JP2569133B2 - A method for detecting cancer by measuring basic fetal protein in urine - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for detecting cancer by measuring basic fetal protein in urine.

[Prior Art] Basic Fetoprotein (abbreviated as BFP) was discovered by one of the present inventors in the serum, intestinal and brain tissues of human fetuses, and is already known in the literature described below. Is a basic protein. In contrast to the known fetal protein, which is an acidic protein, this protein is called basic fetal protein because it is basic. Furthermore, one of the present inventors has established a radioimmunoassay (abbreviated as RIA) for the protein, and found that measurement of the protein in serum is useful for diagnosing cancer, determining its course, and determining the therapeutic effect. . In particular, it has been found that the protein is useful only for diagnosis of cancer of a specific organ like α-fetoprotein (abbreviated as AFP), and is useful for differential diagnosis between non-cancer and cancer, that is, diagnosis of presence or absence of cancer.

Regarding the above-mentioned conventional findings, the following literature (1)
See (7).

(1) Masaru Ishii et al .: Research on Feto-Neoplastic Antigen. Article of the 34th Annual Meeting of the Japanese Cancer Society, p.173 (197
Five).

(2) Masaru Ishii: A new fetal protein present in various malignant tumors
Study on basic fetoprotein. History of Medicine, 100
(3), 344-346 (1977).

(3) Ishii Masaru: Basic fetoprotein (basic fetal protein). History of Medicine, 106 (5), 273-281 (19
78).

(4) Ishii, M.: A new carcinoembryonic protein ch
aracterized by basic property.Scand.J.Immunol., 8
(Suppl. 8), 611-620 (1978).

(5) Ishii, M .: Characterization of basic fetopro
tein and clinical usefulness of BFP for immunodiag
nosis of human cancer.Carcino-Embryonic Proteins.
Chemistry, Biology, Clinical Applications, Vol.1, Lehm
ann, F, -G. (ed.), Elsevier / North-Holland Biomedic
al Press, Amsterdam, 333-340 (1979).

(6) Ishii, M., Nishimura, K, .Hattori, M., Kanda, Ya
nd Ishihara, A .: Postoperative surveillance in patie
nts with stomach cancer and monitoring of immun
o-and polychemotherapy in patients with leuke
mia by basic fetoprotein.Carcino-Embryonic Protei
ns.Chemistry, Biology, Clinical Applications, Vol.2
Lehamnn, F.-G. (ed.), Elsevier / North-Holland Bio
medical Press, Amsterdam, 603-606 (1979).

(7) Ishii, M.: Clinical usefulness of basic feto
protein for immunodiagnosis of human cancer.Compen
dium of Assay for Immunodiagnosis of Human C
ancer, Development in Cencer Research, Heraberman, R.
B. (ed.), Vol. 1, Elsever / North-Holland Biomedical
Press, New York, 45-50 (1979).

(8) The 6th Study Group for Tumor Markers (October 20, 1986)
(Japan, Sapporo) Program and Abstracts, 111 pages (9) The 6th Study Group for Tumor Markers (October 20, 1986)
(Nippon, Sapporo) Records pp. 248-250 Since the amount of BFP in serum of cancer patients is very small, it is necessary to establish a highly sensitive measurement method for its quantification, mainly using the RIA method and the EIA method. Method (enzyme immunoassay)
Has been developed. For details of the RIA method, refer to the above-mentioned references (3) to (7), and for details of the EIA method, refer to the following references (10) to (11).

(10) Masaru Ishii et al .: Basic Fetoprotein, Current Clinical Function Test-Its Practice and Interpretation. Japanese clinical 37: 1536-1539,197
9.

(11) Masaru Ishii: Basic Fetoprotein (Basic Fetoprotein)
toprotein). Laboratory test 24 (8): 931-936,1980.

Thereafter, the present inventors succeeded in obtaining a monoclonal anti-basic fetal protein antibody. For preparation and performance of the monoclonal antibody, see the following reference (12).

(12) Masaru Ishii et al .: Production of Monoclonal Anti
−Basic Fetoprotein (BFP) and Usefulness of Monocl
onal Anti-BFP for Immunodiagnosis of Human Cance
r, Tumor Research Vol.18 (Special Issue) .1983, p75
~ 86.

By the way, when the reaction specificity of the above monoclonal antibody to BFP was examined using the EIA method and the MO method,
Has at least three different antigenic determinants on the molecule, and it has been found that monoclonal antibodies are classified into three types corresponding to each antigenic determinant. When the three antigenic determinants are A, B, and C, and the monoclonal antibodies corresponding to the respective antigenic determinants are arranged, for example, the following is obtained.

Based on the above findings, the present inventors, by sandwich method using these monoclonal antibodies, BF in serum
A method for accurately measuring P has been established (Japanese Unexamined Patent Publication No.
0-80768).

[Problems to be solved by the invention] However, all the measurements introduced above
It was done for BFP. On the other hand, there is no evidence that BFP was detected in urine of healthy persons and patients with various diseases.

In addition, conventionally known tumor markers, such as carcinoembryonic antigen (abbreviated as CEA) and AFP, have no significant difference between their healthy and cancer patients with respect to their urinary measurements, In particular, a tumor marker that is effective in diagnosing urological cancer has not been known so far.

The present inventors have also found that BFP is present in urine of healthy persons and various disease patients.
Have been found, and various studies have been conducted on the usefulness of the protein as a tumor marker, resulting in the present invention.

[Means for Solving the Problems] That is, an object of the present invention is to provide a measurement method which is extremely useful for diagnosing various cancers, especially urological cancer, and monitoring the progress of treatment, and the like. It is characterized by detecting BFP inside.

The measuring method of the present invention is an immunological measuring method such as EIA method and R
Any of the IA method and the like can be used, but the EIA method is preferable in terms of safety, measurement sensitivity, and the like.

Further, as a more preferable specific example of the present invention, an EIA method by a sandwich method using a monoclonal antibody as the first antibody and the second antibody can be mentioned.

Hereinafter, the present invention will be described in detail based on the EIA method (sandwich method) using the monoclonal antibody as a preferred embodiment.

As described above, FBP measured by the present invention is a tumor marker with low organ specificity.

The antibasic fetal protein monoclonal antibody according to the present invention may be prepared, for example, as follows. First, antibody-producing cells are prepared from BALB / c mice sensitized with BP, and P3-X63-Ag8-U1 is used as myeloma cells, and cell fusion is performed between them and a modified method of Herzenberg et al. Next, the antibody titer from the fused cells selected by HAT medium was labeled with ¹²⁵ I.
It is measured by a plate banding assay using BFP. Fused cells showing high antibody titer and having good cell growth are cloned by limiting dilution to obtain several cell lines producing high antibody titer. Finally, each cell line was transplanted into the mouse intraperitoneal cavity, and the ascites obtained was subjected to ammonium sulfate precipitation, dialyzed, and then subjected to DEAE
If the IgG components are collected by cellulose, several kinds of monoclonal antibodies according to the present invention can be obtained.
In order to classify the obtained monoclonal antibodies corresponding to different antigenic determinants of BFP, MO method and EIA shown below
The law may be implemented.

MO method A mixture of two types of monoclonal antibodies in a 1: 1 ratio was prepared for all combinations of monoclonal antibodies, and the MO method was used to observe the formation of sedimentation lines and the presence or absence of fusion with BFP of these mixtures. I do. When a clear sedimentation line is observed, the monoclonal antibodies belonging to the combination belong to groups that react with different antigenic determinants, and conversely, when no sedimentation line is observed, they belong to a group that reacts with the same antigenic determinant. If the generation of the sedimentation line is unclear and the judgment is uncertain, the next EI
Determined by Method A.

EIA method The measurement reagent prepared and carried out in the same manner as in Example 1 except that two kinds of monoclonal antibodies arbitrarily selected in place of K1 and 5C2 in the description of Example 1 were used for all combinations of monoclonal antibodies. prepare. Perform the EIA operation and read the absorbance of the color value. When there is color development, the monoclonal antibodies of the combination belong to groups that react with different antigenic determinants, and when there is no color development, they belong to groups that react with the same antigenic determinant.

As described above, when grouping is performed by the MO method and the EIA method, as described above, BFP has three different antigenic determinants, and monoclonal antibodies are classified into three groups that react with each other.

The components of the entire measurement system are solid phase, solid phase coating antibody (first antibody), BFP (standard antigen and test urine), labeling antibody (second antibody), enzyme and substrate. As the solid phase, wells of a microtiter plate for enzyme immunoassay or plastic beads may be used. Prior to the measurement, one of the monoclonal antibodies according to the present invention is arbitrarily selected as a solid phase coating antibody (first antibody), and a 0.1M sodium carbonate buffer (pH 9.0) is adjusted so that the OD _{280 nm} as the protein concentration becomes 0.050. ), Put in a well for polystyrene enzyme immunoassay or plastic beads, and leave at 4 ° C. overnight to coat the solid phase surface with the first antibody. As the standard antigen, purified BFP was prepared by subjecting ascites obtained from a liver cancer patient to a purification treatment, and the BFP was prepared to a predetermined concentration using a standard antigen diluent.

As the labeling antibody (second antibody), the monoclonal antibody according to the present invention that reacts with an antigenic determinant different from the antigenic determinant that reacts with the first antibody is selected. Examples of enzymes include alkaline phosphatase, glucose oxidase,
Peroxidase, beta-galactosidase and the like can be used. Prior to the measurement, an enzyme is bound to the labeling antibody with a binding agent such as glutaraldehyde to obtain an enzyme-labeled antibody, which can be prepared in advance as a part of a reagent for carrying out the measurement method of the present invention. The substrate may be appropriately used depending on the selected enzyme. For example, when alkaline phosphatase is selected as the enzyme, p-nitrophenyl phosphate or the like may be used.

The measurement may be performed according to a normal procedure in the EIA method. Therefore, as shown in the Examples below, a standard antigen or test urine is added to the wells coated with the first antibody and incubated, followed by addition of an enzyme-labeled antibody, for example, a second antibody-alkaline phosphatase-labeled antibody, followed by incubation. A substrate, for example, p-nitrophenyl phosphate may be added to the mixture and incubated, and the amount of the substrate decomposed may be measured using a spectrophotometer.

The first antibody to be coated on the solid phase may be a single type of monoclonal antibody, or may be a mixture of a plurality of types of monoclonal antibodies. The point is that the first antibody and the second antibody only need to react with different antigenic determinants.Each antibody is composed of a single type of monoclonal antibody or composed of multiple types of monoclonal antibodies. This is not a requirement to be particularly limited in the present invention.

The case where a monoclonal antibody is used has been described above. However, in the present invention, the antibody is not limited to a monoclonal antibody, and a conventional polyclonal antibody can also be used.

Therefore, another object of the present invention is to provide a measurement kit used for measuring urinary BFP. The kit includes a first antibody, a labeled second antibody, a solid phase and a standard basic fetal protein antigen.

The present invention will be described more specifically with reference to the following examples.

Example 2 Saturated ammonium sulfate (4.05M) was added to each 5 ml of mouse ascites fluid containing two types of monoclonal antibodies K1 and 5C2, which react with different antigenic determinants of BFP, to a final concentration of 1.8 M, and incubated at room temperature for 2 hours. Stir. 20 at 12,000 rpm
Separate the precipitate and supernatant by centrifugation for minutes. The sediment is dialyzed against the dissolved buffer. Next, apply to a DEAE-cellulose column,
The IgG component is eluted with a 0.1 M phosphate buffer (hereinafter abbreviated as PB) (pH 8.0). Elute IgG components by collodion bag (MW120
Concentrate at 00) to prepare K1 and 5C2. OD _280nm as protein concentration in 0.1M sodium carbonate buffer (pH 9.0)
Was dissolved to be 0.05, poured into the wells of a microtiter plate for enzyme immunoassay, allowed to stand overnight, drained, washed with 0.1 M sodium carbonate buffer (pH 9.0), and dried in a well dryer. And the solid-phased first antibody.

On the other hand, the production of the enzyme-labeled antibody was performed as follows. 0.1M phosphate buffer containing 1.25% glutaraldehyde (pH
6.8) Dissolve horseradish peroxidase in
After reacting for an hour, it is dialyzed against saline. This solution and a monoclonal antibody basic fetal protein antibody dialyzed against saline in advance and 5C2 solution are mixed in equal amounts, and a 1M carbonate buffer (pH 9.5) is added to a ratio of 5%. 4 ℃, 2
Let stand for 4 hours. Next, a 0.2 M lysine solution is added at a ratio of 5%, left for 2 hours at room temperature, and dialyzed against saline. Then, it was dialyzed against a 0.05 M phosphate buffer (pH 7.4) to prepare a horseradish peroxidase-labeled anti-basic fetal protein antibody.

Separately, a reaction diluent, a coloring solution, a reaction stop solution, and a labeled antigen diluent are prepared as follows.

The reaction diluent was normal calf serum, normal mouse serum (inactivation treatment at 56 ° C for 30 minutes), EDTA 3Na, sodium chloride, and sodium azide in 0.05M Tris-HCl buffer (pH 8.0), respectively.
It is prepared by containing 11.8%, 2%, 10 mM, 0.15 M and 0.1%.

The color developing solution was 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS).
It is made to contain 3 mg / ml in 1 M citrate buffer (pH 4.0), and further prepared by adding hydrogen peroxide to a ratio of 0.02%.

Reaction stop solution contains 0.01% sodium azide
Use 0.1 M citrate phosphate buffer (pH 4.0).

The standard antigen dilution was normal calf serum (inactivated at 56 ° C, 30
Min), EDTA 3Na, sodium chloride, sodium azide
18.1%, 10 mM, in 0.05 M Tris-HCl buffer (pH 8.0)
It is adjusted to contain 0.1%.

First, a sample and a standard antigen of known concentration are diluted 11-fold with a reaction diluent, and 100 μl
And incubated at 37 ° C. for 1 hour. After washing with saline, 100 µl of the optimal concentration of enzyme-labeled antibody was added, followed by incubation at 37 ° C for 1 hour. After washing again with saline, 100 µ of a coloring solution was added, and the mixture was incubated at 37 ° C for 1 hour. 0.01% sodium azide solution 100μ
To stop the reaction, measure the OD _{420 nm} absorbance, and
Values were calculated.

Specimens consisted of 26 healthy subjects, 45 benign urinary disease cases,
There are a total of 142 cases, including 27 other benign disease cases, 27 urinary cancer cases, 6 other cancer cases, and 11 recurrent cases after urological cancer surgery. For urine, urine should be collected at any time and added with sodium azide.
Samples stored at ° C. were used, and the serum used was a sample of the same case collected at the same time as urine collection.

The mean value of urinary BFP in 26 healthy subjects was 2.6 ng / ml, and the mean value + 2SD was 7.3 ng / ml. The cut-off value was set to 20 ng / ml based on the measurement results of benign diseases and cancer cases. Meanwhile, serum BFP
The cut-off value was 75 ng / ml.

The positive rates of urinary BFP and serum BFP were 11% and 0% for benign urological diseases, 0% and 18.5% for other benign diseases, 48% and 33% for urological cancer, and 17% and 33% for other cancer diseases, respectively. The percentage was 27% and 25%, respectively, after surgery for urological cancer. Urinary BFP has a positive rate of urinary cancer of 70% (7/10) of bladder cancer and ureteral cancer
100% (2/2), prostate cancer 25% (2/8), renal cancer 33% (2 /
6), testicular cancer is 0% (0/1), and the urinary tract cancer of bladder cancer and ureteral cancer has a significantly higher positive rate than serum BFP. The above results are shown in FIG. 1 and FIG. In the figure, U and S indicate a urine BFP value and a serum BFP value, respectively.

There was no correlation between urine BFP and serum BFP.
The positive rate of both urinary BFP and serum BFP by Combination assay was improved to 70% in urological cancer. By breakdown, renal cancer improved to 83% (5/6), prostate cancer to 50% (4/8), and testicular cancer to 100% (1/1).

In addition, the effect of hematuria on BFP was examined, but there was no correlation between red blood cell count in urine sediment and urinary BFP.
Hemolysis urine was observed in two cases of cystitis and benign prostatic hyperplasia,
Urinary BFP values were 164.7 ng / ml and 14.9 ng / ml, respectively.

[Effect] Thus, urine BFP shows a high positive rate in urological cancer,
In particular, it is considered to be a tumor marker highly specific to bladder cancer and ureteral cancer. Furthermore, Combinati of the method of the present invention with serum BFP
The on-assay markedly improved the positive rate in renal and prostate cancers, indicating high utility in the diagnosis of urological cancer.

As is clear from the above description, the measurement method of the present invention can provide extremely useful data in screening, diagnosis and follow-up of treatment for cancer, particularly urological cancer.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows urine and serum BF in healthy subjects and benign urological diseases
The measurement results of P are shown. FIG. 2 shows the measurement results of urine and serum BFP in urological cancer.

Claims (7)

(57) [Claims] 1. A method for detecting cancer, comprising measuring basic fetal protein contained in urine by immunoassay using an anti-basic fetal protein antibody. 2. The method according to claim 1, wherein said cancer is a urinary system cancer. 3. The method according to claim 2, wherein the urinary system cancer is bladder cancer, prostate cancer, ureteral cancer or kidney cancer. 4. The method according to claim 1, wherein said immunoassay is a sandwich method. 5. The method according to claim 4, wherein the anti-basic fetal protein antibodies are a first monoclonal antibody and a labeled second monoclonal antibody, each of which reacts with a different antigenic determinant of the basic fetal protein. Method. 6. The method according to claim 5, wherein said first monoclonal antibody is immobilized on a solid phase. 7. The method according to claim 5, wherein said second monoclonal antibody is labeled with an enzyme.