JP2003004748A - Reagent for diagnosing pancreas cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a high-specificity pancreas cancer marker which shows a high positive ratio even to pancreas cancers including Lewis a- and shows a low pseudopositive ratio to chronic pancreatitis and good hepatic diseases. SOLUTION: A monoclonal antibody for recognizing a Sialyl Lewis (a) radical and a Sialyl Lewis c radical, and a monoclonal antibody for recognizing an MUC-1 antigen are combined, thereby enabling diagnosing the pancreas cancer irrespective of whether or not a Lewis blood type antigen is positive in addition to distinguishing the chronic pancreatitis and the pancreas cancer from each other.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a soluble sugar present in serum by combining a monoclonal antibody that recognizes both Sialyl Lewis a group and Sialyl Lewis c group with a monoclonal antibody that recognizes MUC-1 antigen. Chain antigen (MU
C-1 type mucin antigen) to a diagnostic agent for pancreatic cancer capable of qualitative or quantitative determination.

[0001]

2. Description of the Related Art Koprowoski (Somatic Cell Genet.,; 5,
The development of CA19-9 by 957-972, 1979) and others contributed greatly to the diagnosis of pancreatic cancer, and opened a new path in the research and development of tumor markers, Dupan-2 (Metzgar et a
l; Cancer Res., 42, 601, 1982), CA-50 (Lindholm e
t al; Int Arch Allergy Appl Immunol. 71, 178-181,
1983), Span-1 (Chung et al .; Gaiji, 87, 236, 1986)
Pancreatic cancer markers, such as, have been put to practical use one after another. However, these tumor markers showed a high false positive rate not only in chronic pancreatitis but also in benign diseases such as chronic hepatitis and cirrhosis, and had a great problem in specificity. CA19-9 is a blood group sugar chain antigen
Lewis a- negative for pancreatic cancer (Kawa et al; Br.J. Cancer.,
64, 899-902, 1991), so simultaneous testing with other markers is necessary, and since it shows a high positive rate in gastric cancer, colorectal cancer, etc., it is by no means a pancreatic cancer-specific marker. From the above, it has been earnestly desired to develop a highly specific tumor marker which shows a high positive rate for pancreatic cancer including Lewis a-pancreatic cancer alone and a low false positive rate for chronic pancreatitis and benign disease. There is.

[0002]

[Problems to be Solved by the Invention]
It is to develop a highly specific pancreatic cancer marker showing a high positive rate for pancreatic cancer including s a -pancreatic cancer and a low false positive rate for chronic pancreatitis and hepatic benign disease.

[0003]

Means for Solving the Problems The trigger of the present invention begins with the analysis of the clinical results of two completely different tumor markers. That is, tumor marker ST-272 created and studied by Sekine et al. (Japanese clinical; 48, 1028-1030, 1990)
The positive rate for pancreatic cancer (86%) was that of CA19-9 (7%).
2%). On the other hand, the positive rate for the pancreatic cancer of the tumor marker LISA101 (12th Tumor Marker Study Group record; No.2, P150, Nagoya) created and studied by Kono et al. Was similar to that of CA19-9, The false positive rate for liver benign disease (chronic hepatitis / cirrhosis) was clearly low, and it was a highly specific marker. From these clinical results, 2
By combining the monoclonal antibodies used for various types of tumor markers, each had clinical characteristics, namely, the positive rate for pancreatic cancer was higher than that of CA19-9, and the false positive rate for chronic pancreatitis and hepatic benign disease was CA19. It was considered possible to find a pancreatic cancer marker with a specificity lower than -9. Furthermore, since both ST-272 and LISA101 are high molecular weight sugar chain antigens, it was considered possible to establish a measurement system by reacting with various monoclonal antibodies.

Therefore, KL-6, which similarly recognizes a sugar chain antigen,
(Hiroshima University Medical Journal; 33, 971-997, 1985), a combination of monoclonal antibodies, ST-272 (solid phase) -ST-272
(Label) -ST-272 (solid phase) -KL-6 (Label) -LISA 101 (solid phase) -K
Five types of L-6 (labeled), LISA101 (solid phase) -ST-272 (labeled), ST-272 (solid phase) -LISA101 (labeled) were examined. Characteristic clinical specimens (Stage I-II, Stage III-IV pancreatic cancer, Lewis
s a-Pancreatic cancer, chronic pancreatitis Sui stagnation positive, chronic pancreatitis Sui stagnation negative)
-272 (solid phase) -KL-6 (labeled) showed a result exceeding the sensitivity and specificity of CA19-9. As a result of examining the formulation of this measurement system and increasing the number of clinical specimens (pancreatic cancer, other gastrointestinal cancer, chronic pancreatitis, hepatic benign disease), the results showed that the sensitivity and specificity of CA19-9 were exceeded. Low positivity rate for CA19-9 Lewis
All cases of sa-pancreatic cancer were positive above the cutoff value, and all cases of chronic pancreatitis and hepatic benign disease were negative below the cutoff value.

The epitopes of the monoclonal antibody used in this assay system are represented by three synthetic sugar chain antigens (BioCarb Chem).
ST-272 antibody was Sialyl.
Sialyl Le reacts with both Lewis a and Sialyl Lewis c
It recognized different epitopes from CA19-9, which reacts only with wis a, and Dupan-2, which reacts only with Sialyl Lewis c. S
The reaction with ialyl Lewis c confirmed the positive results in all cases of Lewis a-pancreatic cancer. The KL-6 is
It did not react with any of Sialyl Lewis a, Sialyl Lewis c, and Lewis a. KL-6 is the MUC-1 antigen (Stehel et al. T
hird international workshop on lung tumor and diff
erentiation antigens. Int J Cancer (Suppl 8): 6-2
6, 1994.). This assay system is based on the MUC-1 type mucin antigen (Rajvir et al., Cancer R) produced by the human pancreatic cancer-derived cell line Capan-2.
es, 53: 1437-1443, 1993.) and has a molecular weight of 20 as well as the antigen present in the serum of patients with pancreatic cancer and chronic pancreatitis.
Reacted with two kinds of antigens of more than 0,000 and about 400,000 (gel filtration method). On the other hand, both solid-phase and labeled antibody Span-1 (Chung et al .: Nikkan, 88: 89-95, 1987, Sialyl Lewis a and Sialyl
The sandwich method using (recognizing Lewis c) is a MUC-2 type mucin antigen (Niv et al: Int. J. Cancer, 50: 147-152, 1992.) produced by human colon cancer-derived cell line LS-174T.
The reaction was completely different from that of this measurement system. From these results, the present inventors found that Sialyl Lew such as ST-272
Using a monoclonal antibody that recognizes both isa group and Sialyl Lewis c group, and a monoclonal antibody that recognizes MUC-1 antigen such as KL-6, can detect pancreatic cancer with higher sensitivity and specificity than CA19-9. I thought it could be done.

That is, the present invention is as follows: A reagent for diagnosing pancreatic cancer, which comprises a monoclonal antibody that recognizes both a Sialyl Lewis a group and a Sialyl Lewis c group, and a monoclonal antibody that recognizes an MUC-1 antigen.
Here, the Sialyl Lewis a group is a sugar chain represented by the formula I, S
The ialyl Lewis c group is a sugar chain represented by formula II. See also Sialy
l Lewis a group is a monoclonal antibody CA19-9 (Science; 2
12, 55-58, 1981), KMO1 (Japanese Journal: 82 1572-1579, 198)
5) As an antigen that reacts with Sialyl Lewis c group,
Monoclonal antibody Dupan-2 (FEBS Lett; 353, 48-52,
1994), CA-50 (Biochem Biophys Acta; 834, 110-117,
1985) was also identified as an antigen that reacts with.

[Chemical 1]

[Chemical 2] The MUC-I antigen is the monoclonal antibody Ma552, Ma69.
5, DF3, FH6 (Tumor Biol; 19 (suppl), 134-146, 199
8), KL-6 (Int. J. Cancer; Supplement 8, 81-83, 19
94) identified as the antigen that reacts with. 2. The pancreatic cancer diagnostic reagent according to 1, wherein the monoclonal antibody that recognizes both the Sialyl Lewis a group and the Sialyl Lewis c group is the ST-272 antibody. Here, the ST-272 antibody is patent 213286.
The antibody described in 3. In addition, the producer cells have been deposited with the FERM P-18375 number at the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. 3. A monoclonal antibody that recognizes the MUC-1 antigen is KL-6
The reagent for diagnosing pancreatic cancer according to 1, which is an antibody. Here, the KL-6 antibody is the antibody described in Patent 2011158. 4. A reagent for pancreatic cancer diagnosis, which comprises ST-272 antibody and KL-6 antibody as constituent components.

5. Any one of the following Step I or Step II, Step I: (1). Sialyl Lewis a group and Sialyl Lewi
solid phase immobilization of monoclonal antibody recognizing both sc groups, (2) reacting appropriately diluted test serum, (3) reacting labeled MUC-1 antigen recognizing monoclonal antibody Step, (4). Measuring the amount of label,
Step II: (1). A step of immobilizing a monoclonal antibody that recognizes MUC-1 antigen, (2). A step of reacting an appropriately diluted test serum, (3). A labeled Sialyl Lewis a group Sialyl L
A method for inspecting pancreatic cancer, which comprises the step of reacting a monoclonal antibody that recognizes both ewis c groups, and (4). measuring the amount of the label. 6. The method for detecting pancreatic cancer according to 5, wherein the monoclonal antibody that recognizes both the Sialyl Lewis a group and the Sialyl Lewis c group is the ST-272 antibody. 7. A monoclonal antibody that recognizes the MUC-1 antigen is KL-6
The method for examining pancreatic cancer according to 5, which is an antibody. 8. The monoclonal antibody that recognizes both Sialyl Lewis a group and Sialyl Lewis c group is ST-272 antibody, and MUC-1
The method for examining pancreatic cancer according to 5, wherein the monoclonal antibody that recognizes the antigen is a KL-6 antibody.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. 1. Acquisition of monoclonal antibody that recognizes both Sialyl Lewis a group and Sialyl Lewis c group. Acquisition of monoclonal antibody that recognizes MUC-1 antigen, 3. A method for diagnosing pancreatic cancer using the monoclonal antibody will be described in order.

1. Acquisition of monoclonal antibody that recognizes both Sialyl Lewis a group and Sialyl Lewis c group
-2 antibody (reference), Span-1 (reference), CA19-9 (reference) and the like are known, and monoclonal antibodies that recognize both the Sialyl Lewis a group and the Sialyl Lewis c group are prepared by these methods. From cloned monoclonal antibody producing clones
It can be obtained by using alyl Lewis a and Sialyl Lewis c biotinylated probes (Seikagaku Corporation) and selecting clones that react with both. Span-1
Antibodies are known to recognize both Sialyl Lewis a and Sialyl Lewis c groups (Pancreas; 9, 692-697, 1
994). Alternatively, it is also possible to purify a sugar chain antigen from pancreatic cancer cells or a culture supernatant thereof and immunize with the antigen to produce a monoclonal antibody. In particular,
Culture supernatant of pancreatic cancer cells, such as Capan-2 cells, was added to Sepharose C
Gel filtration with L-2B to obtain a v
SDS-P was dissolved in the fraction of oid volume and molecular weight of about 400,000.
Sialyl Lewisa and Sialyl Lea were cloned from monoclonal antibody-producing clones obtained by purifying glycoproteins that migrate to molecular weights of 1000KD and 500KD by AGE and immunizing mice with the antigens.
It can be obtained by selecting a clone that reacts with both using a wisc biotinylated probe (Seikagaku Corporation).

A monoclonal antibody that recognizes both the Sialyl Lewis a group and the Sialyl Lewis c group, preferably ST-2
72 antibody. Patent 213286 is a method for producing ST-272 antibody
ST-272 producing ST-272 antibody described in 3 above.
Shares in the National Institute of Advanced Industrial Science and Technology
Deposited under the number ERM P-18375. ST-272 antibody is ST-
From the culture supernatant of 272 strains or from the ascites of the mouse that has been grown in the abdominal cavity of the mouse, for example, Sepharose CL
It can be obtained by purification by gel filtration using -6B. Method for producing monoclonal antibody, ELIS for selecting desired clone from produced clones
The method of obtaining the antibody described in this patent, such as the method of A, is, for example, Ed Harlow, David Lane, Antibodies A Laboratory.
It can be performed according to the method described in Manual, Cold Spring Harbor Laboratory (1988).

2. Acquisition of monoclonal antibody that recognizes MUC-1 antigen As a monoclonal antibody that recognizes MUC-1 antigen, Ma5
52, DF3, FH6 (TumorBiol; 19 (suppl 1), 134-146, 199
8), KL-6 (Int. J. Cancer; Supplement 8,81-83, 199
4) is known and can be produced by the method described in the document. Ma552 and DF3 are commercially available from Cosmo Bio Inc. and DAKO Inc., respectively.

Among the monoclonal antibodies that recognize the MUC-1 antigen, the KL-6 antibody is preferable. A method for producing KL-6 antibody is described in patent 2011158. Cups or beads coated with the KL-6 antibody and KL-6 antibody labeled with an enzyme or ruthenium (Ru) are commercially available from Sanko Junyaku Co., Ltd. as Etest KL-6 or Picolumi KL-6.

3. Method for diagnosing pancreatic cancer using the monoclonal antibody The present invention quantifies an antigen that binds to both a monoclonal antibody that recognizes both the Sialyl Lewis a group and the Sialyl Lewis c group, and a monoclonal antibody that recognizes the MUC-1 antigen. Thus, although the invention relates to a method and a reagent for inspecting pancreatic cancer, it is preferable to inspect by a sandwich method. In the sandwich method, 1).
When the monoclonal antibody that recognizes both the Sialyl Lewis a group and the Sialyl Lewis c group is immobilized and the monoclonal antibody that recognizes the MUC-1 antigen is labeled, or 2). MUC-
1 Immobilize a monoclonal antibody that recognizes the antigen on Sialy
Two cases of labeling a monoclonal antibody that recognizes both the Lewis Lewis group and the Sialyl Lewis c group can be considered, but the present invention is not limited to either case. There are various methods for labeling, such as labeling with a radioisotope, labeling with a compound that emits electrochemiluminescence, fluorescent labeling, enzyme labeling, and biotin labeling, but the present invention is not limited to these examples. .

As the standard antigen, serum of pancreatic cancer patients, pleural effusion of patients with cancerous pleurisy, culture supernatant of pancreatic cancer cell lines and the like can be used, but pancreatic cancer cell lines are preferably used in consideration of reproducibility and the like. It is desirable to use the culture supernatant. For example, a pancreatic cancer cell line, for example, purified from the culture supernatant of Capan-2 cells, preferably subjected to gel filtration with Sepharose CL-2B to give a molecular weight of about 20,
Glycoproteins that are solute in a fraction with a void volume of 00,000 or more and a molecular weight of approximately 400,000, and that are electrophoresed on SDS-PAGE to a molecular weight of 1000 KD and 500 KD can be purified and used as a standard antigen. For the purified standard antigen, titer is determined based on the standard serum of the pancreatic cancer patient as a reference, and Unit is defined.

Below, Sialyl Lewis a group and Sialyl Lewis
Immobilize a monoclonal antibody that recognizes both c groups,
The sandwich method for labeling a monoclonal antibody that recognizes the MUC-1 antigen will be described step by step. 1). Immobilize a monoclonal antibody that recognizes both Sialyl Lewis a group and Sialyl Lewis c group on beads or cups. The beads may be microbeads, in which case magnetic microbeads are preferred. The solid phase may be bound by covalent bond or non-covalent bond. Normally, bovine serum albumin (BSA) is used to block nonspecific binding sites on beads or cups.
Blocking is performed with a protein such as casein and a surfactant such as Tween 20. 2). Samples, if necessary, bovine serum albumin (BSA)
Dilute with a buffer containing proteins such as casein and surfactants such as Tween 20, and add to beads or cups. Also, a known amount of pancreatic cancer-related antigen (standard antigen) is similarly diluted and added. 3) .Beads or cups are washed with a buffer containing a detergent such as Tween 20, if possible, and then diluted with a buffer containing a protein such as bovine serum albumin (BSA) or casein, or a detergent such as Tween 20, if possible. -1 Add a labeled monoclonal antibody that recognizes the antigen. 4). Wash the beads or cup with a buffer containing a detergent such as Tween 20, if possible, and then measure the radioactivity for a label, the radioactivity for a radiolabel, and the enzyme activity for an enzyme label. If it is a biotinylated label, labeled avidin is further added, and measurement is performed by a method according to the label. 5). Prepare a calibration curve from a known amount of standard antigen and calculate the amount of pancreatic cancer-related antigen contained in the sample. Through the above steps, the pancreatic cancer-related antigen in the sample is quantified.

The present invention further relates to the Sialyl Lewis a group and Sialyl
A monoclonal antibody that recognizes both Lewis c groups, and
It also relates to a pancreatic cancer test reagent containing a monoclonal antibody that recognizes the MUC-1 antigen as a constituent. As an example, a test reagent is, for example, 1) .A monoclonal antibody-coated cup that recognizes both a Sialyl Lewis a group and a Sialyl Lewis c group, or a monoclonal antibody-coated bead that recognizes both a Sialyl Lewis a group and a Sialyl Lewis c group, 2). Labeled monoclonal antibody that recognizes MUC-1 antigen, 3). Pancreatic cancer-related antigen standard solution with known concentration, 4). Diluent, 5). Further, in the case of an enzyme label, 6). Chromogenic substrate and 7). Reaction stop solution may be contained. In another configuration, the test reagent is, for example, 1) .MUC-1 antigen-recognizing monoclonal antibody-coated cup, or MUC-1 antigen-recognizing monoclonal antibody-coated beads, 2) .Sialyl Lewis
Labeled monoclonal antibody that recognizes both a group and Sialyl Lewis c group, 3). Pancreatic cancer-related antigen standard solution with known concentration,
It is a reagent containing 4). Diluent and 5). Further, in the case of an enzyme label, 6). Chromogenic substrate and 7). Reaction stop solution may be contained.

[0017]

According to the present invention, Sialyl Lewis a group and Sialyl
A monoclonal antibody that recognizes the yl Lewis c group, and MUC-
Monoclonal antibody that recognizes 1 antigen is combined,
In addition to distinguishing between chronic pancreatitis and pancreatic cancer, it became possible to diagnose pancreatic cancer regardless of whether Lewis blood group antigens were positive.

[0018]

EXAMPLES The present invention will be described below with reference to specific examples, but the present invention is not limited thereto. [Example 1] Establishment of pancreatic cancer-related antigen measurement system 1. Purification of ST-272 antibody and KL-6 antibody The ST-272 producing strain or the KL-6 producing strain is inoculated into the abdominal cavity of BALB / C mice administered with pristane. Ascites fluid was collected after 1-2 weeks and clarified, and ST-272 antibody was applied to Sepharose CL.
-6B gel filtration for IgM fractionation, KL-6 antibody for protein
The IgG fraction was purified by A Affinity column and used as ST-272 antibody and KL-6 antibody.

2. Preparation of standard antigen As the standard antigen, pleural effusion of a patient with cancerous pleurisy, which had been previously centrifuged at 10,000 rpm for 10 minutes, was used. The antigen concentration was determined by the LISA1-6 antigen (Cance
r Res. 49: 3412-3419, 1989).
Standard antigen diluent (7%) to give 0, 1000, 100, 10 U / ml
It was diluted with BSA, 0.15 M NaCl, 50 mM Tris-HCl pH 7.5) to obtain a standard antigen solution.

3. Preparation of antibody-bound beads The purified ST-272 antibody was added to P at a concentration of OD ₂₈₀ = 0.25.
Dilute with BS. 1 ml of this antibody solution was washed with PBS 5 times in advance, and 1 ml of Dynabeads M-450 Epoxy (DYNAL)
(30 mg / ml) and stir at room temperature for 20 hours to allow the antibody to bind to the beads. The supernatant is collected, 1 ml of PBS containing 1% bovine serum albumin is added, and the mixture is stirred and reacted at room temperature for 2 hours for blocking. Then add the antibody-conjugated beads to 0.
After washing 5 times with PBS containing 1% bovine serum albumin, 1 ml of PBS containing 0.1% bovine serum albumin is added and stored at 4 ° C.
The recovered antibody solution was measured for OD ₂₈₀ and Dynabeads M-450.
Determine the amount of antibody bound to Epoxy (μg / mg). Usually 2
4 μg / mg is bound.

4. Preparation of ruthenium (Ru) -labeled antibody The purified KL-6 antibody was dialyzed against PBS and OD ₂₈₀ = 2.8
To prepare. Ruthenium (II) tris (bipy
13.6 μl (1:20) of ridyl) -N-hydroxy succinimide ester (IGEN: hereinafter ruthenium or Ru) is mixed, and stirred and reacted at room temperature for 30 minutes. Next, 25 μl of 2M glycine is added, and the mixture is stirred and reacted at room temperature for 10 minutes. The mixture is charged on a Sephadex G-25 column (Pharmacia) and separated with running buffer PBS pH 6.0.
Fractions containing IgG are collected and used as a ruthenium-labeled antibody. The prepared ruthenium-labeled antibody is a protein quantification kit (PIERCE
Antibody). Also, the OD _{450 of the} ruthenium-labeled antibody is measured, and the number of ruthenium bound to IgG1 molecule is determined from the value and the antibody concentration. Typical ruthenium bond numbers are about 5-10. Binding of other antibodies to beads or labeling with ruthenium was performed in the same manner.

[Example 2] Measurement of pancreatic cancer-related antigens in clinical samples Measurement of pancreatic cancer-related antigen in serum The pancreatic cancer-related antigen in patient serum was measured by ECL. The following reagents were prepared prior to the measurement. (1). Standard antigen: Pleural fluid obtained from a lung cancer patient was serially diluted 10-fold with 7% bovine serum albumin (10 to 100).
00u / ml). (2) .ST-272 antibody-bound bead solution: 1% BSA, 0.1% normal mouse serum, 0.01% Tween20, 0.01 M EDTA-2Na, 0.15 M NaC
l, Tris buffer pH 7.5 (hereinafter referred to as reaction solution)
It was adjusted to 0.75 mg / ml. (3) .Ru-labeled KL-6 antibody solution: 10% normal rabbit serum, 0.1% normal mouse serum, 0.01% Tween20, 0.01 M EDTA-2Na, 0.15 M NaC
It was adjusted to 0.75 μg / ml with Tris buffer pH 7.5. 25 μl each of the serum sample and the standard antigen solution of each concentration were placed in the reaction tube of the automatic measuring device Picorumi 8220 (manufactured by Sanko Junyaku Co., Ltd.).
Then, add 175 μl of the reaction solution and set it in the reaction tube rack. Reaction tube rack, ST-272 antibody-bound bead solution,
The Ru-labeled KL-6 antibody solution was set in an automatic measuring device Picorumi 8220 to perform automatic measurement. The steps of automatic measurement are as follows. First, 25 μl of antibody-bound beads is added to the reaction tube, and the first reaction is carried out for about 9 minutes with intermittent stirring. After the liquid in the reaction tube is removed by suction, it is washed twice with the washing liquid. After washing, 200 μl of Ru-labeled antibody solution is added, and the second reaction is carried out while intermittently stirring for about 9 minutes. After the liquid in the reaction tube is removed by suction, it is washed twice with the washing liquid. After washing, add 300 μl of luminescent electrolyte and measure the amount of luminescence. A standard curve is prepared from the standard antigens measured simultaneously, and the antigen concentration in the sample is determined. For comparison, the amount of CA19-9 in the same sample was requested to an external clinical laboratory center and measured.

2. Setting of cut-off value by ROC analysis Compared to 77 cases of pancreatic cancer, 84 healthy subjects, 96 cases of chronic pancreatitis, 185 cases of benign liver disease including chronic pancreatitis, and 269 cases of healthy people and benign liver disease including chronic pancreatitis The cutoff value was examined. The relationship between the cutoff value and the diagnostic efficiency in each control disease is shown in Table 1.

[Table 1] From the above results, the cutoff value is
It was set to 400 U / ml. The cut-off value of CA19-9 measured for comparison was set to 37 U / ml.

3. Comparison of positive rates in healthy subjects and various diseases ST-272 was used as a solid phase antibody and KL-6 was used as a labeled antibody in the sandwich method (hereinafter referred to as S-STKL) with a cutoff value of 400 U /
Table 2 shows the positive rates in various diseases when set to ml. For comparison, the positive rate of CA19-9 is also shown.
As shown in Table 2, the positive rate of S-STKL was low in healthy subjects and benign diseases, and was about 20% in cancer diseases other than pancreatic cancer, but was high in pancreatic cancer at 80.5%. It was Also S-STKL
Also showed 100% positive rate for Lewis a-pancreatic cancer. On the other hand CA19
-9 was similarly low in healthy subjects and benign diseases, and high in 77.9% in pancreatic cancer, but 31.8% in bile duct / bile duct cancer and 2 in gastric cancer.
7.7%, colon cancer 47.1%, lung cancer 30.0%, which were higher than those of S-STKL. In Lewis a-pancreatic cancer, the positive rate was as low as 40%. From the above results, S-STKL was useful for distinguishing between pancreatic cancer and chronic pancreatitis, and was particularly effective for detecting Lewis a-pancreatic cancer.

[Table 2]

4. Distribution of S-STKL value in healthy subjects and various diseases The distribution of S-STKL values in healthy subjects and various diseases is shown in FIG. Serum S-STKL levels were high in pancreatic cancer, biliary tract / bile duct cancer, gastric cancer, colorectal cancer, and lung cancer, and there was a clear discrepancy in the distribution of concentrations between healthy subjects and benign diseases. 5. Relationship between S-STKL Value and Positive Rate of Pancreatic Cancer by Progression Level As shown in FIG. 2, the S-STKL value was positively increased along with the positive rate along with the progression level of pancreatic cancer. From these results, S-STKL has characteristics as a tumor marker, and is expected to be useful for follow-up observation such as determination of therapeutic effect and prediction of recurrence.

Example 3 Epitope Analysis of ST-272 Antibody Epitope analysis of ST-272 antibody used in this measurement system was performed. For analysis, three synthetic sugar chain antigens Sialyl Lewis a (sialyllacto-N-fu manufactured by BioCarb Chemicals (Sweden) were used.
copentaose-human serum albumin Conjugate), Sialy
l Lewis c (sialyllact-N-tetraose-human serum album
in Conjugate), Lewis a (lacto-N-fucopentaose-huma
ST-272 (solid phase) -ST-272 (labeled) -KL-6 (solid phase) -KL as an antigen sample using a 2-fold serial dilution of three synthetic sugar chain antigens -6 (sign)
ST-272 (solid phase) -KL-6 (labeled) was measured by an ECL measurement system. CA19-9 and Dupan-2 were measured by outsourcing.

As shown in FIG. 3, the ST-272 antibody (a) was labeled with Sialy.
I recognized both Lewis a and Sialyl Lewis c, and did not react with Lewis a at all. The KL-6 antibody (b) did not react with any of the three types of synthetic sugar chain antigens, and the ST-272 of this measurement system was used.
Similarly, (solid phase) -KL-6 (label) (c) did not react with any of the three synthetic sugar chain antigens. On the other hand CA19-9
(D) strongly reacted with Sialyl Lewis a as in previous reports and did not react with Sialyl Lewis c at all.
Dupan-2, like previous reports, has Sialyl Lewis c
Only reacted with. This reactivity is the result of Kawa et al. (Pancres
9, 692-697, 1994). CA19-9 (d)
However, while Sialyl Lewis a and Dupan-2 (e) react only with Sialyl Lewis c, the ST-272 antibody shows Sialyl Lewis a
Was found to recognize both Sialyl Lewis c and Sialyl Lewis c.

[Example 4] Study on antigen reacting with S-STKL 1. Reaction with human pancreatic cancer-derived cell line antigens In order to find an antigen that reacts with S-STKL, 13 types of human pancreatic cancer-derived cell lines (KP-1, KP-2, KP-3, KP-4, PANC-1, BxPC-
3, AsPC-1, PSN-1, HPAC, MIA pacaII, Capan-1, Capan
-2, SUIT-2) culture supernatant was measured by the ECL method. As a result, Capan-2 produced a high concentration of an antigen that reacts with S-STKL. Rajvir et al. Reported that Capan-2 highly produces the MUC-1 type mucin antigen (C
ancer Res, 53: 1437-1443, 1993), the antigen recognized by this assay system was considered to be the MUC-1 type mucin antigen. 2. Antigen that reacts with S-STKL in Capan-2 culture supernatant, pancreatic cancer patient serum, chronic pancreatitis patient serum Capan-2 culture supernatant, pancreatic cancer patient serum, chronic pancreatitis patient serum are separated by Sepharose CL-2B gel 740 mm) filtered (Buff
er: 0.01 M PBS pH 7.2, flow rate: 10 ml / hr, fraction: 2 ml /
tube). As a result, Capan-2 culture supernatant, pancreatic cancer patient serum, and chronic pancreatitis patient serum had two kinds of antigens having a molecular weight of 2 million or more and about 400,000, which were detected by S-STKL.

[0029]

[Brief description of drawings]

[Figure 1] Distribution of S-STKL levels in healthy subjects and various diseases

[Fig. 2] Relationship between the positive rate of pancreatic cancer and the S-STKL value

FIG. 3 Epitope analysis of ST-272 antibody

Claims (8)

[Claims] 1. A pancreatic cancer diagnostic reagent comprising a monoclonal antibody that recognizes both a Sialyl Lewis a group and a Sialyl Lewis c group, and a monoclonal antibody that recognizes the MUC-1 antigen. 2. The reagent for diagnosing pancreatic cancer according to claim 1, wherein the monoclonal antibody that recognizes both the Sialyl Lewis a group and the Sialyl Lewis c group is the ST-272 antibody. 3. The reagent for diagnosing pancreatic cancer according to claim 1, wherein the monoclonal antibody that recognizes the MUC-1 antigen is KL-6 antibody. 4. A reagent for diagnosing pancreatic cancer, which comprises ST-272 antibody and KL-6 antibody as constituent components. 5. Any one of the following step I or step II, step I: (1). Sialyl Lewis a group and Sialyl Lewis c
Immobilizing a monoclonal antibody that recognizes both groups, (2); reacting an appropriately diluted test serum,
(3). Reacting a monoclonal antibody that recognizes the labeled MUC-1 antigen, (4). Measuring the amount of the label, step
II: (1). Immobilizing a monoclonal antibody that recognizes MUC-1 antigen, (2). Reacting an appropriately diluted test serum, (3). Labeled Sialyl Lewis a group and Sialyl Lewis
A method for inspecting pancreatic cancer, comprising the step of reacting a monoclonal antibody that recognizes both c groups, (4). measuring the amount of label. 6. The method for detecting pancreatic cancer according to claim 5, wherein the monoclonal antibody that recognizes both the Sialyl Lewis a group and the Sialyl Lewis c group is the ST-272 antibody. 7. The method for examining pancreatic cancer according to claim 5, wherein the monoclonal antibody that recognizes the MUC-1 antigen is KL-6 antibody. 8. A monoclonal antibody that recognizes both Sialyl Lewis a group and Sialyl Lewis c group is ST-272 antibody, and a monoclonal antibody that recognizes MUC-1 antigen is KL-6.
The method for testing pancreatic cancer according to claim 5, which is an antibody.