JP3155562B2 - Body - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antibody against a product of the p53 gene (p53 protein), which is called a tumor suppressor gene. The antibody of the present invention can be used as a diagnostic agent for cancer.

[0002]

2. Description of the Related Art The p53 protein is a phosphoprotein localized in the nucleus, and was originally discovered as a protein associated with the Simian virus 40T antigen. Thereafter, it was found that the p53 protein binds to the adenovirus E1B protein having a molecular weight of 58 kd and the heat shock protein HSP70. Became. This p53 protein was abnormally expressed in 40% of breast tumors and 30% of colon cancer tissues in human tumors. It is believed that p53 protein accumulation in these tumor tissues and in cancer cell lines is caused by association with the SV40 Large T antigen, the E1B58 kd protein or mutations in the p53 gene and protein. In addition, the latest findings suggest that p53 protein negatively regulates cell growth in normal cells, and it has been reported that inactivation or mutation of p53 protein results in tumorigenesis of cells. Therefore, this p5
Antibodies to the three gene products can be used as cancer diagnostics.

[0003]

Accordingly, an object of the present invention is to provide an antibody against the p53 gene product that can be used as a diagnostic agent for cancer.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a peptide consisting of a specific amino acid sequence in the p53 protein has high antigenicity, and an antibody against the antigen is expressed in p53 in cancer tissue. They found that they specifically bind to proteins and completed the present invention. That is, the present invention provides an antibody obtained by using a peptide having the following amino acid sequence (hereinafter, may be simply referred to as an antigen peptide) as an antigen. Cys-Phe-Thr-Glu-Asp-Pro-Gly-Pro-Asp-Glu-Ala-Pro-Ar
g-Met-Pro-Glu-Ala

[0005] As described above, the antigen of the present invention is obtained by using the above-mentioned peptide as a corresponding antigen. The amino acid sequence of the antigen peptide corresponds to the amino acid sequence of positions 54 to 69 of the human p53 protein, and is an amino acid sequence obtained by adding cysteine to the amino terminus in order to bind a carrier protein (Molecular and Cellular Biology, July 19).
85, p.1601-1610).

Hereinafter, a method for preparing the antibody of the present invention will be described. First, the antigen peptide is prepared. this is,
It can be easily carried out using a commercially available automatic peptide synthesizer.

Next, the antigen peptide is bound to a suitable carrier. As the carrier, various conventionally known carrier proteins, for example, keyhole limpet hemocyanin, albumin, thyroglobulin and the like can be used. For binding of the carrier protein to the antigen peptide, a conventionally known method, for example, a method using cycinimide can be used.

[0008] The animal is then immunized with the antigen peptide bound to the carrier. Mice, rabbits, rats, sheep, and the like can be used as animals to be immunized, and immunization can be performed by a conventional method.

Next, blood is collected from the immunized animal to obtain an antiserum. The method of obtaining the antibody of the present invention from the obtained antiserum may be any conventionally known method. For example, the human gastric cancer cell line MKN-1 in which a large amount of the p53 gene product is accumulated is solubilized, the protein is transferred to nitrocellulose by Western blotting, and the antiserum collected is used to detect the protein band. Protein with a molecular weight of 53
000 daltons.

Alternatively, it is also possible to fuse the lymphocytes of the animal immunized as described above with myeloma cells, select a hybridoma that specifically produces the antibody of the present invention, and obtain the antibody of the present invention as a monoclonal antibody therefrom. it can. The immunoglobulins obtained in the following examples according to the above method had the following properties. (1) Type of immunoglobulin: IgG (2) Molecular weight: 150 × 10 ³ (3) Reacts specifically with antigen peptide and p53 protein.

[0011]

Industrial Applicability According to the present invention, there is provided an antibody which specifically reacts with an antigen peptide and p53 protein accumulated in cancer tissue. The antibody of the present invention can stain only nuclei of cancer cells in cancer tissues, and is effective for diagnosis of cancer.
In addition, the appearance of autoantibodies against p53 has been reported in the blood of cancer patients overexpressing the p53 gene product,
It is clear that the p53 protein itself flows out into the blood. Therefore, the antibody of the present invention can be used to express p5 in cancer patient serum.
It can be used for cancer diagnosis by measuring 3 proteins or measuring autoantibody titer against p53.

[0012]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the Examples of the present invention are not limited to these. Example 1 (1) Preparation of antigen protein Antigen peptide was synthesized by a solid phase method using a 9013 automatic peptide synthesizer manufactured by Beckman, and then purified by high performance liquid chromatography. The peptide structure of the obtained peptide was hydrolyzed with 1N hydrochloric acid at 120 ° C. overnight, and was measured using an amino acid analyzer. It was confirmed that the peptide structure was in agreement with the theoretical value. A carrier protein was bound to this peptide as follows. That is, 10 mg of antigen peptide and 3 mg of bovine thyrogloline are dissolved in 1 ml of distilled water, and 30 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added thereto. And dialyzed thoroughly against distilled water. Whether or not the antigen peptide and the carrier protein are bound is determined by SDS
Confirmed by electrophoresis.

(2) Preparation of antibody The carrier obtained in (1) and the peptide conjugate (100
μg of the peptide) was injected into the back of the rabbit with Freund's complete adjuvant. 100μ weekly thereafter
g of peptide-equivalent antigen protein was mixed with Freund's incomplete adjuvant and immunized a total of eight times in the same manner. On day 10 after the final immunization, whole blood was collected to separate the antiserum. The IgG fraction was isolated from the antiserum by affinity chromatography using protein A. That is, protein A Sepharose CL4B (Pharmacia) 2
ml was packed into a column and equilibrated with 10 ml of a 1.5 M glycine solution (pH 8.7) containing 3 M NaCl (binding buffer). The serum was diluted with the same amount of the binding buffer and passed through the column to bind IgG to protein A, and then the column was washed with 30 ml of the binding buffer. Next, it was eluted with 0.1 M glycine hydrochloride (pH 3.0) to obtain an IgG fraction. The purity of the purified antibody was determined by SDS electrophoresis.

(3) Properties of Anti-p53 Gene Product Antibody (i) Immunospecificity of Antibody Human Gastric Cancer Cell Line MKN- Overexpressing p53 Gene
1 was thoroughly washed with a phosphate buffer and a RIPA buffer (1% N
P40, 0.1% sodium deoxycholate, 0.15M Na
Cl, 1 mM phenylethylsulfonyl fluoride,
This was suspended and dissolved in 50 mM Tris-HCl (pH 7.4) at 4 ° C. for 20 minutes. This lysate was centrifuged at 120,000 g for 30 minutes, and the obtained supernatant was used as a lysate. Laemmli lysate
After electrophoresis on a 12% SDS polyacrylamide gel according to the above method, all proteins were electrically transferred to a nitrocellulose membrane using a Western blotting device manufactured by Tefco. After immersing this membrane in a phosphate buffer containing 3% bovine serum albumin at room temperature overnight, the membrane was immersed in 0.05%
The plate was washed with a phosphate buffer (TPBS) containing Tween20, and reacted with an anti-p53 gene product antibody. Similarly, the membrane was washed, and a goat biotin-conjugated rabbit IgG (H + L) antibody (manufactured by Vector) was incubated. Color development was performed using a streptavidin-biotin peroxidase kit from Vector, using 4-chloronaphthol as a substrate. The molecular weight of the detected protein is 53 kd, which is consistent with the molecular weight of the p53 gene product. In addition, anti-p53
When a similar Western blot was performed after the gene antibody was absorbed by the antigen peptide, this 53 kd band was not detected, which proved that the antibody was a protein specifically recognized.

(Ii) Tissue staining with anti-p53 gene product antibody Acetone-fixed paraffin sections or frozen sections of human gastric cancer tissues were prepared and reacted with the anti-p53 gene antibody at 4 ° C. overnight. The glass plate is washed with a phosphate buffer, and a goat biotin-conjugated rabbit IgG (H + L) antibody (manufactured by Vector)
After reacting at room temperature for 1 hour, the plate was washed with a phosphate buffer, and color development was performed using 3,3′-diaminobenzidine tetrahydrochloride as a substrate using a streptavidin-biotin peroxidase kit from Vector. As a result, only the nuclei of cancer cells stained, and the nuclei of normal cells did not stain. In addition, Protein A Sepharose CL4B
The same results were obtained when the present antibody purified by column chromatography packed with (Pharmacia) was biotinylated and stained using a biotinylation kit from Pharmacia. The fluorescent label of the purified antibody was 0.
The solution was adjusted to 1 mg / ml with 1M sodium hydrogen carbonate (pH 9.5), mixed with 1 mg / ml of fluorescein isothiocyanate, reacted at 37 ° C. for 30 minutes, and passed through a desalting column PD-10 (Pharmacia). Collected. The antibodies of the present invention have been found to be useful in staining cancer tissue.

(Iii) Quantification of p53 gene product The purified anti-p53 gene product antibody was diluted to a concentration of 10 µg / ml, and 50 µl was spread on an immunoplate (manufactured by Nunc) and allowed to stand at 4 ° C overnight. After washing with TPBS, blocking was performed by incubating at 37 ° C. for 2 hours with a phosphate buffer containing 5% normal rabbit serum. Similarly, TPBS
After washing with 1 μg / ml antigen peptide, serially dilute
0 μl was added to the wells to serve as a standard solution for protein quantification. On the other hand, human neuroblasts solubilized with RIPA buffer
1 × 10 ⁶ Jones were placed in another well as a measurement sample and reacted at 37 ° C. for 1 hour. After washing the plate,
g / ml biotin-conjugated anti-p53 gene product antibody
The reaction was carried out at a temperature of 1 hour for further washing. Color development was performed using O-phenylenediamine dihydrochloride as a substrate using a streptavidin biotin peroxidase kit from Vector. The reaction is stopped with 1N sulfuric acid and the absorbance at 490 nm is measured using Bio-Rad's microplate reader model.
Measured at 3560. As a result, a standard curve of serially diluted antigen peptide was obtained. Since p53 protein has 394 amino acids and antigen peptide has 17 amino acids, p
53 protein amounts were estimated (FIG. 1). By this method, the amount of p53 protein in the sample cell line could be calculated.

[Brief description of the drawings]

FIG. 1 is a diagram showing an antigen peptide standard curve for performing p53 protein quantification.

──────────────────────────────────────────────────続 き Continuation of front page (56) References Eur. J. Biochem. , Vol. 159, no. 3 (1986) p. 529-534 (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 16/32 BIOSIS (DIALOG) CA (STN) JICST file (JOIS) MEDLINE (STN) REGISTRY (STN) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. An antibody obtained by using a peptide having the following amino acid sequence as an antigen. Cys-Phe-Thr-Glu-Asp-Pro-Gly-Pro-Asp-Glu-Ala-Pro-Ar
g-Met-Pro-Glu-Ala