JPH0785085B2 - Fibronectin measurement reagent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel reagent for measuring fibronectin, and more particularly to a novel reagent for measuring non-fragmented fibronectin and / or fragmented fibronectin.

[Conventional technology]

In recent years, cancer and fibronectin (hereinafter abbreviated as FN)
The relation of is attracting attention. FN is a glycoprotein with a molecular weight of 440,000 on the cell surface and in blood, and is involved in cell adhesion, migration, differentiation, proliferation, canceration, metastasis, wound, healing, inflammation and the like. 1974 Yamada et al. [Proc.Nat.Acad.Sci.USA] etc. 71 Proceedings of National Academy of Sciences of the USA
Vol., P. 3492 (1974)], it was found that when cells become cancerous, FN, which is a glycoprotein on the cell surface, disappears or decreases. However, it has been pointed out that in some cancers, serum FN concentration is elevated, and FN fragmentation is also occurring at the same time [Cancer Research (Canc
er Res.) 39, 4341 (1979)]. Furthermore, it has been shown that cancerous cells produce an enzyme having FN-degrading activity [Cell, Vol. 48, p. 193] (198).
7)].

In addition, as a result of examination using a monoclonal antibody in colon cancer, the present inventors have shown that FN production is increased as compared with normal cells (Japanese Patent Application No. 61-307760).

From the above, it is presumed that FN is associated with cancer development and proliferation in some system.

On the other hand, regarding the relationship between FN and other diseases, many studies such as lung disease, liver disease, colorectal disease, infectious disease, collagen disease, kidney disease, diabetes, thyroid disease, skin disease, DIC, and shock have been conducted. However, in these studies, fragmented FN (hereinafter abbreviated as fFN) and non-fragmented FN (hereinafter abbreviated as nFN) were measured as the total amount without distinction, especially in blood containing a large amount of FN. There were many unclear points in the measurement of medium concentrations.

[Problems to be solved by the invention]

For the above reasons, a novel reagent for measuring FN has been desired. An object of the present invention is to provide a novel measuring reagent capable of highly sensitive and highly accurate measurement by distinguishing the amount of fFN and the amount of nFN.

[Means for solving problems]

The present invention can be summarized as follows.The present invention is an invention relating to a fibronectin measurement reagent, in a reagent for measuring fragmented fibronectin containing a fragment that is not recognized by FN4 or FN9 and is recognized by FN10 or FN30 in a test sample. An antibody that recognizes the fragment but has a different antigen recognition site is a constituent component.

The present inventors have previously obtained a novel monoclonal antibody with a clear binding domain for FN (Japanese Patent Application No. 61-308311).
In addition, it was found that staining the tissue section and cells of the excised tissue with this monoclonal antibody clearly stains more strongly than normal cells / tissues, and that the amount of FN serves as a marker for detecting human cancer. A detection method was developed (Japanese Patent Application No. 61-307760).

The present inventors have conducted further studies and developed a measurement reagent for distinguishing and quantifying fFN amount and nFN amount by using a domain-specific antibody against FN. Furthermore, we discovered that a large amount of fFN, which is not found in normal people, appears in the urine of cancer patients,
It was confirmed that cancer can be diagnosed by using the novel FN measuring reagent according to the present invention, and the present invention has been completed.

In the present specification, the domain means an antigen recognition site.

Human nFN for obtaining a domain-specific antibody against FN in the present invention is, for example, human blood plasma, by a method known per se, for example, gel filtration, ion exchange chromatography, affinity chromatography alone or by a method. It can be obtained by separating and refining it in combination [Ann.NYAcad.S (Ann.NYAcad.S.
ci), No. 312, page 256 (1978)]. Further, human fFN is, for example, human nFN obtained by the above method, for example, thermolysin, trypsin, by a method known per se.
Limited or complete degradation using proteases such as thrombin, plasmin, chymotrypsin alone or in combination, and separation using various separation methods such as gel filtration, ion exchange chromatography, affinity chromatography, etc. It can be obtained by purification [Journal of Biological Chemistry (J. Biol. Chem.) Volume 260, page 5105 (1985)].
In addition, for example, urine of cancer patients such as lung cancer, gastric cancer, colon cancer, breast cancer, pancreatic cancer, etc. can be purified and collected as a raw material. Furthermore, since the entire amino acid sequence of human FN has been recently determined, a synthetic peptide having an amino acid sequence of a specific fragment is also used.

On the other hand, a domain-specific antibody against human FN, for example, using the above human fFN as an antigen, a mammal other than human, for example, a guinea pig, a rabbit, a rat, a mouse, a goat, etc. After immunization according to the procedure described above, blood is collected to obtain antiserum, and the antibody is separated. In obtaining the antibody, for example, 0.1 to 1 mg of human fFN described above is dissolved in 0.1 to 5 ml of physiological saline, and the same amount of complete.
After adding Freund's adjuvant and sufficiently emulsifying, it is injected subcutaneously or intradermally in the mammal to be used, such as rabbit and mouse, and is injected several times every 1 to 3 weeks for immunization.
Then, after a fixed period from the day of the final immunization, blood was collected and human fFN was collected.
An antiserum containing an antibody against is obtained.

In addition, as the animal used in this case, any animal may be used as long as it has an antibody-producing ability, and in order to obtain a large amount of antibody, a large animal is preferably used, and usually a rabbit or a goat is used, but It is not limited. Furthermore, in order to obtain anti-human fFN antibody from anti-serum containing anti-human fFN antibody obtained from these animals, it is possible to carry out by a method of antibody purification means which is usually used. Then, it may be purified and collected by ion exchange chromatography or gel filtration. To further purify it to antipurity, it may be obtained by adsorbing it by affinity chromatography using an insolubilized carrier on which human fFN is immobilized as a base material, and then causing elution. As another method, splenocytes of non-human mammals immunized with human fFN as an antigen and myeloma cells are fused, and monoclonal antibody-producing cells against human fFN are separated from the fused cells, and the fused cells are isolated. There is a method for producing an anti-human fFN monoclonal antibody using, and particularly a method using a mouse as a mammal is often used [Nature 256, 495 (1975)]. A domain-specific antibody against human FN is obtained as described above.

On the other hand, a domain-specific antibody against human FN can be obtained by using the above human nFN as an antigen.

That is, fusion was performed using splenocytes and myeloma cells of a mammal other than human immunized with human nFN as an antigen,
From this fused cell, a clone producing a monoclonal antibody against human nFN is isolated. The monoclonal antibody thus obtained is a domain-specific antibody that reacts only with a specific domain of human FN. Mice are often used as animals, and the present inventors have proposed an anti-human FN monoclonal antibody whose binding domain is already clear (Japanese Patent Application No. 61-308311).

By using the domain-specific antibody against human FN obtained as described above, it is possible to discriminate and quantify nFN and fFN, which was not possible with conventional anti-human FN polyclonal antibodies.
FIG. 1 is a schematic diagram showing the structure of fibronectin and a particularly generated fragment of fibronectin digested with thermolysin. That is, if antibodies specific for I and VI, which are typical fragments of FN shown in FIG.
It is possible to measure the amount of N only, and for example, if an antibody specific to only fragment IV is combined, the total amount of nFN and fFN can be measured, and therefore, if the amount of nFN is subtracted from the total amount of nFN and fFN, then fFN
The amount of fN can be determined, and the decomposition rate of FN can be known from the ratio of the amount of fFN to the total amount of nFN and fFN. These effects
This is the first to be achieved by using a domain-specific antibody against FN, and in particular, when a monoclonal antibody is effective and only a polyclonal antibody that does not have the conventional domain-specificity is used, nFN and fFN are distinguished in this way. It was impossible to measure. As an assay method using such a domain-specific antibody against FN, immunoassay methods well known in the art, that is, enzyme immunoassay methods, radioimmunoassay immunoturbidimetric methods, latex agglutination methods, and hemagglutination methods are known. , SRID method (immunodiffusion method) is used. Among them, the enzyme immunoassay method is the most practical in terms of sensitivity and simplicity. That is, the anti-human FN domain-specific antibody is treated with an insoluble carrier such as polystyrene beads, glass beads, or polystyrene microtiter plate, and covalently bonded or physically adsorbed to these carriers to insolubilize the anti-human FN domain-specific antibody bound thereto. Get the antibody. On the other hand, the anti-human FN domain-specific antibody is labeled with an enzyme using a conventionally known method. For example, an optimal compound for the enzyme used (for example, m-maleimide ester for β-galactosidase, periodic acid for peroxidase), and then an antibody are coupled to this reaction product to obtain an enzyme-labeled anti-human FN domain-specific antibody.

The thus obtained anti-human FN domain-specific antibody-binding carrier (insoluble antibody) and enzyme-labeled anti-human FN domain-specific antibody (labeled antibody) were used to measure urinary fFN and urinary nFN. Compared with the above, it was found that fFN was specifically increased in the urine of cancer patients, and it was shown to be useful for the diagnosis of cancer.

〔Example〕

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Quantification of nFN and fFN by EIA method (1) The present inventors previously proposed (Japanese Patent Application No. 61-308311).
No.) Anti-human FN monoclonal antibody-producing hybridoma FN
4, FN9, FN10, and FN30 strains were intraperitoneally injected into Balb / C mice, which had been pre-stained, at about 2 × 10 ⁷ cells / mouse,
An ascites tumor was made, and ascites was collected 10 days later. The ascites was subjected to ammonium sulfate fractionation and ion exchange chromatography to collect the IgG fraction according to a conventional method to obtain anti-human FN monoclonal antibody. The fragment specificity of each monoclonal antibody is shown in Table 1.

(2) Anti-human FN domain-specific antibody FN obtained in (1) above
Polystyrene balls (manufactured by Sekisui Chemical Co., Ltd.) in 20 ml of 0.1 M phosphate buffer (pH 8.0) containing 4 mg and 4 mg of FN30, respectively.
100 particles (having a particle size of 6.35 mm) were added and reacted at 5 ° C. for 16 hours and 37 ° C. for 1 hour to immobilize the antibody on the beads. After thoroughly washing the beads with physiological saline, 1% bovine serum albumin (BS
A), 0.05% sodium azide, 0.9% NaCl containing 10 mM phosphate buffer (pH 7.4) and left at 5 ° C. for overnight to obtain anti-human FN domain-specific antibody-bound beads.

(3) Anti-human FN domain antibody FN obtained in (1) above
9 and FN10 were each provided with a peroxidase (Boehringer-Mannheim) by the method of Nakane et al. [Journal of Histochemistry and Cytochemistry (J. Histochem. Cytochem.) Vol. 22, page 1084 (197).
4)] to obtain a labeled antibody. Ie 10m
g peroxidase is dissolved in 2 ml of purified water, and 0.2 ml of 0.1 M potassium periodate is added. After reacting for 20 minutes at room temperature, it is dialyzed against 1 mM acetate buffer (PH4.0) overnight at 4 ° C. 0.2M carbonate buffer (pH 9.5) was added to this to adjust the pH to 9
Adjust to ~ 9.5. On the other hand, anti-human FN domain specific antibody 2 mg
Is dissolved in 1.5 ml of phosphate buffered saline (pH 7.4),
It was dialyzed against mM carbonate buffer (pH 9.5) overnight at 4 ° C., mixed with the above periodate-treated peroxidase and reacted at room temperature for 2 hours, and then sodium borohydride (4 mg / ml) was added and reacted at 4 ° C for 2 hours, and then fractionated by gel filtration using Ultrogel AcA22 (manufactured by LKB) equilibrated with phosphate buffered saline (pH 7.4). Fractions with matching peroxidase activity and antibody activity were collected, and sodium merthiolate was added at a final concentration of 0.001%.
And stored at 4 ° C.

(4) Anti-human FN polyclonal antibody Using the anti-human FN polyclonal antibody (manufactured by Institute of Medical Biology), the insolubilized antibody and the labeled antibody were obtained in the same manner as in (2) and (3) above.

(5) Production of human nFN and human fFN Human nFN used was human blood-derived FN (Funakoshi). On the other hand, human fFN was prepared by the method of Borsi et al. [Analytical Biochemistry.
Vol., P. 335 (1986)]. Ie 500 mg
Human blood plasma-derived FN (Funakoshi Co., Ltd.) was treated with 52 mM Tris-HCl (pH 7.6, 0.5 mM EDTA, 50 mM NaCl, 2.5 mM)
Thermolysin (manufactured by Sigma) in 500 ml containing CaCl ₂ )
Was added to a final concentration of 5 μg / ml and digested for 4 hours at 22 ° C. EDTA was added to a final concentration of 5 mM to stop the reaction. This reaction product and nFN by SDS-polyacrylamide electrophoresis, nFN does not contain fFN and the thermolysin digest did not contain nFN,
It was confirmed that it was fragmented into 6 species.

The amount of protein is the absorption coefficient from the absorbance at 280 nm. [Mosesson et al. Journal of Biological Chemistry Vol. 245, p. 5728 (1970)]
Was calculated using.

(6) Measurement of nFN and fFN The EIA was performed as follows. Put 200μm of sample into the tube, put insolubilized antibody beads in the tube one by one, and perform the first incubation at 37 ° C for 1 hour. Next, wash the beads three times with 3 ml of physiological saline, and add labeled antibody solution (300
200μ in a tube containing beads, 37
Perform the second ink incubation at ℃ for 1 hour. Next, wash the beads 3 times with 3 ml of physiological saline, transfer the beads to another tube, and add the coloring reagent 300 μ (o-phenylenediamine 1 mg / ml, 0.01% of H ₂ O ₂ , 0.1M buffer of citric acid). pH
(Dissolved in 5.0), and react at 37 ℃ for 30 minutes, 1
The reaction was stopped by adding 1 ml of NH ₂ SO ₄ . The absorbance at a wavelength of 492 nm was measured. Table 2 shows the results of measurement of nFN and fFN obtained in (5) above by this method. As a result, nFN can be measured with the combination of FN4 and FN9, that is, the combination of domain-specific antibodies that recognize fragment I and fragment VI, but fFN
Was shown to be unmeasurable at all. It was also shown that the detection sensitivity was poor in the combination of polyclonal antibodies.

In addition, create a model specimen that mixes nFN and fFN in appropriate amounts,
The fractional quantification of nFN and fFN and the decomposition rate of FN in the sample were calculated.
Table 3 shows the results of using the above-mentioned examples for the calibration curve.
In the combination of FN30 and FN10, the total amount of nFN and fFN is calculated,
In the combination of FN4 and FN9, the amount of nFN alone was obtained, and it was shown that the fragmentation rate can be obtained by the following formula, and it was possible to separately quantify nFN and fFN.

Example 2 Measurement of urinary FN from cancer patients (1) Detection by the blot immunoassay method Early morning urine of 10 healthy subjects and 10 cancer patients was collected, and 10 ml of each was collected.
Was dialyzed against purified water at 4 ° C. overnight and lyophilized,
The dried product was dissolved in 0.1 ml of 10 mM phosphate buffer (pH 7.2) and subjected to SDS-polyacrylamide gel electrophoresis,
Next, the fractionated proteins are electrically transferred onto a nitrocellulose membrane by Western blotting. Using various anti-human FN domain-specific antibodies and anti-human FN polyclonal antibodies as the primary antibody of the protein adsorbed on the nitrocellulose membrane 2
As a coloring solution of peroxidase-labeled anti-mouse Ig antibody and peroxidase-labeled anti-rabbit Ig antibody (both manufactured by Amasyamu Co., Ltd.) as a secondary antibody, 0.1 M containing 1 mg / ml 4-chloro-1-naphthol and 0.01% H ₂ O _2. It was detected by an enzyme immunostaining method using phosphate buffer (pH 6.5). The results are shown in Table 4, and in healthy humans, no matter which primary antibody was used, only the nFN band having a molecular weight of 220K was detected.
The fFN of K could not be detected. On the other hand, when anti-human FN domain-specific antibody (FN4, FN9, FN10, FN30) was used, 220K nF
The fFN present at 5K to 200K along with the N band was clearly sought only in cancer patients and showed a better detection rate than with the anti-human FN polyclonal antibody. From the above results, it was revealed that fFN, which does not appear in healthy individuals, is clearly present in the urine of cancer patients, and that it is clearly detected by anti-human FN domain-specific antibody.

(2) Measurement by San-Germany EIA method Early morning urine was collected from 10 healthy subjects and 10 cancer patients, and Example 1 was obtained.
The amount of FN in the sample was measured in the same manner as in. At the same time, the amount of creatinine in urine was measured using a commercially available kit (creatinine test Wako: Wako Pure Chemical Industries, Ltd.), and in order to correct the amount of urine, the ratio of the amount of FN to the amount of creatinine (FNμg / g
・ Cr).

Fig. 2 shows the case where FN30 was used as the insolubilized antibody and FN10 was used as the labeled antibody, Fig. 3 was the case where FN4 was used as the insolubilized antibody and FN9 was used as the labeled antibody, and Fig. 4 is the case where both polyclonal antibodies were used. FIG. 5 is each graph showing a case where the decomposition rates of FN in the samples are compared. The vertical axis shows the FN amount (μg / g ・ C
r) or fragmentation rate (%). As a result, in the combination of FN30 and FN10, the amount of urinary FN in cancer patients was obviously higher than that in healthy subjects, and in particular, cases with metastasis (white circles in the figure) showed abnormally high values. On the other hand, FN
There was no clear difference in the combination of 4 and FN9, that is, the amount of nFN between healthy subjects and cancer patients. Furthermore, when the polyclonal antibody was combined, the cancer patients showed higher values than the healthy subjects, but the difference was smaller than that of the combination of FN-30 and FN10. Moreover, when the fragmentation rate of FN in the sample was calculated, it was widely distributed as 15 to 85% in healthy people, but in cancer patients,
It was over 90%. From the above results, it was revealed that a large amount of fragmented FN appears in the urine of cancer patients, and by measuring this, it is possible to diagnose metastatic activity even during cancer diagnosis. It was

〔The invention's effect〕

As described in detail above, according to the present invention, fFN and n
A measurement reagent capable of separately measuring FN was provided. The measurement test of the present invention enables cancer diagnosis and FN fragmentation rate measurement.

[Brief description of drawings]

Fig. 1 is a schematic diagram showing the structure of fibronectin and the fragment produced when fibronectin is digested with thermolysin, and Fig. 2 shows the results obtained using FN30 antibody and FN10 antibody.
The figure shows the results of FN4 antibody and FN9 antibody, and Fig. 4 the polyclonal antibody, and the results of measuring the urinary FN levels of healthy subjects and cancer patients. Fig. 5 shows healthy subjects and cancer. It is a graph which shows the result of having calculated the fragmentation rate of FN in a patient's urine.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroko Yokota 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Minami Shuzo Co., Ltd. (72) Kaoru Katayama 3-4-1 Seta, Otsu City, Shiga Prefecture Central Brewery Co., Ltd. (72) Ikuno Kato, Inventor Ikunobu Kato 3-4-1 Seta, Otsu City, Shiga Prefectural Laboratories Co., Ltd. (56) Reference JP-A-57-79455 (JP, A)

Claims (1)

[Claims] 1. FN10 which is not recognized by FN4 and FN9 in a test sample
Alternatively, a reagent for measuring fragmented fibronectin containing a fragment recognized by FN30, which comprises an antibody that recognizes the fragment but has a different antigen recognition site as a constituent component.