JP3165828B2 - Malignant tumor detection method and kit - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and a kit for detecting a malignant tumor.

[0002]

2. Description of the Related Art Cadherin is a cell membrane surface molecule having a transmembrane domain having a molecular weight of about 100,000, and is a protein which exerts a function dependent on coexisting Ca ²⁺ ions with respect to cell-cell adhesion. Until now, many molecular species have been discovered in cadherin, and among them, the three oldest isolated and identified molecular species, E-cadherin, N-cadherin, and P-cadherin, are well known. It is known that each of these three molecular species exhibits the property of selectively binding only the same molecular species, and it is considered that the homogenous binding mechanism is one of the characteristics of the cadherin family. These cadherin molecular species are known to be important intercellular adhesion molecules that work on tissue construction, particularly in the process of development and differentiation of living organisms, and are used in specific organs in vivo such as nerve tissue, heart and placenta. In many cases, certain molecular species are mainly distributed, and it is considered that they exert a cell adhesion function in the formation of each tissue [M. Takeichi, Science, No. 251
Vol., Pp. 1451-1455 (1991)]. Of these three representative cadherins, E-cadherin is a molecule that is mainly expressed in epithelial cells and epithelial tissues in humans and mice, etc., and is localized in tissues of various diseases using immunohistochemical staining and the like. Are examined [Y. Shimoyama et al., Cancer Research (Cancer R)
esearch), Vol. 49, pp. 2128-2133 (198
9)]. As a result of these studies, many phenomena in which the expression level of E-cadherin is reduced particularly in malignant tumor tissues have been found. Also, in animal experiments, among the malignant tumor cell lines, strains with low E-cadherin expression level and weak cell-cell adhesion are higher in malignancy and higher in comparison with strains with high E-cadherin expression level and strong cell-cell adhesion. There is a tendency to form metastatic foci with probability [U. H. Frixsen (U.S.A.)
H. Frixen) et al., Journal of Cell Biology, 113, 173-
185 (1991)]. From these results, it is estimated that there is a close relationship between the amount of E-cadherin displayed on the cell surface and the metastatic performance and malignancy of malignant tumors.

On the other hand, cadherin having essentially a transmembrane region in a molecule has a property of being solubilized only in a solvent containing a surfactant or the like, and has a property of dissolving cells in a solvent containing no surfactant. Part of the extracellular region of the cadherin molecule is extremely easily cleaved by protease in extracts and the like, and the cut low-molecular-weight cadherin-degraded fragments having a molecular weight of about 80,000 are solubilized in an aqueous solvent containing no surfactant or the like. It is already known that it can exist [M. J. Feelock (MJ Wheelock) et al.
Journal of Cellular Biochemistry (Jo
urnal of Celluar Biochemistry), Vol. 34, 187
Pp. 202 (1987)]. However, it has not been known at all that cadherin hydrolyzate generated by protease is present in biological fluids such as humans and animals.
This is also due to the fact that there was no conventional technique for measuring the concentration of a soluble cadherin containing a cadherin hydrolyzate as a main constituent molecule in a solution and calculating the content.

[0004]

The relationship between the expression level of E-cadherin on cells, which has been analyzed for a long time, and diseases such as malignant tumors has also been examined by histochemical analysis using antibodies which have been conventionally used as an E-cadherin detection technique. Methods such as detection, flow cytometry, western blotting, and immunoprecipitation are all based on qualitative methods, which involve the danger of collecting tissue biopsy material from patients, preparation of reagents and equipment, etc. However, it is extremely difficult to actually apply clinical applications such as diagnosis of diseases in that it takes time. An object of the present invention is to establish a method for simply quantifying soluble E-cadherin in a body fluid, and to provide a method and a kit for detecting a malignant tumor using the method.

[0005]

SUMMARY OF THE INVENTION To summarize the present invention, a first aspect of the present invention relates to a method for detecting a malignant tumor, comprising measuring the amount of soluble E-cadherin in a body fluid and comparing the measured value with a healthy value. Features. Further, the second invention of the present invention relates to a kit for detecting a malignant tumor, which is a kit for detecting a malignant tumor by measuring the amount of soluble E-cadherin in a body fluid and comparing the value with a healthy value. And an antibody reactive with soluble cadherin in body fluid.

[0006] In consideration of the above-mentioned problems of the prior art, the present inventors have established a method for simply measuring the trace concentration of soluble E-cadherin in a body fluid in a solution. The present inventors have found that a certain amount of body fluid soluble E-cadherin is present, and found that the amount of body fluid soluble E-cadherin is significantly increased in host diseases, particularly malignant tumors, and completed the present invention. In addition, reagents for use in the method of the present invention are collectively made into a kit, thereby enabling more convenient measurement.

[0007] The body fluid soluble E-cadherin to be measured in the present invention includes all molecules derived from E-cadherin molecules, such as degraded or non-degraded products of E-cadherin which are soluble and present in body fluids, and are mainly It is composed of a decomposition product of E-cadherin having a molecular weight of about 80,000.

In the present invention, the method for measuring soluble E-cadherin in body fluid includes, for example, a method using an antibody reactive with soluble E-cadherin in body fluid. Examples of the method include any method capable of measuring body fluid soluble E-cadherin, such as enzyme immunoassay (EIA), radioimmunoassay, fluoroimmunoassay, immunoturbidimetry, latex agglutination, etc. EIA is preferred.

[0009] The antibody having reactivity to the body fluid soluble E-cadherin may be either a polyclonal antibody or a monoclonal antibody, and the antibody-derived animal may be any of animals generally used as experimental animals such as mice, rats, rabbits and the like. However, it is particularly desirable to use a mouse-derived monoclonal antibody because specific reactivity to the antigen is assured and mass production is easy. Examples of mouse-derived monoclonal antibodies include HEC
D-1 (Takara Shuzo), SHE78-7 (Takara Shuzo)
In addition to commercially available products such as
HE13-6 and SHE3-10. These four
Since certain monoclonal antibodies recognize different sites on the E-cadherin molecule, the EIA system can be constructed in any combination. For example, HECD-1 and SHE3
An EIA system can be constructed with a combination of -10 and a combination of SHE78-7 and SHE3-10. Also, SHE
13-6 shows strong reactivity to body fluid soluble E-cadherin and only weak reactivity to non-fragmented E-cadherin on cells. Therefore, by using SHE13-6, it is possible to more uniformly measure body fluid soluble E-cadherin in a body fluid sample. These antibodies may be appropriately fragmented, labeled,
Processing such as immobilization and modification may be performed.

The body fluid used in the present invention includes all samples that can be collected in ordinary extracorporeal diagnostic medicine, such as serum, plasma, urine, cerebrospinal fluid, amniotic fluid, ascites fluid, and lymph fluid. An extract is also included, but serum and urine are preferred because they are used frequently and collection is easy.

The present inventors have proposed sandwich type EIA as a method for measuring body fluid soluble E-cadherin,
One of two different monoclonal antibodies reactive to cadherin is adsorbed on a solid phase such as a microplate,
The other was successfully established by labeling the other with a peroxidase enzyme and combining the two. The body fluid soluble E-cadherin measurement EIA method enables the quantification of body fluid soluble E-cadherin in a solution having a concentration as low as several ng / ml, and no such body fluid soluble E-cadherin measurement method has been reported. In addition, the present inventors can use the body fluid soluble E-cadherin measurement EIA to detect body fluid soluble E-cadherin in serum, plasma, and urine of healthy human adults in a range of several ng / ml to several μg / ml. I found that. Heretofore, the fact that humoral soluble E-cadherin is present in this type of body fluid derived from humans or animals has not been found.

Further, the present inventors have developed these inventions, and have developed the distribution of the amount of soluble E-cadherin in the body fluid of several tens of disease patients and the amount of soluble E-cadherin in the body fluid of several tens of healthy persons. The distribution was compared and analyzed statistically, finding a phenomenon in which the value significantly increased in patients with malignant tumors, and was found to be extremely useful as a tumor marker for diagnosing diseases and grasping disease states. In addition to showing that serum can be used as a body fluid sample, it was also shown that urine, which can be more easily collected, can be used as a body fluid sample. As described above, there has been no report of using body fluid soluble E-cadherin as a simple tumor marker existing in body fluid.

[0013] In addition, the body fluid soluble E-cadherin measurement can be performed more easily by preparing reagents such as an immobilized antibody solution, a labeled antibody solution, a standard solution, and a substrate solution used in the method of the present invention. be able to. The reagent contained in the kit may be in the form of a solution or a lyophilized product.

As described above in detail, the present invention enables the measurement of trace amounts of soluble E-cadherin in body fluids, which was impossible in the prior art, by using body fluids used for general clinical tests such as serum and urine. The monitoring of disease with a novel tumor marker that does not exist has now become possible.

[0015]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of immunogen A placenta collected from a healthy pregnant woman at the time of delivery was placed in a 20 mM Tris physiological buffered saline (150 mM NaCl, 5 mM CaC).
l ₂ , 2 mM NEM, 0.3 mM PMSF, 0.2%
The plate was washed five times with NaN ₃ (pH 7.6) (hereinafter abbreviated as TBS) to remove adhering blood. 500 g of the washed placenta was shredded in a homogenizer together with 1 liter of TBS. After sufficient fragmentation, the extract supernatant was separated from insolubles by high-speed centrifugation (2000 g × 3).
0 minutes). Next, 10 mg of an anti-human E-cadherin mouse monoclonal antibody HECD-1 (manufactured by Takara Shuzo) was adsorbed on 2 g of bromocyan-activated sepharose (manufactured by Pharmacia) to prepare HECD-1 antibody-immobilized sepharose. The prepared HECD-1 antibody gel was filled in a glass column, and the column was washed well with TBS. Subsequently, the placenta extraction supernatant prepared above was passed through this column, and then the column was sufficiently washed with 1 liter of TBS. After washing the column, the antigen substance adsorbed on the antibody column was eluted with TBS containing 8 M urea. The eluted protein solution was placed in a dialysis tube and dialyzed overnight in 5 liters of TBS at 4 ° C. After complete removal of urea by dialysis, the eluted protein solution is again
The mixture was passed through an ECD-1 antibody-immobilized column, and the antigen was eluted from the antibody column by the same operation as described above. The eluate was concentrated to a protein concentration of about 1 mg / ml using a Collodion Pack (manufactured by Sartorius), and dialyzed against 5 liters of physiological saline containing ₂ mM CaCl ₂ at 4 ° C. overnight. The obtained protein solution mainly contains an antigenic substance that strongly reacts with HECD-1, which is an anti-human E-cadherin mouse monoclonal antibody. The protein antigen is referred to as standard E-cadherin). 500 μg of this standard E-cadherin was dispensed into tubes and stored frozen at −80 ° C. or lower until use as an immunogen or standard substance.

(2) Preparation of monoclonal antibody Standard E-cadherin 500 obtained in Example 1- (1)
μg was mixed with an equal amount of Freund's complete adjuvant (manufactured by Difco), and the mixture was equally administered intraperitoneally to five 6-week-old Balb / c mice (manufactured by CLEA Japan). Four weeks later, 500 μg of standard E-cadherin was mixed with an equal amount of Libya adjuvant TDM (manufactured by Livi), and the mixture was divided into five equal immunized mice and administered intraperitoneally. Four weeks later, the latter administration operation was performed again on the five mice. Three days later, spleens were excised from two of the mice, and cell fusion was performed according to the method of Keller and Milstein to produce hybridoma cells. Using standard E-cadherin as an antigen, hybridomas producing monoclonal antibodies reactive with standard E-cadherin were selected by ELISA. That is, a solution was prepared by diluting standard E-cadherin in TBS to a final concentration of 10 μg / ml.
An appropriate amount was dispensed into a 6-well microplate (Nunc), allowed to stand at 4 ° C. for 24 hours, and then all the solutions in the plate were discarded. A phosphate-buffered saline solution containing 1% bovine serum albumin (hereinafter referred to as phosphate buffered saline) (Referred to as BSA / PBS), and the plate was allowed to stand at 37 ° C. for 1 hour. Thereafter, all the solutions in the plate were discarded, and the plate was washed twice with a phosphate buffered saline (hereinafter, referred to as PBS), and the culture supernatants of all the hybridoma cells prepared as described above were respectively removed from each plate. Added to the wells. The plate was allowed to stand at room temperature for 1 hour and then washed three times with PBS. Next, an appropriate amount of a solution obtained by appropriately diluting an anti-mouse IgG peroxidase-labeled antibody solution (manufactured by Kappel) with BSA / PBS was added to each well of the plate. After the plate was again allowed to stand still at room temperature for 1 hour, the plate was washed three times with PBS, and the residual liquid was sufficiently removed. An ABTS substrate solution (manufactured by Amersham) was prepared according to the instructions for use, and an appropriate amount was added to each well of the plate after washing, and allowed to stand at room temperature for 15 minutes. Thereafter, the 96-well microplate was placed on a plate reader (manufactured by Flow) to calculate the color development of each well as the absorbance at a wavelength of 410 nm, and a hybridoma cell line that had stronger color development than the background was selected. In this way, 19 strains were selected as cell lines that produce antibodies that cause a strong antigen-antibody reaction using standard E-cadherin as an antigen.
These 19 hybridoma cells were twice subjected to limiting dilution cloning, screened again by the above-described ELISA method, and finally selected as the most appropriate two hybridoma cell clone strains, and monoclonal antibodies produced by these cells were obtained. To SHE13-6 and SH
It was named E3-10. SHE13-6, SHE3-1
No. 0 was confirmed by indirect enzyme antibody staining to strongly react with the surface of human skin cancer cell A431, which is already known to produce E-cadherin in the same manner as HECD-1 and SHE78-7. However, after the A431 cell surface was sufficiently washed with physiological saline or the like to remove the remaining soluble E-cadherin from the cell surface, the reaction of these antibodies to the cell surface E-cadherin was examined by indirect enzyme antibody staining. However, it was confirmed that SHE13-6 showed only a weak reaction on the A431 cell surface.

Then, SHE13-6, SHE3-10
Similarly to HECD-1 and SHE78-7, it was also confirmed by the same indirect enzyme antibody staining method that it did not produce E-cadherin and did not react at all with the surface of HeLa cells known to produce N-cadherin. Furthermore, SHE13
-6, SHE3-10 is standard E-cadherin with an appropriate amount SD
HECD-1, SHE78-7 were separated by S-polyacrylamide gel electrophoresis and subjected to Western blotting using a product electrically transferred to a nitrocellulose membrane.
The reaction was confirmed to react with a protein having a molecular weight of about 80,000 in the same manner as in the above.

Another property common to the cadherin family is that when cadherin-expressing cultured cells are dispersed with a protease such as trypsin,
In the presence of 2 to 10 mM calcium ions, cadherin is not decomposed and remains in its original molecular form,
Under the absence of calcium ions, such as in the presence of 10 mM EDTA, it has been confirmed that cadherin is degraded and molecules disappear [M. Takei (M. Take
ichi) et al., Developmental Biology (Develo
pmental Biology), Vol. 87, pp. 340-350 (1
981)]. SHE13-6 was treated with a 0.02% trypsin solution (manufactured by Flow Co.) containing 10 mM CaCl ₂ , A4.
31 shows a low reactivity to E-cadherin having a molecular weight of about 100,000 in cell extract, and does not react with any substance in A431 cell extract treated with 0.02% trypsin solution (manufactured by Flow) containing 10 mM EDTA. And SDS polyacrylamide gel electrophoresis and Western blotting.

SHE3-10 is HECD-1, SHE
0.02% containing 10 mM CaCl ₂ as in 78-7
Trypsin solution-treated A431 cell extract has a molecular weight of about 1
Shows strong reactivity to 100,000 E-cadherin and 10 mM ED
It was confirmed by SDS polyacrylamide gel electrophoresis and Western blotting that it did not react with anything in the A431 cell extract treated with a 0.02% trypsin solution containing TA. In addition, SHE3-10 is capable of producing SH against human placenta-derived soluble E-cadherin having a molecular weight of about 80,000.
It was confirmed by ELISA that it had the same reactivity as E13-6. Furthermore, SHE3-10 shows strong reactivity with A431 cell surface whose surface has been sufficiently washed,
HE13-6 was confirmed to exhibit weak reactivity by immunoflow cytometry.

From these results, SHE13-6, SH
E3-10 was confirmed to be a mouse monoclonal antibody having reactivity to standard E-cadherin, like HECD-1 and SHE78-7. The SHE13-6-producing hybridoma was designated and named Hybridoma SHE13-6, and was deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology (FERM P-1).
3389). The SHE3-10-producing hybridoma was designated and named Hybridoma SHE3-10, and was deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (FERM P-14144).

(3) Confirmation of Specificity of Monoclonal Antibody Hybridoma SHE13-6 (FERM P-13389) was administered to Balb / c mouse 10 previously administered with pristen (manufactured by Wako Pure Chemical Industries, Ltd.).
Weekly (manufactured by CLEA Japan, Inc.) Proliferate in large quantities in the abdominal cavity, collect ascites from mice about 10 days after administration, and use methods commonly used for antibody purification, such as ammonium sulfate precipitation or ion exchange chromatography. And purified SHE13-6 was obtained. SHE13-6 was labeled with peroxidase (Boehringer Mannheim) by the periodate method. Using a 96-well microplate adsorbing labeled E-cadherin as a solid phase, SHE3-10, SH
By reacting E78-7 or HECD-1 with the adsorbed antigen, the aforementioned enzyme-labeled SHE is dose-dependent.
It is determined whether binding of 13-6 to the adsorbed antigen is inhibited.
When examined by the LISA method, no such competitive inhibition was found between the two antibodies. That is, SHE13-6 is the other three antibodies, namely SH
Sites that do not compete with the binding of E3-10, SHE78-7, and HECD-1 to the standard E-cadherin antigen and are therefore distinct from the recognition sites of SHE3-10, SHE78-7, and HECD-1 on the E-cadherin molecule Confirm that you recognize. Purified SHE3-10 prepared from Hybridoma SHE3-10 (FERM P-14144), and SHE78-
7 were also examined in the same manner, and it was confirmed that none of them did not compete with the other three types of monoclonal antibodies except themselves for antigen binding. Therefore, SHE13-
6, SHE3-10, SHE78-7, and HECD-
1 confirmed that all recognized different sites on the E-cadherin molecule.

(4) Construction of a sandwich EIA system HECD-1 and SHE13-6 were respectively immobilized on an antibody,
A sandwich EIA was constructed as an enzyme-labeled antibody. First, a solution containing HECD-1 at a final concentration of 1 mg / ml is prepared in PBS, and sodium azide is added to a final concentration of 0.1% to prepare a solid-phase antibody solution. The solid phase antibody solution was diluted 100-fold with a coating buffer (PBS containing 0.1% sodium azide) to a final concentration of 10 μg / ml.
Is added to each well of a 96-well microplate (module plate F-16, Nunc), sealed, and allowed to stand at 4 ° C. for 24 hours. Next, the solution was discarded from the plate, and blocking solution (BSA / PBS containing 0.1% sodium azide) was added to each well of the plate.
Add 00 μl each, seal and leave at 37 ° C. for 1 hour. The plate was washed twice with PBS, and the sample to be measured or standard E-cadherin was 800, 400, 200, 100, 5
A standard solution containing 0.0,0 ng / ml was added to each well of the plate.
Add 00 μl each. After allowing to stand still at room temperature for 1 hour, each well of the plate was washed three times with PBS, and peroxidase-labeled SHE13-6 was appropriately diluted with BSA / P.
Dilute with BS and add 100 μl to each well of the plate. After standing still for 1 hour at room temperature, PBS
Wash each well of the plate three times with. The solution was thoroughly removed from each well of the plate, and 1 tablet of orthophenylenediamine dihydrochloride (Sigma) was added to 10 ml of a substrate solution (citrate buffer containing 0.01% hydrogen peroxide, pH 5.0). After dissolution (final concentration: 1 mg / ml), the substrate solution is added to each well of the plate in an amount of 100 μl, and the plate is allowed to stand for 15 minutes.
Next, 100 μl of 1N sulfuric acid solution was added to each well of the plate to stop the color reaction by the enzyme, and the color of each well of the 96-well plate was immediately quantified by absorbance measurement at a wavelength of 492 nm using a plate reader (Flow). I do. A calibration curve is prepared from the absorbance and the indicated concentration of a standard E cadherin solution of 0 to 800 ng / ml, and the concentration of body fluid soluble E-cadherin is calculated from the absorbance of each measurement sample solution using the calibration curve. A typical calibration curve is shown in FIG. That is, about 0
Measurement was possible in a low concentration range of 800 ng / ml. In addition, even when SHE78-7 and SHE3-10 were respectively used as an immobilized antibody and an enzyme-labeled antibody, it was possible to construct a sandwich EIA in exactly the same manner as described above.

(5) Measurement of Body Fluid Samples 51 samples of malignant patient serum, 18 samples of healthy adult serum, 21 samples of malignant tumor urine, 20 samples of healthy adult urine
Example 1 by Combination of D-1 and SHE13-6
The body fluid soluble E-cadherin concentration was measured by the method described in (4). The urine is urine at any time, urine creatinine is simultaneously measured with a creatinine test Wako (manufactured by Wako Pure Chemical Industries) kit, and the amount of body fluid soluble E-cadherin in the urine is measured as the level of body fluid soluble E-cadherin per g urine / creatinine. (Mg / g creatinine). Also,
The sample was diluted 100 times with BSA / PBS in both serum and urine for measurement. FIG. 2 shows the distribution of the body fluid soluble E-cadherin concentration in each serum, and FIG. 3 shows the distribution of the body fluid soluble E-cadherin level in each urine. As shown in FIG. 2, the malignant tumor positive rate was 51% when the upper limit of the normal value of the body fluid soluble E-cadherin concentration in the serum was set to 3 μg / ml (26 cases positive / 5 in all cases).
All of the healthy adults were negative (0 cases / 18 cases). In addition, as shown in FIG. 3, the positive rate of malignant tumor was 57% (12%) when the upper limit of the normal value of the soluble E-cadherin in body fluid in urine was determined to be 11.7 mg / g · creatinine.
Positive cases / out of all 21 cases), and healthy adults were all cases negative (0 cases positive / out of all 20 cases). Thus, the malignant tumor could be detected by comparing the amount of body fluid soluble E-cadherin in serum and urine with the normal value.

Example 2 Construction of Kit As shown in Table 1, EIA for measuring body fluid soluble E-cadherin
A kit was constructed. Among them, the enzyme-labeled antibody was prepared by enzymatically labeling purified SHE13-6 with peroxidase, and a solution containing the labeled antibody at a final concentration of 1 mg / ml IgG was added to BSA / PB.
The mixture was diluted 1000-fold with S, sealed in a glass vial bottle at 11 ml per bottle, and lyophilized as it was. The sample diluent is B containing 0.1% sodium azide.
SA / PBS was sterilized by filtration through a 0.22 μm-diameter membrane filter to prepare a filter. The standard product is Example 1
The standard E-cadherin prepared in (1) was diluted with BSA / PBS containing 0.1% sodium azide and 800 ng /
A solution having a final concentration of 1 ml was prepared, and 1 ml of this standard solution was sealed in one glass vial and lyophilized to prepare a solution. The others described in Example 1- (4) were used.

[0026]

[Table 1] Table 1 ─────────────────────────────────── <Contents> <Capacity> <Number> Solid phase antibody solution 220 μl × 1 vial Coating buffer 11 ml × 2 vial Blocking solution 11 ml × 2 vial Enzyme-labeled antibody (lyophilized product) for 11 ml × 1 vial standard product (lyophilized product) for 1 ml × 1 vial Sample diluent 11 ml × 2 vials Orthophenylenediamine tablets 2 tablets Substrate lysis solution 11 ml × 2 vials 96-well microplate 1 piece ──────────────────────── ───────────

[0027]

As described above in detail, according to the present invention, a rapid and simple method for measuring body fluid soluble E-cadherin in body fluid has been established, and a novel method and kit for detecting a malignant tumor has been provided.

[Brief description of the drawings]

FIG. 1 shows a sandwich EIA in Example 1- (4).
4 is a graph showing a calibration curve used in the system.

FIG. 2 shows the body fluid soluble E in serum measured by the method of the present invention.
It is a figure which shows the measurement result of a cadherin concentration.

FIG. 3 is a graph showing the results of measurement of a body fluid soluble E-cadherin level in urine measured by the method of the present invention.

Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) G01N 33/574 G01N 33/53 BIOSIS (DIALOG) JICST file (JOIS)

Claims (4)

(57) [Claims] 1. A method for detecting a malignant tumor, comprising measuring the amount of soluble E-cadherin in a body fluid and comparing the measured value with a healthy value. 2. The method according to claim 1, wherein the amount of body fluid soluble E-cadherin is measured using an antibody reactive with body fluid soluble E-cadherin. 3. The method according to claim 1, wherein serum, plasma, or urine is used as a body fluid. 4. A kit for detecting a malignant tumor by measuring the amount of soluble E-cadherin in a body fluid and comparing the measured value to a healthy value, wherein the antibody is reactive with the soluble E-cadherin in body fluid. A kit for detecting a malignant tumor, comprising: