JPH07309895A - New protein, method for detecting the same and diagnostic - Google Patents

Abstract

PURPOSE:To obtain a new protein usable as a marker for urinary cancers including renal cancer and bladder cancer for which no cancer marker has been known, to provide a method for detecting this protein, and to obtain an urinary cancer diagnostic capable of detecting this protein. CONSTITUTION:This protein is found in the urine of urinary cancer patients, cannot be detected in the urine of healthy man, being about 80kDa in molecular weight and 5.20-6.50 in isoelectric point, and making an antigen-antibody reaction with anti-cathepsin B monoclonal antibody SBMI-1 (FERM P-13683), SBM2-2 (FERM P-13682), or RB-1 (FERM P-13685). The second objective method is such that this protein in a specimen is immunologically detected or quantified using an antigen (or an antigen-binding fragment thereof) making an antigen- antibody reaction with this protein. And, the third objective urinary cancer diagnostic contains an antibody (or an antigen-bonding fragment thereof) making an antigen-antibody reaction with this protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein that can be used as a cancer marker for urinary system cancer, a method for detecting the protein, and a urinary system cancer diagnostic agent that can detect the protein.

[0002]

2. Description of the Related Art Among urinary system cancers, specific cancer markers for kidney cancer and bladder cancer are not known. In the case of bladder cancer, urine cytology has been used for screening. Measurement of serum prostate-derived acid phosphatase (PAP) is known as a marker for prostate cancer (activity measurement, EI
A, RIA).

[0003]

An object of the present invention is to provide a novel protein which can be used as a marker for urinary system cancer including kidney cancer and bladder cancer for which a cancer marker has not been known. It is also an object of the present invention to provide a method of detecting the protein. Further, an object of the present invention is to provide a urinary system cancer diagnostic agent capable of detecting the protein.

[0004]

[Means for Solving the Problems] The inventors of the present invention prepared a monoclonal antibody against human cathepsin B, and used it to immunoassay components in various biological specimens. It was found that an unknown protein that reacts with the B antibody in an antigen-antibody manner but is not human cathepsin B is specifically present in the urine of patients with urinary system cancer, and further characterized the protein to complete the present invention.

That is, the present invention exists in the urine of a patient with urinary system cancer, is not detected in the urine of a healthy person and has a molecular weight of about 8
The anti-cathepsin B monoclonal antibody SBM1-1 has a 0 kDa and an isoelectric point between 5.20 and 6.50.
(FERM P-13683), SBM2-2 (FER
MP-13682) and RB-1 (FERM P-1)
3685) and a protein that reacts with an antigen-antibody.
The present invention also provides a method for immunologically detecting or quantifying the above-mentioned protein of the present invention in a sample using an antibody or an antigen-binding fragment thereof that reacts with the above-mentioned protein of the present invention by an antigen-antibody reaction. Further, the present invention provides a urinary system cancer diagnostic agent comprising an antibody or an antigen-binding fragment thereof that reacts with the protein of the present invention as an antigen-antibody.

The present invention will be described in detail below.

As described above, the protein of the present invention is present in the urine of patients with urinary system cancer, but it is not detected in the urine of healthy individuals. Therefore, the protein of the present invention can be used as a marker for urinary system cancer. Here, the urinary system cancer includes renal cancer, bladder cancer and prostate cancer.

The molecular weight of the protein of the present invention is about 80 kDa as measured by the Western blotting method described in detail in Examples below. Therefore, this protein is a substance different from cathepsin B, though it reacts with the anti-cathepsin B antibody in an antigen-antibody manner. Further, the protein of the present invention has an isoelectric point between 5.20 and 6.50.

The protein of the present invention is an anti-cathepsin B monoclonal antibody SBM1-1 (FERM P-136).
83), SBM2-2 (FERM P-13682) and RB-1 (FERM P-13685). Anti-cathepsin B monoclonal antibody SBM1-
1 is 8 out of the amino acid sequence of human procathepsin B
Leu-Pro-, which is the amino acid sequence from 0 to 92
Ala-Ser-Phe-Asp-Ala-Arg-G
A synthetic peptide of lu-Gln-Trp-Pro-Gln was used as a carrier protein for bovine serum albumin (B
It is a monoclonal antibody obtained by using the one bound to SA) as an immunogen and reacts with human cathepsin B in an antigen-antibody reaction. Anti-cathepsin B monoclonal antibody SBM2
-2 is an amino acid sequence of human procathepsin B,
Leu- which is the 309th to 309th amino acid sequence
Val-Ala-Asn-Ser-Trp-Asn-T
hr-Asp-Trp-Gly-Asp-Asn-Gl
It is a monoclonal antibody obtained by using a synthetic peptide of y-Phe bound to BSA as a carrier protein as an immunogen and reacts with human cathepsin B in an antigen-antibody reaction. The anti-cathepsin B monoclonal antibody RB1 is a monoclonal antibody obtained by using genetically engineered human procathepsin B as an immunogen, and since it reacts with human cathepsin B in an antigen-antibody reaction, its corresponding epitope is It exists in the mature part (mature part) of procathepsin B.

Since the protein of the present invention reacts with the anti-human cathepsin B monoclonal antibody as described above, it is clear that it has an amino acid sequence which is at least partially common to human cathepsin B. Since the molecular weight is about 28 kDa, the molecular weights are quite different. Since the protein of the present invention has an amino acid sequence that is at least partially common to human cathepsin B, it goes without saying that it reacts with the anti-human cathepsin B polyclonal antibody in an antigen-antibody reaction.

The protein of the present invention (hereinafter sometimes referred to as p80) can be isolated by subjecting urine centrifugal supernatant to Western blotting, as described in detail in Examples below. That is, the centrifugation supernatant of urine is subjected to electrophoresis, the pattern is transferred onto nitrocellulose filter paper, etc., and the labeled human cathepsin B monoclonal antibody is reacted to identify the band in which p80 is present,
This can be isolated by eluting it from filter paper by a conventional method and separating p80 from the antigen-antibody complex.

When p80 is used as an immunogen, it can be used to prepare an antibody that reacts with an antigen-antibody, and such an antibody has a use as a diagnostic agent for urinary system cancer as described later. In addition, p80 has a use as a reagent for detecting or quantifying p80 in a sample by a competitive method.

As described above, the present invention also detects or quantifies the protein of the present invention in a sample immunologically using an antibody or an antigen-binding fragment thereof that reacts with the above-mentioned protein of the present invention in an antigen-antibody manner. Provide a way. The antibody used here may be any antibody as long as it reacts with the protein of the present invention in an antigen-antibody manner, such as the anti-human cathepsin B monoclonal antibody or anti-human cathepsin B described above.
Polyclonal antibodies can be used. Further, it may be a monoclonal antibody or a polyclonal antibody obtained by a conventional method using the protein of the present invention as an immunogen. Although an antibody may be used for detection, its antigen-binding fragment, that is, Fab fragment or F (a
b ′) ₂ fragments can also be used.

The immunoassay method itself for detecting or quantifying an antigen in a sample using an antigen-antibody reaction is well known in this field, and any well-known method can be adopted. That is, a sandwich method, a competition method, an agglutination method, or the like can be used as the measurement format, and an enzyme label, a radiolabel, a fluorescent label, or the like can be used as the label. Furthermore, a Western blotting method as described in detail in the Examples below can also be used.

Among the above-mentioned methods, a preferred and simpler and widely used sandwich method will be described in more detail, but it is merely an example and the present invention is not limited to this.

A monoclonal antibody (for example, the above-mentioned anti-human cathepsin B monoclonal antibody) (first antibody) that reacts with p80 as an antigen-antibody is immobilized on an appropriate insoluble carrier (for example, a plastic container) (hereinafter, this is referred to as immobilized antibody).

Next, the surface of the insolubilized carrier is coated with a suitable substance (eg BSA) in order to avoid non-specific adsorption between the insoluble carrier and the reagent or sample to be measured. The insoluble carrier on which the first antibody is immobilized thus obtained is incubated with a sample. The specimen may be diluted, in which case dilution is generally performed with phosphate buffered saline (PBS). The immobilized antibody binds to p80 in the sample during this incubation (if present in the sample). Then, after washing with a suitable washing liquid,
A solution of a second antibody, which is labeled with an appropriate labeling substance (for example, an enzyme) and reacts with p80 in an antigen-antibody reaction, is contacted with p80 bound to a solid phase antibody in an insoluble carrier for a certain period of time and temperature to react with the second antibody. After washing this with an appropriate washing solution, the amount of the labeling substance labeled with the second antibody that is bound to the solid phase antibody on the insoluble carrier through p80 and is present is measured. Thus, p80 in the sample can be detected or quantified by the value.

Examples of the insoluble carrier used here include polymeric substances such as polystyrene, polyethylene, polypropylene, polyester, cross-linked dextran and polysaccharides, as well as paper, glass, metal, agarose and combinations thereof. The shape can also be various shapes such as a tray shape and a spherical shape. Further, as the labeling substance of the labeled antibody, it is advantageous to use an enzyme, a fluorescent substance, a luminescent substance, a radioactive substance or the like.

As the enzyme, peroxidase, alkaline phosphatase, β-D-galactosidase, as the fluorescent substance, fluorescein isothiocyanate, etc., as the luminescent substance, isorcinol, lucigenin, etc., and as the radioactive substance, ¹²⁵ I, ¹³¹ I, ¹⁴ C,
³ H or the like can be used. However, these are not limited to the exemplified ones, and other substances can be used as long as they can be used in the immunoassay method.

When peroxidase is used as the labeling enzyme, hydrogen peroxide is used as the substrate and o as the color-developing agent.
-Phenylenediamine, 5-aminosalicylic acid and the like, when using alkaline phosphatase as a labeling enzyme, o-nitrophenyl phosphate or the like as a substrate, and when using β-D-galactosidase as a labeling enzyme, 4-methyl as a substrate Umbelliferyl-β-D-galactopyranoside and the like can be used.

A urine sample is preferable as the sample. The urine sample may be urine as it is, but is preferably a centrifugal supernatant of urine or a diluted product thereof. Urinary system cancer can be diagnosed by detecting or quantifying p80 in a urine sample.

The above-mentioned antibodies (monoclonal antibodies and polyclonal antibodies) used in the method of the present invention, antigen-binding fragments thereof and those labeled with the above-mentioned labeling substances, or they are further solid-phased. The bound product can be used as a diagnostic agent for urinary system cancer. The diagnostic agent can be used in the immunoassay method described above,
If p80 is detected in a urine sample using this, it can be diagnosed that there is a high possibility that the cancer is of the urinary system.

[0023]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 Detection of p80 in urine by Western blotting The urine of healthy humans and urine of urological cancers was centrifuged at 3000 × g for 20 minutes, and the supernatant was used for SDS electrophoresis buffer (1.5% SDS, Tris-hydrochloric acid buffer containing 1.5% 2-mercaptoethanol, pH 8.9) was mixed in a volume of 2 to 1 volume and heat-treated at 100 ° C. for 2 minutes, and then S was added.
DS electrophoresis was performed. After electrophoresis, transferred to a Western blotting membrane according to a conventional method, reacted with SBM1-1 antibody, SBM2-2 antibody, RB1 antibody, anti-human cathepsin B polyclonal antibody, and then reacted with alkaline phosphatase-labeled secondary antibody, Color was developed. As a result, a band having a molecular weight of about 80 kDa was specifically detected in the urine of patients with urological cancer, but was not detected in the urine of healthy humans. Table 1 below shows the relationship between the types of urological cancer and the p80 positive rate.

[0025]

[Table 1]

Example 2 Detection of p80 in urine by sandwich ELISA method The p80 in urine cancer patients and normal human urine was detected by the following method.
Was measured to examine its usefulness as a cancer marker.

1) Preparation of Urine Sample Urine of urinary system cancer patient and healthy person was centrifuged at 3000 × g for 20 minutes, and the supernatant was neutralized with phosphate buffer or 2-5 times with phosphate buffer. The urine sample was diluted with.

2) Preparation of Assay Plate The SBM1-1 antibody was diluted with PBS to a concentration of 2 μg / ml, and 100 μl of this was dispensed into each well of a 96-well microplate (Sumitomo Bakelite Co., Ltd.) at 30 ° C.
The antibody was allowed to bind to the plate by allowing to stand for 2 hours or overnight at 4 ° C. PBS containing 0.05% Tween (PBS-T
After washing with (ween), 320 μl of Block Ace solution (manufactured by Dainippon Pharmaceutical Co., Ltd.) was dispensed into each well and left overnight at 4 ° C. for blocking.

3) Immunoassay 100 μl of the urine sample prepared in 1) was added to the well of the assay plate prepared in 2) and reacted at 37 ° C. for 1 hour. After washing with PBS-Tween, a known method (enzyme-linked immunosorbent assay, edited by Eiji Ishikawa, Institute of Medicine, Tokyo, 198).
According to 2), 100 μl of peroxidase-labeled anti-human cathepsin B polyclonal antibody (diluted with PBS-Tween to a concentration of 1 to 5 μg / ml) was added and reacted for 1 hour. The assay plate is then placed on PBS-T.
After washing with ween, peroxidase substrate solution 100μ
1 was added to develop color. The substrate solution was prepared by adding 5 μl of 30% hydrogen peroxide solution to a solution prepared by dissolving 10 mg of o-phenylenediamine in 25 ml of 0.1 M citric acid-phosphate buffer (pH 5.0). After 20 minutes (selectable in the range of 10 to 30 minutes), 50 μl of 2M sulfuric acid was added to stop the reaction, and the absorbance at 492 nm was measured.

By this method, urine of healthy subjects (5 samples), urine of prostate cancer patients (7 samples), urine of kidney cancer patients (8 samples) and urine of bladder cancer patients (5 samples) were measured. The results are shown in Table 2.

[0031]

[Table 2] * Specify a sample as positive if it exceeds the average of healthy people + 2x standard deviation.

Example 3 Measurement of Isoelectric Point of p80 The isoelectric point of p80 in urine was determined by using the Pharmacia Fast System. 4 μl of urine from a patient with renal cancer was separated on an IEF gel (pI5-8) manufactured by Pharmacia for about 30 minutes, and then transferred to a Western blotting membrane according to the manual of First System. The transfer membrane was reacted with an anti-human cathepsin B polyclonal antibody, and then reacted with an alkaline phosphatase-labeled secondary antibody to develop color. As a result, multiple bands were detected from pI5.2 to 6.5.

From these results, urinary p80 was pI5.2-
It was found to have multiple isoelectric points over 6.5.

As described above, the method for detecting p80 in urine of the present invention has a high positive rate in urine of patients with urinary system cancer, and is therefore useful for diagnosing urinary system cancer.

[0035]

INDUSTRIAL APPLICABILITY The present invention provides a novel protein useful as a marker for urinary system cancer. In addition, a method for detecting or quantifying the same and a urinary system cancer diagnostic agent for detecting the above protein are provided. Therefore, the present invention is expected to greatly contribute to the diagnosis of urinary system cancer.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12P 21/08 9161-4B (C12P 21/08 C12R 1:91)

Claims (7)

[Claims] 1. It exists in the urine of a urinary system cancer patient, is not detected in the urine of a healthy person, and has a molecular weight of about 80 kDa,
It has an isoelectric point between 5.20 and 6.50, and has anti-cathepsin B monoclonal antibody SBM1-1 (FERM P-
13683), SBM2-2 (FERM P-1368)
2) and a protein that reacts with RB-1 (FERM P-13685) in an antigen-antibody reaction. 2. A method for immunologically detecting or quantifying the protein of claim 1 in a sample using an antibody or an antigen-binding fragment thereof that reacts with the protein of claim 1 in an antigen-antibody manner. 3. The method according to claim 2, wherein the antibody is an anti-human cathepsin B antibody. 4. A urinary system cancer diagnostic agent comprising an antibody which reacts with the protein according to claim 1 in an antigen-antibody reaction or an antigen-binding fragment thereof. 5. The diagnostic agent according to claim 8, wherein the antibody is an anti-human cathepsin B monoclonal antibody. 6. The antibody is SBM1-1 (FERM P
-13683), SBM2-2 (FERM P-136
82) and RB-1 (FERM P-13685). 7. The diagnostic agent according to claim 8, wherein the antibody is an anti-human cathepsin B polyclonal antibody.