JPH06324046A - Method and kit for detecting malignant tumor - Google Patents

Abstract

PURPOSE:To simply determine cadherin soluble in humor and to detect malignant tumor by using an antibody which is reactive to the cadherin soluble in humor. CONSTITUTION:E cadherin soluble in humor which is to be measured includes any molecules originating from E molecules such as soluble, decomposed or non decomposed substances of E cadherin present in humor. The antibody reactive to the cadherin soluble in humor is either polyclonal or monoclonal. Moreover, any kind of animals generally used in laboratory experiments, e.g. mice, rats, rabbits, etc., may be employed as an animal originating from the antibody. A monoclonal antibody originating from a mouse is especially desirable because of its sure specific reaction to an antigen and mass-production easiness. In addition, if reagent and the like such as a solidification antibody liquid, a marker antibody liquid, a calibrator solution, and a matrical solution are set in a kit, the measurement, becomes easier.

Description

Detailed Description of the Invention

[0001]

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

[0002]

Cadherin is a cell membrane surface molecule having a transmembrane domain having a molecular weight of about 100,000, and is a protein that exerts a function dependent on coexisting Ca ²⁺ ions in cell-cell adhesion. Up to now, many molecular species have been discovered in cadherin, and among them, three species of E-cadherin, N-cadherin, and P-cadherin are well known as the oldest isolated and identified molecular species. It is known that each of these three kinds of molecular species has a property that only the same molecular species are selectively bound to each other, and the homologous binding mechanism is considered to be one of the characteristics of the cadherin family. These cadherin molecular species are known to be important intercellular adhesion molecules that act in tissue construction particularly in the process of development and differentiation of living organisms, and they are found in specific organs in the living body such as nerve tissue and heart and placenta. In many cases, a certain molecular species is mainly distributed, and it is considered that the cell adhesion function is exerted in the formation of each tissue [M. M. Takeichi, Science, No. 251
Vol. 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, and is localized in tissues of various diseases using immunohistochemical staining. Have been investigated [Y. Y. Shimoyama et al., Cancer Research
esearch), 49, 2128-2133 (198).
9)]. From the results of these studies, many phenomena in which the expression level of E-cadherin is decreased particularly in malignant tumor tissues have been found. Also in animal experiments, compared with the malignant tumor cell lines with high E-cadherin expression and strong intercellular adhesion, the strain with low E-cadherin expression and weak intercellular adhesion have higher malignancy and higher. A tendency to form metastatic lesions has been found [U. H. Frixen (U.
H. Frixen) et al., Journal of Cell Biology, Vol. 113, 173-.
185 (1991)]. From these results, it is presumed that there is a close relationship between the amount of E-cadherin displayed on the cell surface and the metastatic ability and malignancy of malignant tumors.

On the other hand, cadherin, which essentially has a transmembrane region inside the molecule, has a property of being solubilized only in a solvent containing a surfactant and the like, and in a solvent containing no surfactant, cells are solubilized. A part of the extracellular region of the cadherin molecule in the extract is very easily cleaved by protease, and the cleaved low molecular cadherin fragment with a molecular weight of around 80,000 is solubilized in an aqueous solvent containing no surfactant. It is already known that it can exist [M. J. Feellock (MJ Wheelock) et al.
Journal of Cellular Biochemistry (Jo
urnal of Celluar Biochemistry), 34, 187.
~ 202 (1987)]. However, it has not been known at all that a cadherin degradation product produced by a protease exists in a biological fluid of humans or animals.
This is also due to the fact that there has been no conventional technique for measuring the concentration of soluble cadherin in solution, which has cadherin degradation products as its main constituent molecules, and calculating the content.

[0004]

The relationship between the expression level of E-cadherin on cells and the diseases such as malignant tumors, which has been analyzed for a long time, is also histochemically determined by the antibody which has been conventionally used as a technique for detecting E-cadherin. Detection methods, flow cytometry, Western blotting methods, immunoprecipitation methods, etc. are all qualitative methods, and there is a risk in collecting tissue biopsy materials from patients, preparation of reagents and equipment, etc. It is extremely difficult to apply clinically such as diagnosis of a disease because it takes time. An object of the present invention is to establish a method for easily quantifying body fluid soluble E-cadherin, and to provide a method and a kit for detecting a malignant tumor using the method.

[0005]

Means for Solving the Problems To summarize the present invention, the first invention of the present invention relates to a method for detecting a malignant tumor, which comprises measuring the amount of body fluid soluble E-cadherin and comparing the measured value with a healthy value. Characterize. The second invention of the present invention also relates to a kit for detecting a malignant tumor, which is a kit for detecting a malignant tumor by measuring the amount of body fluid soluble E-cadherin in body fluid and comparing the measured value with a healthy value. , An antibody having reactivity with body fluid soluble E-cadherin.

In consideration of the above-mentioned problems of the prior art, the present inventors established a method for simply measuring a trace amount of a body fluid-soluble E-cadherin in a solution, and using the measurement method, the body fluid of humans and animals. The present inventors have found that a certain amount of body fluid soluble E-cadherin is present in E. coli, and found that the amount of body fluid soluble E-cadherin significantly increases in host diseases, particularly malignant tumors, and completed the present invention. In addition, the reagents for use in the method of the present invention were put together into a kit to enable easier measurement.

The body fluid-soluble E-cadherin to be measured in the present invention includes all molecules derived from the E-cadherin molecule such as a decomposed product or a non-decomposed product of E-cadherin which is soluble and exists in the body fluid, and these are mainly It is composed of a decomposed product of E-cadherin with a molecular weight of around 80,000.

Examples of the method for measuring body fluid-soluble E-cadherin in the present invention include a method using an antibody reactive with body fluid-soluble E-cadherin. Examples of the method include enzyme immunoassay (EIA), radioimmunoassay, fluoroimmunoassay, immunoturbidimetric method, latex agglutination method, and any method that can measure body fluid-soluble E-cadherin. EIA is preferred.

The antibody reactive with body fluid-soluble E-cadherin may be either a polyclonal antibody or a monoclonal antibody, and the antibody-derived animal may be any animal commonly used as an experimental animal such as mouse, rat or rabbit. However, it is particularly preferable to use a mouse-derived monoclonal antibody because it has a specific reactivity with an antigen and is easily mass-produced. HEC is an example of a mouse-derived monoclonal antibody.
D-1 (Takara Shuzo), SHE78-7 (Takara Shuzo)
In addition to commercially available products such as
HE13-6 and SHE3-10 are mentioned. These 4
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 is strongly reactive with body fluid soluble E-cadherin and weakly reactive with unfragmented E-cadherin on cells. Therefore, by using SHE13-6, it is possible to measure the body fluid soluble E-cadherin in the body fluid sample more uniformly. These antibodies are appropriately fragmented, labeled, and
Treatments such as immobilization and modification may be performed.

The body fluid used in the present invention includes all samples that can be collected in the usual in vitro diagnostic medicine such as serum, plasma, urine, cerebrospinal fluid, amniotic fluid, ascites fluid, and lymph fluid, and a washing solution with a tissue or cell solvent or the like. An extract is also included, but serum and urine are preferable because they are particularly frequently used and can be collected easily.

The present inventors used sandwich type EIA as a method for measuring body fluid-soluble E-cadherin, and
One of two different monoclonal antibodies reactive with cadherin is adsorbed on a solid phase such as a microplate,
The other was labeled with a peroxidase enzyme, and the combination was successfully established for the first time. The body fluid-soluble E-cadherin measurement EIA method can quantify body fluid-soluble E-cadherin in a solution having a low concentration of several ng / ml, and no body-fluid-soluble E-cadherin measurement method of this type has been reported so far. Further, the present inventors can detect body fluid-soluble E-cadherin in serum, plasma, and urine of healthy human adults in the range of several ng / ml to several μg / ml using this EIA for measuring body fluid-soluble E-cadherin. I found that. Heretofore, the fact that body fluid soluble E-cadherin is present in this type of body fluid of human or animal origin has not been found.

Furthermore, the present inventors have developed these inventions and distributed the amount of body fluid soluble E-cadherin in body fluids of tens of patients with disease and the amount of body fluid soluble E-cadherin in body fluids of tens of healthy people. The distributions were compared and statistically analyzed, and a phenomenon in which the value was significantly increased in patients with malignant tumor was found, and it was found to be extremely useful as a tumor marker for diagnosing disease and grasping the medical condition. It was shown that serum can be used as a body fluid sample, and that urine, which can be collected more easily, can be used as a body fluid sample. Thus, there have been no reports of using body fluid-soluble E-cadherin as a simple tumor marker present in body fluid.

Further, by preparing reagents such as a solid phase antibody solution, a labeled antibody solution, a standard solution and a substrate solution which are used in the method of the present invention together as a kit, the body fluid soluble E-cadherin can be measured more easily. be able to. The reagents included in the kit may be in the form of solution or lyophilized product.

As described in detail above, according to the present invention, it becomes possible to measure a trace amount of body fluid soluble E-cadherin in body fluid, which has been impossible in the past, and it is possible to use body fluid used in general clinical tests such as serum and urine. It has become possible to monitor disease with a novel tumor marker that does not exist.

[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 Placenta collected from a healthy pregnant woman at the time of parturition was treated with 20 mM Tris physiological buffered saline (150 mM NaCl, 5 mM CaC).
l ₂ , 2 mM NEM, 0.3 mM PMSF, 0.2%
The adhered blood was removed by washing 5 times with pH 7.6 containing NaN ₃ (hereinafter abbreviated as TBS). 500 g of washed placenta was minced with 1 liter of TBS in a homogenizer. After sufficient fragmentation, the extract supernatant and insoluble matter were separated by high-speed centrifugation (2000 g x 3
0 minutes). Next, 10 mg of the anti-human E-cadherin mouse monoclonal antibody HECD-1 (Takara Shuzo) was adsorbed on 2 g of bromocyanine-activated Sepharose (Pharmacia) to prepare HECD-1 antibody-immobilized Sepharose. The prepared HECD-1 antibody gel was filled in a glass column, and the column was thoroughly washed with TBS. Subsequently, the placenta extract supernatant prepared above was flowed through this column, and then the column was thoroughly washed with 1 liter of TBS. After washing the column, the antigen substance adsorbed on the antibody column was eluted with TBS containing 8M urea. The eluted protein solution was placed in a dialysis tube and dialyzed overnight in 5 liters of TBS at 4 ° C. After the urea was completely removed by dialysis, the eluted protein solution was again added to the H
After flowing through the ECD-1 antibody-immobilized column, the antigen substance 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 Collodion Pack (manufactured by Sartorius), and dialyzed against 5 liters of physiological saline containing ₂ mM CaCl ₂ overnight at 4 ° C. 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, and the antigenic substance is mainly composed of a human E-cadherin degradation product (hereinafter, this The protein antigen is referred to as standard E-cadherin). This standard E-cadherin was dispensed into tubes in an amount of 500 μg each, and frozen and stored under conditions of −80 ° C. or lower until used as an immunogen or a standard substance.

(2) Preparation of Monoclonal Antibody Standard E-cadherin 500 obtained in Example 1- (1)
µg was mixed with Freund's complete adjuvant (manufactured by Difco) in an equal amount, and the mixture was divided into five aliquots of 6 6-week-old Balb / c mice (manufactured by CLEA Japan, Inc.) and administered in aliquots. After 4 weeks, 500 μg of standard E-cadherin was mixed with an equal amount of Libi adjuvant TDM (manufactured by Libi), and the mixture was again divided into 5 immunized mice and intraperitoneally administered. After a further 4 weeks, the latter administration procedure was repeated for 5 mice. Three days after that, spleens were extracted from two of the mice, and cell fusion was performed according to the method of Keller and Milstein to prepare hybridoma cells. Using the standard E-cadherin as an antigen, a hybridoma producing a monoclonal antibody reactive with the standard E-cadherin was selected by the ELISA method. That is, standard E-cadherin was diluted in TBS to prepare a solution having a final concentration of 10 μg / ml.
An appropriate amount was dispensed to a 6-well microplate (Nunc), and the plate was allowed to stand at 4 ° C for 24 hours. Then, all the solution in the plate was discarded, and phosphate buffered saline containing 1% bovine serum albumin (hereinafter The plate was filled with BSA / PBS) and allowed to stand at 37 ° C. for 1 hour. Then, all the solution in the plate was discarded, and the plate was washed twice with phosphate buffered saline (hereinafter referred to as PBS), and the culture supernatants of all the hybridoma cells prepared as described above were added to each plate. Added to the holes. The plate was allowed to stand at room temperature for 1 hour as it was, and then washed 3 times with PBS. Then, 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 allowing the plate to stand still at room temperature for 1 hour again, the plate was washed 3 times with PBS to sufficiently remove the residual liquid. An ABTS substrate solution (manufactured by Amersham) was prepared according to the instruction manual, an appropriate amount was added to each hole of the plate after the washing, and the plate was allowed to stand at room temperature for 15 minutes. Then, this 96-well microplate was applied to a plate reader (manufactured by Flow Co.), and the color development of each well was calculated as the absorbance at a wavelength of 410 nm, and a hybridoma cell line in which strong color development was obtained compared to the background was selected. In this way, 19 strains were selected as cell lines producing antibodies that cause a strong antigen-antibody reaction with standard E-cadherin as an antigen.
These 19 strains of hybridoma cells were subjected to limiting dilution cloning twice, and again screened by the above-mentioned ELISA method to finally select the 2 most suitable hybridoma cell clone strains, and the monoclonal antibody produced by these cells. To SHE13-6 and SH respectively
It was named E3-10. SHE13-6, SHE3-1
It was confirmed by an indirect enzyme antibody staining method that 0 strongly reacts with the surface of human skin cancer cell A431 which is already known to produce E-cadherin like HECD-1 and SHE78-7. However, after the A431 cell surface was thoroughly washed with physiological saline or the like to remove the remaining soluble E-cadherin from the cell surface layer, the reaction of these antibodies with the cell surface E-cadherin was examined by an indirect enzyme antibody staining method. However, it was confirmed that SHE13-6 shows only a weak reaction on the surface of A431 cells.

Then, SHE13-6, SHE3-10
It was confirmed by the same indirect enzyme antibody staining method that, like HECD-1 and SHE78-7, did not react with HeLa cell surface known to produce N-cadherin without producing E-cadherin. Furthermore, SHE13
-6, SHE3-10 is standard E cadherin SD in appropriate amount
HECD-1, SHE78-7 was separated by S-polyacrylamide gel electrophoresis, and Western blot analysis was carried out using the product electrically transferred to a nitrocellulose membrane.
It was confirmed to react with a protein having a molecular weight of about 80,000 in the same manner as in.

Further, as a property common to the cadherin family, when a cultured cell expressing cadherin is 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 shape.
It has been confirmed that cadherin is decomposed and the molecule disappears in the absence of calcium ions such as in the presence of 10 mM EDTA [M. Takei (M. Take
ichi) et al., Developmental Biology (Develo
pmental Biology), 87, 340-350 (1
981)]. SHE13-6 is a 0.02% trypsin solution (made by Flow Co.) containing 10 mM CaCl ₂ treated A4.
31 It shows a weak reactivity to E-cadherin having a molecular weight of about 100,000 in the cell extract, and it does not react with any substance in the 0.02% trypsin solution containing 10 mM EDTA (manufactured by Flow Co.) A431 cell extract. , SDS polyacrylamide gel electrophoresis and Western blotting.

SHE3-10 is HECD-1, SHE
0.02% containing 10 mM CaCl ₂ as in 78-7
Molecular weight of trypsin solution treated A431 cell extract is about 1
Reacts strongly with 0,000 E-cadherin, 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 0.02% trypsin solution containing TA. Moreover, SHE3-10 was compared to SH with respect to soluble E-cadherin derived from human placenta having a molecular weight of about 80,000.
It was confirmed by an ELISA method that it had a reactivity equivalent to that of E13-6. Furthermore, SHE3-10 shows a strong reactivity to the surface of A431 cells whose surface is sufficiently washed,
It was confirmed by the immunoflow cytometry method that HE13-6 exhibits weak reactivity.

From these results, SHE13-6, SH
It was confirmed that E3-10 is a mouse monoclonal antibody reactive with standard E-cadherin, similar to HECD-1 and SHE78-7. The SHE13-6 producing hybridoma was designated and named Hybridoma SHE13-6 and deposited at the Institute of Biotechnology, Institute of Industrial Science (FERM P-1).
3389). The SHE3-10 producing hybridoma was designated and named as Hybridoma SHE3-10 and deposited at the Institute of Biotechnology, Institute of Industrial Science (FERM P-14144).

(3) Confirmation of specificity of monoclonal antibody Balb / c mouse 10 in which Hybridoma SHE13-6 (FERM P-13389) was pre-administered with pristen (manufactured by Wako Pure Chemical Industries, Ltd.)
Ascites was collected from mice after a large amount of abdominal growth (CLEA Japan, Inc.) intraperitoneal growth, and about 10 days after administration, and a method commonly used for antibody purification such as ammonium sulfate salting out method or ion exchange chromatography. Purification was carried out to obtain purified SHE13-6. 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 an adsorbed antigen, the enzyme-labeled SHE described above is dose-dependently produced.
Whether or not the binding of 13-6 to the adsorbed antigen is inhibited is determined by E
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 for the binding of E3-10, SHE78-7, and HECD-1 to standard E-cadherin antigens, and thus are different from the recognition sites of SHE3-10, SHE78-7, and HECD-1 on the E-cadherin molecule. Confirmed to recognize. Purified SHE3-10 prepared from Hybridoma SHE3-10 (FERM P-14144), and SHE78- described above.
Similar investigations were carried out for 7 as well, and it was confirmed that none of them competed with the other 3 types of monoclonal antibodies except themselves for antigen binding. Therefore, SHE13-
6, SHE3-10, SHE78-7, and HECD-
It was confirmed that all of 1 recognized different sites on the E-cadherin molecule.

(4) Construction of sandwich EIA system HECD-1 and SHE13-6 are immobilized antibodies, respectively.
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 the final concentration of 0.1% to prepare a solid-phase antibody solution. The solid phase antibody solution was diluted 100-fold with coating buffer (PBS containing 0.1% sodium azide) to give a final concentration of 10 μg / ml.
200 μl each of the antibody solution of (1) 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. Then, discard the solution from the plate and add blocking solution (BSA / PBS containing 0.1% sodium azide) 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 measurement sample or standard E-cadherin was washed with 800, 400, 200, 100, 5
Add 1 standard solution containing 0.0 ng / ml to each well of the plate.
Add 00 μl each. After allowing to stand at room temperature for 1 hour, each well of the plate was washed 3 times with PBS, and peroxidase-labeled SHE13-6 was appropriately added to BSA / P.
Dilute with BS and add 100 μl to each well of the plate. After leaving it still at room temperature for 1 hour, PBS
Wash each hole of the plate 3 times. Carefully remove the solution from each hole in the plate and add 1 tablet of ortho-phenylenediamine dihydrochloride (Sigma) to 10 ml of substrate solution (citrate buffer containing 0.01% hydrogen peroxide, pH 5.0). After dissolution (final concentration 1 mg / ml), 100 μl of the substrate solution was added to each well of the plate, and the plate was 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 development reaction by the enzyme, and the color development of each well of the 96-well plate was immediately quantified by absorption measurement at a wavelength of 492 nm with a plate reader (FLOW). To do. Standard E-cadherin A calibration curve is prepared from the absorbance of a standard solution of 0 to 800 ng / ml and the indicated concentration, 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
It was possible to measure in the low concentration range of 800 ng / ml. Even when SHE78-7 and SHE3-10 were used as the immobilized antibody and the enzyme-labeled antibody, respectively, it was possible to construct the sandwich EIA in exactly the same manner as above.

(5) Measurement of body fluid samples HEC for 51 cases of malignant tumor serum, 18 cases of healthy adult serum, 21 cases of malignant tumor patient urine, and 20 cases of healthy adult urine
Example 1 by a combination of D-1 and SHE13-6
The body fluid soluble E-cadherin concentration was measured by the method described in (4). Of these, urine is urine at any time, and creatinine in the urine is measured at the same time using a creatinine test Wako (manufactured by Wako Pure Chemical Industries) kit, and the amount of body fluid soluble E-cadherin in urine is measured as the body fluid soluble E-cadherin value per urine g / creatinine. It was calculated as (mg / g · creatinine). Also,
Both serum and urine samples were diluted 100 times with BSA / PBS and measured. The distribution of the body fluid soluble E-cadherin concentration in each serum is shown in FIG. 2, and the distribution of the body fluid soluble E-cadherin value in each urine is shown in FIG. As shown in FIG. 2, when the upper limit of the normal value of the body fluid soluble E-cadherin concentration in serum was set to 3 μg / ml, the malignant tumor positive rate was 51% (26 cases positive / total 5).
In 1 case), all healthy adults were negative in all cases (0 case positive / in all 18 cases). In addition, as shown in FIG. 3, when the normal upper limit of the body fluid soluble E-cadherin level in urine was determined to be 11.7 mg / g.creatinine, the malignant tumor positive rate was 57% (12
Positive cases / out of 21 cases), and healthy adults were negative cases in all cases (0 cases positive / out of 20 cases). Thus, it was possible to detect malignant tumors by comparing the amounts of body fluid soluble E-cadherin in serum and urine with normal values.

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

[0026]

[Table 1] Table 1 ─────────────────────────────────── <Contents> <Contents> <Number> Solid phase antibody solution 220 μl x 1 vial Coating buffer 11 ml x 2 vial blocking solution 11 ml x 2 vial Enzyme-labeled antibody (lyophilized product) 11 ml x 1 vial Standard product (lyophilized product) 1 ml x 1 vial Specimen diluent 11 ml x 2 vials Orthophenylenediamine tablets 2 tablets Substrate dissolution solution 11 ml x 2 vials 96 well microplate 1 sheet ───────────────────────── ───────────

[0027]

INDUSTRIAL APPLICABILITY As described in detail above, 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 malignant tumor have been provided.

[Brief description of drawings]

FIG. 1 Sandwich EIA in Example 1- (4)
It is a graph which shows the calibration curve used by a system.

FIG. 2 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 density | concentration.

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

Claims (4)

[Claims] 1. A method for detecting a malignant tumor, which comprises measuring the amount of body fluid 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 the body fluid. 4. A kit for detecting a malignant tumor by measuring the amount of body fluid soluble E-cadherin in body fluid and comparing the measured value with a healthy value, which is an antibody reactive with body fluid soluble E-cadherin. A kit for detecting a malignant tumor, which comprises: