JPH0643167A - Cancer marker tenascin and its determining method - Google Patents

Abstract

PURPOSE:To detect the presence or state of cancer in a human being by authorizing tenascin as a cancer marker in a living organism body fluid. CONSTITUTION:Human tenascin is prepared and refined from a human cell and human tenascin antibody is obtained using the prepared human tenascin. On the other hand, labeled human tenascin, where a labeled substance which can be detected using highly refined human tenascin is connected, is created. The labeled substance which can be detected, where the detection sensitivity is sufficient for a detection sensitivity needed for the determination method of human tenascin, is selected. An already known method is used to connect the substances which can be detected and the human tenascin. Those with different number of combinations between, the substance which can be detected and the human tenascin may be mixed in addition to the original target labeled human tenascin in the labeled human tenascin which is obtained in this manner. Therefore, only the labeled human tenascin is separated from a mixture for increasing determination sensitivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to tenascin as a cancer marker and a method for quantifying the tenascin. More specifically, quantifying tenascin in body fluids, comparing it to a control level, confirming the presence of high levels, tenascin as a cancer marker showing a diagnostic indication as cancer, and a method for quantifying tenascin by immunological measurement. Regarding

[0002]

2. Description of the Related Art Tenascin is a glycoprotein which is an extracellular substance. In the process of producing a monoclonal antibody against type V collagen by Chiquet and Fambrough in 1984,
It was discovered as an antigen of an antibody that exhibits a unique staining property different from V-type collagen [J. Cell Biol. 98, 1926-1936, 1937-1.
946, (1984)].

Today, various physiological actions of tenascin are being revealed. The early discovered function of tenascin is hemagglutination, which has a stronger activity than fibronectin, suggesting that it acts in a regulated manner in the field of tissue formation [Bioscience, Vol. 39]. , August issue (1988)].

Fibronectin [Cell, 53, 383-3] is also used as a non-covalent interaction with other molecules.
90 (1988)] and proteoglycans [Journal of Cell Biology, 98, 1926-1936 (1984)]
Has been reported.

Further, physiological activities such as cell adhesion activity [Journal of Cell Biology, 105, 138a] and growth promoting activity [Cell, 47, 131-139 (1986)] have been clarified. It is thought to play an important role in cell proliferation and morphogenesis.

Tenascin was initially considered to be a carcinoembryonic antigen, after which several monoclonal antibodies against human tenascin were taken and the expression of tenascin in various cancers examined.

Recently, it has been revealed that tenascin is present in serum [Cancer Res., 51, 4853-4858 (199).
1)]. A number of methods have been reported as methods for quantifying tenascin. For example,

E using a monoclonal antibody and a rabbit polyclonal antibody against anti-chicken tenascin
Measurement of tenascin in human osteosarcoma tissue and chicken embryo brain by LISA method [Annals New York Academy of Science]
science), 580, 260-275 (1990)]

By mixing the serum of melanoma patient and the monoclonal antibody and subsequently reacting with tenascin bound to the solid phase, the amount of the monoclonal antibody bound to tenascin on the solid phase after the reaction is measured by the RIA method [Cancer Research ( Cancer Res.) Volume 51, 4853-4858
Page (1991)] is reported.

[0010]

However, these have many problems. That is, the problems in the method (1) are as follows. (A) Since an antibody against chicken tenascin is used, when measuring tenascin in human tissue, the crossover rate has not been determined, and therefore it cannot be concluded that the value is accurate. (B) The amount of tenascin has not been measured in cancer patients based only on the measurement results of the tissue extract and normal human plasma.

The problems with the method (1) are as follows. (A) The inhibition of tenascin bound to the solid phase of the antibody by tenascin in serum is shown as relative inhibition%, and the amount of tenascin is not accurately determined. (B) Although the measured value is a kind of melanoma and the inhibition rate is higher than that of a normal person, there is not much difference in other cancers.

In order to solve the above-mentioned problems, the present inventors prepared human tenascin from a culture solution of human cells,
Rats and rabbits were immunized with the obtained human tenascin as an antigen. From this, anti-human monoclonal antibody and anti-human polyclonal antibody were prepared, and immunoassay reagents were prepared using these antibodies, and the concentration of tenascin in human serum was accurately quantified for the first time, and it was not clear until now. The present invention was completed by discovering that the proportion of high tenascin levels in serum of various cancer patients other than epithelial cancer is significantly higher than that in normal individuals, and that tenascin can be newly used as a cancer marker.

[0013]

The present invention provides a method for detecting the presence and status of cancer in humans by assaying tenascin as a cancer marker in biological fluids. That is, according to the present invention in which tenascin is quantified as a cancer marker, it is possible to detect the possibility of cancer detection or status change of various cancer treatments or recurrence or metastasis of prognosis in diagnosis.

The present invention also provides the above-mentioned method for quantifying human tenascin. For example, regarding an immunoassay using a specific antibody against human tenascin which is a suitable quantification method,
An example thereof will be described in detail.

Human tenascin is described in, for example, JP-A-2-5649.
It can be prepared and purified from human cells by the method described in JP-A-9. Using the human tenascin thus prepared, a human tenascin antibody can be obtained, for example, according to the method described in JP-A-2-219590. That is, human tenascin is injected as it is or after being mixed with an appropriate adjuvant, into the skin, intraperitoneal cavity, or the like of an animal. This operation is repeated at 1 to 2 week intervals for immunization. By hybridizing cells of the immunized animal with myeloma cells, a hybridoma producing a human tenascin antibody can be obtained. The polyclonal antibody can be obtained from the serum of the animal in which antibody production has been confirmed after immunizing the animal with human tenascin as described above.

The antibody to be used can be used in the prepared immunoglobulin fraction itself, but it can also be used as a fraction of F (ab ') ₂ , Fab', Fab or the like in which only the binding site with human tenascin is separated. it can.

On the other hand, highly purified human tenascin is used to prepare labeled human tenascin bound to a detectable labeling substance. It is necessary to select, as the detectable labeling substance, one whose detection sensitivity is sufficient for the detection sensitivity required for the human tenascin quantification method. Specifically, there are various radioisotopes, various enzymes, fluorescent substances, metal ions and the like, and more preferably ¹²⁵ I, ¹³¹ I, β-D-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, malate dehydrogenase, fluorescein. , Methyl umbelliferone, magnesium and the like.

A known method is used for binding these detectable substances to human tenascin. For example, when a radioisotope is used, reagents such as chloramine T and iodogen (registered trademark by Pierce) are used, and in the case of an enzyme, a coupling method using a bifunctional reagent, a periodate oxidation method and the like are used. It can be applied.

In the labeled human tenascin thus obtained, in addition to the originally intended labeled human tenascin,
Detectable substances that differ in the number of bindings to human tenascin, those that have lost their original activity due to partial changes in the detectable substance during the binding procedure to human tenascin, and those that have not been involved in the binding reaction The substance itself or the human tenascin itself, which is not bound to the detectable substance, may be contaminated. Therefore, in order to increase the sensitivity of quantification, it is preferable to separate only the labeled human tenascin of interest from the above mixture. As a method for this purpose, ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like can be used.

In addition to the above, a reagent necessary for quantifying human tenascin is a water-insoluble carrier to which an antibody is directly bound. Further, there is a factor that binds to an antibody that is insolubilized in a water-insoluble carrier. Since the antibody is an immunoglobulin, an anti-immunoglobulin antibody, that is, a second antibody can be used as the factor that binds. In addition, protein A and protein G derived from microorganisms, which are immunoglobulin-binding proteins, can also be used. These binding factors are preferably highly purified by means such as affinity chromatography in order to increase the binding amount of the antibody. As the water-insoluble carrier for insolubilizing the binding factor,
Various polysaccharide gels, particles made of synthetic resin such as polystyrene, beads, test tubes, other small containers, similar forms of glass, metal, etc., as well as synthetic thin films such as nitrocellulose and nylon are suitable. .

As a method for insolubilizing the factor that binds to the water-insoluble carrier, chemical adsorption such as physical adsorption or covalent bonding can be used. For example, in the physical adsorption method, the polystyrene beads and the like can be physically insolubilized by putting the polystyrene beads and the like in a solution of the binding factor of a constant concentration and leaving it for a certain period of time. On this occasion,
By constantly moving the solution of the factor that binds upon insolubilization, the factor that binds to the surface of the water-insoluble carrier can be uniformly insolubilized. Also, the solution of the binding factor is 15-40 ° C.
It can be insolubilized in a short time by keeping it at. In the chemical coupling method, various coupling reagents such as cyanogen bromide, glutaraldehyde, sodium periodate, carbodiimide derivative, silane agent, maleimide derivative, succinimide ester, oxirane compound, tosyl chloride, carbonylimidazole, etc. are used. To be

The factor binding to the antibody thus insolubilized in the water-insoluble carrier is not preferably used as it is for quantifying human tenascin. That is, when used as is, the surface of the water-insoluble carrier or the chemically active reactive group used for the insolubilization of the factor to be bound non-specifically adsorbs and binds the reaction solution component during the quantification, deteriorating the measurement sensitivity. Will bring. In order to prevent this, it is preferable to treat the surface of the water-insoluble carrier after insolubilizing the binding factor. As this treatment method, a method of inactivating the surface of the water-insoluble carrier after thoroughly washing the water-insoluble carrier after insolubilizing the binding factor is preferable. In order to inactivate the surface of the water-insoluble carrier, albumin, gelatin, a solution containing one or more kinds of other proteins, various amino acids, a solution of other compounds having an amino group, a reducing agent, an oxidizing agent, etc. are each used alone. Alternatively, they can be used in combination.

Human tenascin is quantified using the reagent thus prepared. First, a certain amount of biological fluid (for example, cerebrospinal fluid, serum, plasma, tissue extract, cell extract, etc.) is reacted with a certain amount of water-insolubilized carrier to which an antibody is bound and a buffer solution. The total reaction volume at this time is preferably 50 to 1000 μl. The buffer is preferably a buffer having a pH of 4 to pH 8.5 containing 0.01 to 1% of one or more proteins such as albumin and gelatin, and the reaction temperature is preferably 2 to 40 ° C.

The reaction time is an important factor affecting the measurement sensitivity, and considering the operability, it is preferably 10 minutes to 4 days. Then, human tenascin labeled with a detectable substance is added to this reaction solution, and the reaction is further carried out. The reaction temperature and time are preferably 2 ° C. to 40 ° C. and 10 minutes to 4 days as above.

After the reaction, the water-insoluble carrier is washed with water or an appropriate buffer solution, and the detectable substance bound to the water-insoluble carrier is measured. The above operation is also performed on a standard solution containing a known concentration of human tenascin, and the concentration of human tenascin in the biological fluid can be quantified by comparing the measured value of the standard solution with the measured value of the biological fluid.

Of course, it is also possible to quantify human tenascin using a known immunological measuring method other than the above.

The amount of tenascin in the biological fluid thus quantified can be used as an index to detect the presence of cancer in humans. To that end, a statistically significant number of biological fluid samples from clinically established cancer patients, normal patients and patients with benign tumors are quantified by the method of the present invention, and the human tenascin concentration obtained by these measurements is determined. Comparing the levels found in normal samples with those in cancer patients, it was found to be significantly higher in cancer patients. Therefore, it is possible to draw an arbitrary control level line between the normal state and the amount of tenascin in cancer patients. Human tenascin is then quantified according to one of the methods of the invention in an unknown sample, and the potential for cancer in the subject is assessed by determining whether the amount of tenascin is present in a concentration in excess of control levels. Can be defeated.

When cerebrospinal fluid is used as a biological fluid as a sample, tenascin is found in the cerebrospinal fluid of patients with malignant tumors such as glioma, and in diseases other than malignant tumors, tenascin is substantially contained in the cerebrospinal fluid. It turned out that it was not detected. That is, astrocytoma (As
trocytoma patient, Glioblastom
a) Tenascin is detected in the cerebrospinal fluid of patients, brain stem glioma patients, brain tumor patients, etc., and is found in the cerebrospinal fluid of benign diseases such as cerebral infarction patients, cerebral hemorrhage patients, meningitis patients, and cervical spondylosis spondylosis patients. Is virtually undetected. This allows the possibility of cancer to be assessed by measuring tenascin in the cerebrospinal fluid of the patient.

By carrying out such an assay, it is possible to use it as data for judging the effectiveness of treatment of cancer patients, and it can also be used as data for judging recurrence or metastasis.

The embodiments of the present invention will be described in more detail below. The present invention is not limited to these. The antigen and antibody used in this example were prepared as follows.

Antigen Human melanoma cells purified from a culture solution in a Dulbecco's modified Eagle medium / Ham's F12 medium containing 10% fetal bovine serum were used (Japanese Patent Laid-Open No. 2-2021).
56499).

Antibody As a polyclonal antibody, human melanoma cell-derived tenascin was subcutaneously injected into rabbits.
0.5 mg / nead was administered once a week for 3 weeks, and 3 weeks after the final administration, the antiserum was obtained weekly from the ear vein.

Next, the obtained antiserum was salted out with 50% saturated ammonium sulfate,
Ion exchange chromatography with DEAE-cellulose and CNBr-activated Sepharose 4B (Pharmacia)
What was purified by chromatography using an affinity column in which the antigen was bound to was used.

As the monoclonal antibody, a culture solution of hybridomas HTN2-8C9 and HTN2-9B2 obtained by fusing lymphocytes taken out from a rat immunized with human fibroblast cell-derived tenascin with X63Ag8,653 myeloma cells was prepared at 45%. The product was purified by saturated ammonium sulfate salting out and Protein G affinity chromatography (JP-A-2-219590).

[0035]

【Example】

Example 1 (1) Preparation of solid phase bound to antibody Polyclonal antibody or monoclonal antibody was added to pH 7.0.
Was dissolved in the phosphate buffer solution of No. 1 and immersed in polystyrene beads as a solid phase to bind to the surface thereof.

(2) Preparation of various labeled substance-bound antibodies and labeled substance-bound tenascin As a labeling enzyme for the antibody or tenascin, peroxidase or β-D, which is generally used in enzyme immunoassay, is used.
-Labeled with galactosidase or alkaline phosphatase.

The antibody is obtained by treating IgG, F (ab ') ₂ obtained by treating IgG with pepsin, and further treating Fab', IgG obtained by reducing F (ab ') ₂ with a reducing agent, with papain. The Fab obtained by was used. In the case of radiolabeling, ¹²⁵ I was labeled with an antibody or tenascin by a method using chloramine T.

(3) Labeling enzyme activity measuring method The enzyme activity was measured by a coloring method, a fluorescence method and a luminescence method. The labeling enzymes and substrates used are shown in Table 1.

[0039]

[Table 1]

In the table, AMPGD is 3- (4-Methoxyspiro [1,2
-dioxetane-3,2'-tricyclo [3.3.1. ^3,7 ] decan] -4-yl) phe
nyl-β-glactopyranoside, AMPPD is 3- (4-Methoxy
spiro [1,2-dioxetane-3,2'-tricyclo [3.3.1. ^3,7 ] decan]
-4-yl) phenyl-phosphate is shown. (4) Immunoassay The sandwich assay and the competitive assay were performed as the immunoassay.

Sandwich method 10 μl of the sample was bound to antibody-bonded polystyrene beads (diameter 6.5 m
m) 1 pc, 10 mM sodium phosphate buffer 0.5 ml (0.1% B
SA, 0.1 M NaCl) was added, and the mixture was reacted at 37 ° C. for 3 hours. After the reaction was completed, the beads were washed, 0.5 ml of each labeled antibody solution was added, and the mixture was reacted overnight at 4 ° C.

After the reaction was completed, the beads were washed and the labeled substance bound to the beads was measured. Enzyme activity is measured at 37 ℃,
The color development method was measured in 60 minutes, the fluorescence method in 30 minutes, and the light emission method in 30 seconds. Radioactivity was measured by using a scintillation counter.

Competitive method 10 μl of sample was bound to antibody-bonded polystyrene beads (diameter 6.5 m
m) 1 piece, dissolved tenascin labeled with various labeling substances 1
0.5 ml of 0 mM sodium phosphate buffer was reacted at 37 ° C. for 3 hours. After completion of the reaction, the beads were washed and the labeled substance bound to the beads was measured. Enzyme activity was measured at 37 ° C for 60 minutes for color development, 30 minutes for fluorescence, and 30 seconds for luminescence. Radioactivity was measured by using a scintillation counter.

(5) Measurement of standard tenascin One 10 μl solution of standard human tenascin with rabbit anti-human tenascin polyclonal antibody F (ab ′) ₂ bound to 6.5 mm diameter polystyrene beads, 10 mM sodium phosphate buffer (pH 7.0) , 0.1% BSA, 0.1M NaCl) 0.5 ml, and add 37 ℃
And reacted for 3 hours.

After the completion of the reaction, the beads were washed, 0.5 ml of a rat anti-human monoclonal antibody F (ab ') ₂ solution labeled with β-D-galactosidase was added, and the mixture was reacted overnight at 4 ° C. After the reaction, 0.5 ml of a chlorophenol red-β-D-galactopyranoside solution was added as a chromogenic substrate as a substrate,
For the fluorescent substrate, 0.5 ml of 4-methylumbelliferyl-β-D-galactopyranoside solution was added, and the temperature was 37 ° C and 1 respectively.
The enzyme reaction was carried out for a time and 30 minutes, and the enzyme reaction was stopped by adding 2 ml of a 1% galactose solution for the color development method and 2 ml of 0.1 M glycine-NaOH buffer (pH 10.3) for the fluorescence method as a solution for stopping the enzyme reaction. Absorbance at 575 nm for color development, excitation wavelength 3 for fluorescence
The fluorescence intensity was measured at 90 nm and a measurement wavelength of 460 nm. The measurement results of standard human tenascin are shown in FIG. The measurement sensitivity of the coloring method was 1 ng / Tube, and that of the fluorescence method was 0.1 ng / Tube.

(6) Quantification of the amount of tenascin in serum The amount of tenascin in normal and cancer patients was quantified. The amount of serum used in the assay system was 10 μl, which was measured by the same method as in (5), the enzyme activity was measured by the fluorescence method, and the fluorescence intensity of each sample was read from the fluorescence intensity of the standard line to obtain the amount of tenascin.

It was confirmed that the proportion of high tenascin levels in serum of cancer patients was significantly higher than that in normal subjects. The result is shown in FIG.

Example 2 The amount of tenascin in the ascitic fluid of various brain tumor patients was measured in the same manner as in Example 1. The results are shown in Table 2.

[0049]

[Table 2]

As a result, tenascin was detected in the ascites of glioma patients with malignant tumors. Also, cerebrospinal fluid of patients with cervical spondylotic spondylosis (11 samples), cerebrospinal fluid of meningitis patients (10 samples), cerebrospinal fluid of patients with cerebral infarction (9 samples), cerebrospinal fluid of patients with spinocerebellar deformity (6 samples). Specimens), cerebrospinal fluid of cerebral sclerosis patients (3 specimens), cerebrospinal fluid of neurotic patients (2 specimens), cerebrospinal fluid of cerebral hemorrhage patients (1 specimen), cerebrospinal fluid of polyneuropathy patients (1 specimen), Parkinson's Fluid (1 sample), dementia patient (1 sample), Huntington disease patient (1 sample), syringomyelia patient (1 sample), bulbar spinal muscular atrophy patient From the cerebrospinal fluid of patients with other benign diseases such as
Not detected.

Industrial Applicability According to the present invention, human tenascin can be accurately quantified, and the amount of tenascin in a biological fluid that has been quantified is compared with a control level to confirm the presence of high levels of tenascin, It has become possible to give a diagnostic indication as cancer.

[Brief description of drawings]

FIG. 1 shows a calibration curve of standard human tenascin, in which the black circles show the results of the fluorescence method and the white circles show the results of the color development method.

FIG. 2 shows the results of quantification of tenascin in the sera of normal and cancer patients. In the results of cancer patient sera in the figure, black circles indicate lung cancer, black squares indicate pancreatic cancer, white circles indicate liver cancer, white squares indicate colon cancer, and black triangles indicate measurement results of patient serum.

Claims (3)

[Claims] 1. A method for quantifying human tenascin in biological fluid as a cancer marker in vitro. 2. A method for quantifying human tenascin, which comprises immunologically quantifying the human tenascin in vitro by utilizing a cross-reaction of a human body fluid sample containing human tenascin with an antibody specific for human tenascin. . 3. The method according to claim 2, wherein the presence of human tenascin at a concentration higher than the control level is a diagnostic indication of cancer.