)

[Scope of Claims] 1. An enzyme-labeled anti-enolase antibody is reacted with a reaction product obtained by reacting human enolase in a specimen with an anti-enolase antibody-bound solid phase, or human enolase in a specimen and an enzyme-labeled anti-enolase antibody are reacted. Human enolase and enzyme-labeled anti-enolase antibodies were immobilized on the solid phase via the anti-enolase antibody by reacting the reaction product obtained by the reaction with the anti-enolase antibody-bound solid phase, and then bound to the solid phase. In a method for determining the amount of human enolase in a sample by measuring the labeled enzyme activity,
The anti-enolase antibody is an antibody against enolase derived from animals other than primates and has a reactivity to human enolase equivalent to that of an anti-human enolase antibody, or an anti-enolase antibody which has been purified as necessary and has a reactivity to human enolase. A method for quantifying human enolase, which comprises using an anti-enolase antibody that is equivalent to a human enolase antibody. 2. The method for quantifying human enolase according to claim 1, wherein the antibody against animal-derived enolase is an anti-enolase antibody prepared using bovine or porcine enolase. 3. Quantification of human enolase according to claim 1, wherein the antibody against animal-derived enolase is an anti-enolase antibody prepared using rat or rabbit-derived enolase purified by affinity chromatography using human enolase. Method.