JP2001124777A - Method for evaluating usefulness of tumor marker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for evaluating usefulness of a tumor marker of a CA19-9. SOLUTION: A Lewis enzyme protein or Lewis enzyme activity in a saliva is detected to evaluate usefulness of a tumor marker of a CA19-9. The usefulness of the tumor marker of the CA19-9 is recognized in an individual in which the enzyme protein and/or enzyme activity is detected, but the usefulness of the tumor marker of the CA19-9 is not recognized in an individual in which the enzyme protein and/or the enzyme activity is not detected, and another tumor marker is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for evaluating the usefulness of a tumor marker.

[0002]

2. Description of the Related Art In the medical field, various tumor markers have been studied in order to examine their relevance to cancer. Among them, it has been found that some sugar chain antigens are strongly related to cancer. For example, it has been reported that sialyl Lewis a antigen can be a marker for pancreatic cancer, and that sialyl Lewis x antigen can be a marker for lung cancer. A measurement system using an anti-sialyl Lewis a antibody or an anti-sialyl Lewis x antibody is an indicator of pancreatic cancer or lung cancer. It is widely used in clinical settings. Furthermore, in recent years, research has been advanced to accurately detect cancer changes at an early stage, and glycosyltransferases, particularly fucosyltransferases (FUTs), which produce carbohydrate antigens themselves, which are tumor markers, are one of the attracting glycosyltransferases. One.

It has been known that fucosyltransferases (FUTs) can be classified into several groups depending on the position to which fucose is added, corresponding genes have been obtained, and some antibodies against FUT have been obtained (particularly). Kaihei 8-119999). Among them, Lewis enzyme (α- (1,3 / 1,4) fucosyltransferase: Fuc-TIII), which is one of glycosyltransferases, has the activity of adding fucose to the sugar chain terminal by α1-3 bond and α1 It has both activities of adding fucose to the sugar chain terminal by -4 bond, and is known to synthesize sialyl Lewis a antigen, an epitope of one of the tumor markers CA19-9. It is known that the expression level of this sialyl Lewis a antigen generally increases with the occurrence of cancer, and the prognosis is worse for patients who express the sialyl Lewis a antigen strongly. 9 is not synthesized or very low, and in such patients CA19
-9 cannot be used to assess cancer status. That is, the amount of CA19-9 varies greatly between individuals, and further research has been conducted on its application in detecting and evaluating cancer states.

On the other hand, a gene encoding a Lewis enzyme (Le)
Several mutant alleles are known in the
Therefore, in Japanese, at least 3 types of le1, le2, le3
Mutant alleles are known, and le1 and le2 are completely lost.
It may be a gene encoding a living Lewis enzyme
Are known. le1 protein has Leu in transmembrane domain ²⁰
From Arg²⁰Gly in the active domain¹⁷⁰From Ser¹⁷⁰Mutation to
And the le2 protein is Leu²⁰From Arg²⁰Active domain
To Ile³⁶⁷From Lys³⁶⁷Apparently has a mutation to
Has been. In Japanese, Le, le1, le2, le
The ratio of 3 is 68.9%, 24.8%, 5.8%, 0.5% respectively
(Narimatsu, et al., Cancer Re
s., 58, 512-518, 1998).

[0005]

The object of the present invention is to provide a CA19.
It is intended to provide a method for evaluating the usefulness of -9 as a tumor marker.

[0006]

Means for Solving the Problems As described above, the present inventors have reported that the Japanese Lewis enzyme gene can be classified into four alleles, a wild-type allele and mutant alleles of le1, le2, and le3. Was. We have already reported that the products of le1 and le2 are inactive, but as a result of further research, the present inventors have found that the le1 and le2 gene products are susceptible to degradation by proteolytic enzymes and are widely present in living organisms. Degraded by proteolytic enzymes, and found that CA19-9 could not be synthesized. Le3 gene mutation is Leu ²⁰
From is only one place of Arg ^{2 0,} was found to encode a Lewis enzyme incompletely inactivated. Furthermore, the present inventors have newly found that there is a strong correlation between the amount and activity of the Lewis enzyme regardless of the genotype and its final product, a sugar chain antigen.

The present inventors have invented a method for evaluating the usefulness of a tumor marker based on the above new findings. Therefore, the method of the present invention is a method for evaluating the usefulness of a tumor marker by detecting Lewis enzyme protein in saliva and / or Lewis enzyme activity in saliva. In particular, the present invention is a method for determining the usefulness of CA19-9 as a tumor marker by detecting Lewis enzyme protein and / or Lewis enzyme activity in saliva.

[0008]

DETAILED DESCRIPTION OF THE INVENTION In one embodiment of the present invention, Lewis enzyme protein and / or Lewis enzyme activity in saliva is measured and used for evaluating the usefulness of CA19-9 as a tumor marker. That is, for individuals in which Lewis enzyme protein and / or Lewis enzyme activity was detected in saliva, the usefulness of CA19-9 as a tumor marker was recognized.
CA19-9 is adopted as a tumor marker, while it is determined that CA19-9 is not adopted as a tumor marker for an individual in which Lewis enzyme protein and / or Lewis enzyme activity is not detected in saliva.

In the present specification, the Lewis enzyme is α (1,3
/ 1,4) -Fucosyltransferase refers to the enzyme encoded by the Fuc-TIII gene. The Fuc-TIII gene is also described as a Fuc-TIII gene or a Lewis gene, and these are used interchangeably. This Lewis enzyme
It is an enzyme that transfers fucose using GDP-fucose as a substrate. In the present invention, the sample for which the amount and activity of the Lewis enzyme are to be measured can be obtained from saliva of a healthy person or a patient. Lewis enzymes are normally secreted in saliva,
Inactive Lewis enzymes, ie, le1 and le2 proteins, are considered to be present in saliva in a state degraded by proteolytic enzymes, and are suitable for use as a simple sample by a non-invasive method. In addition, Lewis enzyme activity can be measured by a known substrate containing an appropriate label and a known measurement method. Substrates that can be used for such purposes for both α (1,3) fucosyltransferase activity and α (1,4) fucosyltransferase activity are known. Including GDP-fucose and 2'OMeGalβ
1,4GlcNacβBn, 2'OMeGalβ1,3GlcNacβBn, lacto-N-te
traose etc. can be used. After the reaction, the reaction product is separated using an appropriate column or the like, and the enzyme activity can be determined by measuring the amount of fucose incorporated.

On the other hand, when it is necessary to detect or quantify the Lewis enzyme protein, the protein can be detected and / or quantified using a monoclonal antibody specific to the Lewis enzyme, for example, an FTA1-16 antibody. FTA1
Naturally, an antibody having the same specificity as the -16 monoclonal antibody can be used, and other antibodies can also be used. However, the antibody used in the present invention is Lewis which has been degraded in saliva and lost its activity. The antibody must not recognize the enzyme-derived peptide fragment. As a detection method, a known method can be used. For example, the amount of protein can be determined by performing Western blotting. In this case, the full-length Lewis enzyme protein has a molecular weight of 43 kDa
Band. Blots can be quantified, if necessary, by analyzing the signal intensity of the band with a densitometer, which can be compared between samples. As a result, for individuals in which the presence and / or activity of the Lewis enzyme is not detected, sialyl Lewis a
Attempting to measure the antigen is not beneficial, and for such individuals CA19-9 with sialyl Lewis a antigen as epitope is not selected as a tumor marker, other tumor markers known in the art are selected. Will be.
For individuals with sufficient Lewis enzyme activity, CA
19-9 could still be used as one of the useful tumor markers.

[0011]

EXAMPLES Reference Example 1 Preparation of Anti -Fuc-TIII Antibody As the anti-Fuc-TIII antibody, monoclonal antibody FTA1-16 (obtained from Fujirebio) was used. Monoclonal antibody FTA1
-16 is a monoclonal antibody described in JP-A-8-119999, which specifically reacts with Fuc-TIII produced by a conventional method by immunizing a mouse using Le enzyme as an immunogen. . This antibody is a monoclonal antibody produced by a hybridoma having accession number FERM P-14588, which has been deposited with the Biotechnology Research Institute.

Reference Example 2 Expression of Le Enzyme, le1 Enzyme, and le2 Enzyme 1 μg of genomic DNA was extracted from leukocytes of volunteers having Le gene, le1 gene, and le2 gene, and 1 mM of sn1 primer described in SEQ ID NO: 2 and SEQ ID NO: 1 Using 1 mM of the sn2 primer described in No. 3 in a PCR buffer (200 μM
Of each dNTP, 10 mM Tris-hydrochloric acid (pH 8.8), 50 mM potassium chloride, 2.5 mM magnesium chloride, 0.1 mg / ml gelatin, and 10% DMSO for 30 cycles (94 ° C. for 1 minute, 60 ° C. 2 Min, 72 ° C. for 2 minutes) to amplify each gene, and subcloned into pBluescriptSK (−) vector (Stratagene). After confirming the sequence, the expression vector pCDM8 (Seed, B. (1987) Nature 329,
840-842, manufactured by Invitrogen)
Using oRI and HindIII sites for integration, vectors incorporating each gene were inserted into pCDM8-Le, pCDM8-le1, pCDM8-1
Named e2. Plasmid DNA, pCDM8-Le, pCDM8-
COS-1 cells (obtained from RIKEN cell bank) were co-transfected with 10 μg of le1, pCDM8-le2 and 1 μg of an actin promoter-inducible luciferase expression vector by a calcium phosphate coprecipitation method. After 55 hours, the cells were collected and sonicated in a phosphate buffer containing 2% Triton X-100 to obtain a cell extract. (Sequence listing free text) SEQ ID NO: 2: sn1 PCR primer for amplifying Lewis enzyme gene SEQ ID NO: 3: sn2 PCR primer for amplifying Lewis enzyme gene

Reference Example 3 Measurement of Le, le1, and le2 Enzyme Activities Fucosyltransferase activity and luciferase activity of the supernatant obtained in Reference Example 2 were measured. The protein concentration was determined using a DC protein measurement kit (manufactured by Bio-Rad). Fucosyltransferase activity was determined by comparing 50 μg of the protein equivalent extract with the enzyme reaction solution (25 μM GDP-
[ ³ H] fucose (85.1 GBq / mmol, manufactured by DuPont), 50
mM Tris-HCl (pH 7.2), 10 mM sodium azide, 1 mM
0mM manganese chloride, 5mM ATP, 0.5% Triton X-10
0) 100 μl was mixed and measured. For the substrate, 200 μM
2'OMeGalβ1,4 GlcNAcβBn (for α (1,3) fucosyltransferase activity) and 50μM 2'OMeGalβ1,3 GlcNAcβB
n (for α (1,4) fucosyltransferase activity) was used. After reacting at 37 ° C. for 3 to 5 hours, 100 μl of ethanol was added, and the mixture was centrifuged to collect the supernatant. 5 ml of water was added, the substrate was recovered using Sep-Pak plus C18 (manufactured by Waters), and the amount of [ ³ H] fucose incorporated was measured by a liquid scintillation counter. In addition, 5 μl of the cell extract was subjected to 20 mM tricine, 1.07 mM magnesium hydroxide hydroxide, 2.67 mM magnesium sulfate, 0.1 mM ED.
Mix with 200 μl of a reaction solution containing TA, 33.3 mM DTT, 270 μM coenzyme A, 470 μM luciferin, and 530 μM ATP, measure the amount of chemiluminescence for 30 seconds with a Lumat system (Bertoold), and use it for correction of transfection efficiency Was. The radioactivity incorporated into the substrate was corrected by the amount of chemiluminescence, and the respective enzyme activities of Le enzyme, le1 enzyme, and le2 enzyme were determined.
The Le enzyme had an activity 1000 times or more higher than the background level, but the le1 and le2 enzymes had no activity, and the amino acid substitution of both mutant enzymes inactivated the enzyme. It was confirmed.

Example 1 Measurement of Le3 Enzyme Expression and Enzyme Activity 500 patients were screened, and two colorectal cancer patients having the le3 allele were found. Both patients had le1 / le3 type genes. Genomic DNA was extracted from the leukocytes of these two patients in the same manner as described in Reference Example 2, the le3 gene was amplified by PCR, and p
It was subcloned into Bluescript SK (-) vector. After sequencing the entire sequence and confirming that only the 59th base has a mutation from T to G, the expression vector pCDM was used.
8. pCDM8-le3 was obtained. In a manner similar to the method described in Reference Example 3, the cells were introduced into COS-1 cells, and the fucosyltransferase activity was measured. GDP- [
^The reaction was carried out by changing the amount of [3H] fucose and the amount of 2'OMeGalβ1,3GlcNAcβBn, and the Km value and Vmax value were determined. Table 1 shows the results. When the Km / Vmax of the Le enzyme and the le3 enzyme were compared, the Km value for GDP-fucose, and the Km value for 2′OMeGalβ1,3 GlcNAcβBn and Vmax
There was no difference in the x value, and the le3 enzyme had the same activity as the Le enzyme.

Example 2 Preparation of Chimeric Gene A chimeric gene was prepared in order to obtain an enzyme having one amino acid substitution in each of the enzyme active sites. Le gene and le2
Using the EcoRV site of the base sequence at position 336 of the gene, the 3 'side and the 5' side were combined after enzyme cleavage, and SK-Le-le1 and 1067 position A gene whose base is mutated from T to A
K-Le-le2 was prepared. SK-Le-le1 encodes an enzyme in which the amino acid at position 170 changes from Gly to Ser, and SK-Le-le2 encodes an enzyme in which the amino acid at position 356 changes from Ile to Lys. These chimeric genes were inserted into the expression vector pCDM8 at the EcoRI site and Hi
Incorporation using ndIII site, pCDM8-Le-le1 and PCD
M8-Le-le2 was obtained. Similarly, the 59th base is changed from T to G
The le3 gene, which is a mutated gene, is also incorporated into pCDM8,
pCDM8-le3 was obtained. In le3, the 20th amino acid is from Leu to Ar
Encodes the le3 enzyme mutated to g. PCDM8-Le-le1, prepared in Example 2 in the same manner as described in Reference Example 3,
PCDM8-Le-le2 and pCDM8-le3 were introduced into COS-1 cells,
20 mM Hepes buffer containing 1% Triton X-100 (pH
The mixture was sonicated in 7.2) and solubilized. The protein concentration was determined using a DC protein assay kit (Bio-Rad).

Example 3 Sensitivity Test of Chimeric Enzyme with Protein Digestive Enzyme Sequence grade trypsin (promega) was used as a protein digestive enzyme in a cell extract containing 100 μg of protein.
At a concentration of 5, 50, 200, 400, 600 μg / ml on ice.
The mixture was treated for 10 minutes, and the reaction was stopped by adding 10 mM p-phenylmethanesulfonic acid chloride (PMSF). A Laemmli sample buffer was added to the reaction solution, treated at 100 ° C. for 5 minutes, and then subjected to 10% SDS polyacrylamide electrophoresis. Transferred to a PVDF nylon membrane (manufactured by Immobilon), FTA1-16 anti-Fuc-TIII antibody (deposited from the Institute of Biotechnology and Industrial Technology, accession number FERM P-14588, a monoclonal antibody produced by a hybridoma; obtained from Fujirebio) Was used to perform a Western blot. For Western blot, 10 μg / ml as primary antibody
Vector stain elite ABC using FTA1-16 antibody
The reaction was performed using a kit (manufactured by Vector Inc.), and the color was developed using an ECL kit (manufactured by Amersham). Figure 1 shows the results
Shown in As shown in Fig. 1, wild-type Lewis enzyme and le3
For the enzyme, a band corresponding to a molecular weight of 43 kDa corresponding to the molecular weight of the enzyme active region was detected even when the concentration of trypsin was changed, but for the chimeric enzymes Le-le1 and Le-le2, a band corresponding to a molecular weight of 43 kDa disappeared by trypsin digestion. did. From these results, it was determined that when the amino acids at positions 170 and 356 were mutated, the higher order structure could not be formed and the enzyme was digested, which was the cause of the inactivation of the le1 and le2 enzymes having mutations at the same site.

Example 4 Measurement of Sample of Healthy Person A saliva sample was collected from a volunteer of a healthy person. At the same time, blood is collected, leukocytes are separated, and genome D
NA was extracted, and Lewis genotype and Se genotype were also determined by the PCR-RFLP method. The fucosyltransferase reaction was carried out in the same manner as in Reference Example 3, and the α (1,4) fucosyltransferase activity in saliva was measured. Western blot analysis was performed in the same manner as in Example 3 using the FTA1-16 antibody (obtained from Fujirebio). The results are shown in FIG. As shown in FIG. 2, le1 / le
In the saliva from individuals 1 and le1 / le2, no Lewis enzyme reacting with the FTA1-16 antibody was found, and no α (1,4) fucosyltransferase activity could be detected. Le ／
In saliva derived from Le or Le / le1 individuals, a band corresponding to the enzyme active region having a molecular weight of 43 kDa detected in FIG. 3 of Example 3 was observed, and the signal intensity thereof was α (1,4) fucosyltransferase activity. Was correlated. From these results, as in the chimeric enzyme of Example 3, in vivo,
The le1 and le2 enzymes cannot form higher-order structures due to one missense mutation (Gly ¹⁷⁰ at Ser and Ile ³⁵⁶ at Lys) in the enzyme active region, and are sensitive to proteolytic enzymes, and are present in saliva. Not confirmed.

Example 5 Confirmation of Le enzyme and le enzyme in normal tissues The genotype and the type of expressed enzyme in normal tissues were examined. In the same manner as described in Example 3, Western blotting with the FTA1-16 antibody (obtained from Fujirebio) and α (1,4) fucosyltransferase activity were determined. For measurement of α (1,4) fucosyltransferase activity, lacto-N-tetraose (LNT) was used as a substrate. 2
20 mM Hepes buffer containing 7.2% Triton X-100, pH 7.2
Α (1,4) fucosyltransferase activity of tissues dissolved in 5 mM ATP, 10 mM L-fucose, 75 μM GDP
-0.1 M cacodylate buffer containing fucose, 25 mM manganese chloride and 25 mM pyridylaminated LNT as substrate (pH 6.
8) Measured in. After the reaction at 37 ° C. for 2 hours, the enzyme reaction by boiling for 3 minutes was stopped, and the mixture was diluted with distilled water. Reaction solution
After centrifugation at 15000 rpm for 5 minutes, 10 μl of the supernatant was
LC (TSK gel ODS-80Ts column (4.6 × 3
00 mm) (manufactured by Tosoh Corporation). HPCL is TS
Using a K-gel ODS-80Ts column, elution was carried out with a 20 mM ammonium acetate buffer at 35 ° C., and a fluorometer (JASCO
FP-920). The results are shown in FIG. The amount of enzyme in individuals with Le / le1 or Le / le2 type gene is about half of the amount of enzyme obtained in individuals with Le / Le type gene, and almost no enzyme is detected in individuals with le / le type gene. Was. In addition, expression of the enzyme was confirmed in individuals with the le3 / le1 type gene, albeit little. Comparison of the correlation between the amounts of these enzymes detected by Western blotting and α (1,4) fucosyltransferase activity showed a high correlation regardless of genotype.

Example 6 Immunohistochemical test of non-cancerous part of colon and cancerous part For colon cancer patients having genotypes of Le / le1 and le1 / le3, non-cancerous part of 10% formalin fixed (normal Tissue) and cancer specimens, FTA1-16
Immunostaining was performed using 7LE (anti-Lewis a antibody, manufactured by Seikagaku Corporation) and CA19-9 antibody (anti-s Lewis a antibody, manufactured by Centcore) (obtained from Fujirebio). Specimens were treated with methanol containing 0.3% (v / v) H 2 0 2, to block the POD endogenous. FTA1 for each primary antibody
-16, 7LE, using anti-CA19-9 antibody at 10 μg / ml for 12 hours,
The reaction was performed at room temperature. The following reaction was performed using Vectastain Elite A
Using a BC kit (manufactured by Vector), color development is 0.1M Tris
This was performed using 0.1 mg / ml diaminobenzidine tetrahydrochloride in hydrochloric acid buffer (pH 7.6). For nuclear staining, Mayer's hematoxylin was used. In Le / le1 genotype individuals having the Le gene, clear staining images were observed in the absorptive epithelium of the non-cancerous part and the Golgi region of goblet cells, but in the le1 / le3 genotype individuals, the corresponding staining images were very low. It was weak. le3
The enzyme substitutes one amino acid residue at the Golgi transmembrane site (Le
Since u ²⁰ has a mutation) to Arg, compared to the wild-type Le enzyme, the amount localized in the Golgi region is reduced, and judged that no longer observed stained image. In addition, anti-Lewis a
Comparing the staining images with the antibody (7LE), le1 / le3
The staining intensity was significantly reduced in individuals compared to Le / le1 individuals. The le3 enzyme was determined to be unable to efficiently synthesize a Lewis antigen on mucin in large intestine tissue due to ineffective localization to the Golgi region due to mutation of the transmembrane site.

Example 7 Relationship between CA19-9 and Le Genotype in Cancer Patient Tissue A Western blot analysis similar to the method described in Example 3 was performed using a cancer patient tissue. The primary antibody is anti-CA
The antibody 19-9 (Centcore) was used. FIG. 4 shows the results. CA19-9 was positive in patients with the Le / le1 genotype, but no CA19-9 was detected in patients with the le1 / le1 genotype. However, patients with le1 / le3 genotype 2
One of the names showed CA19-9 positive. As shown in Example 6, in the le1 / le3 genotype, the amount of the le3 enzyme localized in the Golgi region is reduced, but CA19-9 is positive due to the action of the le3 enzyme localized in a small amount. It was judged that it became.

[0021]

According to the present invention, the amount of the sialyl Lewis a antigen present in an individual can be estimated by measuring the Lewis enzyme activity or the amount of the Lewis enzyme obtained from the individual, and therefore, the amount of CA19-9 in a specific individual can be estimated. The usefulness as a tumor marker can be evaluated.

[Sequence List] <110> FUJIREBIO INC. <120> A method of evaluating the utility of tumor marker <130> Y1G0652 <140> <141> <160> 3 <210> 1 <211> 361 <212> PRT <213 > Homo sapience <400> 1 Met Asp Pro Leu Gly Ala Ala Lys Pro Gln Trp Pro Trp Arg Arg Cys 1 5 10 15 Leu Ala Ala Leu Leu Phe Gln Leu Leu Val Ala Val Cys Phe Phe Ser 20 25 30 Tyr Leu Arg Val Ser Arg Asp Asp Ala Thr Gly Ser Pro Arg Ala Pro 35 40 45 Ser Gly Ser Ser Arg Gln Asp Thr Thr Pro Thr Arg Pro Thr Leu Leu 50 55 60 Ile Leu Leu Trp Thr Trp Pro Phe His Ile Pro Val Ala Leu Ser Arg 65 70 75 80 Cys Ser Glu Met Val Pro Gly Thr Ala Asp Cys His Ile Thr Ala Asp 85 90 95 Arg Lys Val Tyr Pro Gln Ala Asp Thr Val Ile Val His His Trp Asp 100 105 110 Ile Met Ser Asn Pro Lys Ser Arg Leu Pro Pro Ser Pro Arg Pro Gln 115 120 125 Gly Gln Arg Trp Ile Trp Phe Asn Leu Glu Pro Pro Asn Cys Gln 130 135 140 His Leu Glu Ala Leu Asp Arg Tyr Phe Asn Leu Thr Met Ser Tyr Arg 145 150 155 160 Ser Asp Ser Asp Ile Phe Thr Pro Tyr Gly Trp Leu Glu Pro Trp Ser 16 5 170 175 Gly Gln Pro Ala His Pro Pro Leu Asn Leu Ser Ala Lys Thr Glu Leu 180 185 190 Val Ala Trp Ala Val Ser Asn Trp Lys Pro Asp Ser Ala Arg Val Arg 195 200 205 Tyr Tyr Gln Ser Leu Gln Ala His Leu Lys Val Asp Val Tyr Gly Arg 210 215 220 Ser His Lys Pro Leu Pro Lys Gly Thr Met Met Glu Thr Leu Ser Arg 225 230 235 240 Tyr Lys Phe Tyr Leu Ala Phe Glu Asn Ser Leu His Pro Asp Tyr Ile 245 250 255 Thr Glu Lys Leu Trp Arg Asn Ala Leu Glu Ala Trp Ala Val Pro Val 260 265 270 Val Leu Gly Pro Ser Arg Ser Asn Tyr Glu Arg Phe Leu Pro Pro Asp 275 280 285 Ala Phe Ile His Val Asp Asp Phe Gln Ser Pro Lys Asp Leu Ala Arg 290 295 300 Tyr Leu Gln Glu Leu Asp Lys Asp His Ala Arg Tyr Leu Ser Tyr Phe 305 310 315 320 Arg Trp Arg Glu Thr Leu Arg Pro Arg Ser Phe Ser Trp Ala Leu Asp 325 330 335 Phe Cys Lys Ala Cys Trp Lys Leu Gln Gln Glu Ser Arg Tyr Gln Thr 340 345 350 Val Arg Ser Ile Ala Ala Trp Phe Thr 355 360 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> sn1 PCR primer for amplifying lewis e nzyme gene <400> 2 ctcgaattct aagcaggaga ttgtcatcac tgacc <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> sn2 PCR primer for amplifying lewis enzyme gene <400> 3 ctcaagcttc gtgccgtgat gatctctctc cac

[Brief description of the drawings]

FIG. 1 shows the sensitivity of Lewis enzyme (Le enzyme) to proteolytic enzymes. (A): Le enzyme,
(B): le3 enzyme, (c): Le-le1 chimeric enzyme,
(D): Le-le2 chimeric enzyme.

FIG. 2 shows the results of detection of Lewis enzymes in saliva of Japanese having various Lewis genotypes.
(A): Western blot using FTA1-16. Positive bands corresponding to the solubilized form of the Lewis enzyme are indicated by arrows.
(B): Relative intensity of positive band in Western blot analysis. (C) α1,4 fucosyltransferase activity in saliva.

FIG. 3 shows the correlation between the relative amount of active Lewis enzyme and α1,4 fucosyltransferase activity in normal colon tissue. (A): The amount of Lewis enzyme determined by Western blot analysis with FTA1-16 in normal colon tissue and measuring the relative intensity of the band with a densitometer. (B): Correlation between the amount of Lewis enzyme in normal colon tissue and α1,4 fucosyltransferase activity. Black triangle is Le / Le individual, black triangle is Le / Le
le1 or Le / le2 individuals, white circles indicate le1 / le1 individuals.

[4] Detection of sLe ^a antigens in colorectal cancer tissues by Western blot analysis. Western blot analysis with anti -SLe ^a monoclonal antibody was performed in patients with colorectal cancer tissue with various Lewis genotypes. Lane 1-3: Le / le
1 patient; lanes 4 and 5, le1 / le3 patient; lane 6, le1 / le1 patient.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/10 C12P 21/08 15/02 C12N 5/00 B 15/09 ZNA 15/00 C C12P 21 / 08 ZNAA F term (reference) 4B024 AA12 BA10 BA42 CA04 DA02 EA04 GA11 HA11 HA15 4B050 CC03 DD11 LL03 4B064 AG27 CA10 CA20 CC24 DA14 4B065 AA90X AB01 AB04 BA02 CA25 CA29 CA46 4H045 AA11 CA40 DA76 EA51 FA72

Claims (4)

[Claims] 1. A method for evaluating the usefulness of CA19-9 as a tumor marker, comprising detecting a Lewis enzyme protein in saliva and detecting the presence of Lewis enzyme in saliva. Is considered useful as a tumor marker, and if the presence of Lewis enzyme in saliva is not detected, CA
The above-mentioned evaluation method, wherein 19-9 is judged to be not useful as a tumor marker. 2. The method according to claim 1, wherein the detection of the Lewis enzyme protein is performed using an antibody that does not recognize the degraded Lewis enzyme protein fragment. 3. The method according to claim 1, wherein the detection of the Lewis enzyme protein is performed using a monoclonal antibody FTA1-16 produced by a hybridoma having an accession number of FERM P-14588. 4. A method for evaluating the usefulness of CA19-9 as a tumor marker, comprising detecting Lewis enzyme activity in saliva and detecting the Lewis enzyme activity in saliva.
The above evaluation method, comprising determining that CA19-9 is useful as a tumor marker, and determining that CA19-9 is not useful as a tumor marker when Lewis enzyme activity in saliva is not detected.