JPS62116262A - Method and kit for quantifying terminal peptide of human i-type procollagen c - Google Patents

Abstract

PURPOSE:To enable rapid quantification with high sensitivity, by preparing a monoclonal antibody specifically reacted with the terminal of procollagen C. CONSTITUTION:A specimen is simultaneously reacted with an anti-procollagen C terminal antibody or antibody fragment binding solid phase substance and an enzyme labelled anti-procollagen C terminal antibody or antibody fragment in one process and the sandwich structure of the procollagen C terminal in the specimen is formed between the solid phase antibody or antibody fragment and the enzyme labelled antibody or antibody fragment. Next, the enzyme on the obtained sandwich is reacted with a substrate specific to said enzyme and enzymatic activity is measured. By this method, a procollagen C terminal amount can be rapidly quantified within about 1hr with high sensitivity.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method and kit for quantifying human type I procollagen C-terminal peptide (hereinafter abbreviated as procollagen C-terminal), and more specifically relates to an enzyme The present invention relates to a method for quantifying procollagen C-terminus using an immunopedometric method and a kit for quantifying the procollagen C terminus.

[Conventional technology]

In vivo protein collagen is synthesized within fibroblasts as its precursor procollagen, and is converted into specific peptidases during secretion. The terminal peptide is cleaved and converted into a complete protein molecule.

The main pathology of various fibrosis diseases such as liver fibrosis is excessive synthesis and excessive deposition of collagen in the tissue, and the side effects of collagen formation in the body are important for the diagnosis of persimmon fibrosis diseases such as liver fibrosis. 9 or can serve as an important guideline for treatment.

Even after release from procollagen, procollagen C terminus remains
It remains in the blood for a period of time. Therefore, measurement of the amount of procollagen C in the blood is important in clinical testing in terms of diagnosing fibrosis in tissues, and it has been desired to establish a simple and rapid quantitative method.

Today1, tissue fibrosis was diagnosed by tissue biopsy, measurement of hydroxyproline levels in urine and tissues, blood proline hydroxylase activity, and blood lysine oxidase activity, but the procedures were complicated and However, they often have shortcomings such as problems with accuracy and specificity.

Furthermore, a radioimmunoassay method using rabbit antiserum against procollagen 0-terminus has been recently developed; see Hisaha Koda, Liver, Vol. 25, No. 192, p. 203 (1984).
. In this quantitative method, procollagen C-terminus labeled with radioactivity in advance and the sample are simultaneously reacted with a competitive VC anti-procollagen C-terminus antibody, and anti-stone removal is performed for about 1 to 2 days.
The amount of procollagen C in the sample is quantified by reacting PL with anti-heron IgG antibody for about 1 day to create radioactivity in the immunoprecipitate or radioactivity remaining in the supernatant. .

[Problem that the invention seeks to solve]

The radioimmunoassay described above requires at least two days of operation time.In addition to the complexity of the operation, there are concerns about environmental contamination with radioactive substances and the impact on the human body, and because antiserum is used as an antibody against the target antigen. However, problems arose with the specificity and strength of its reactivity. In order to prepare tfc, its antiserum, and radiolabeled procollagen C terminus, a large amount of procollagen C wood ends had to be purified, which was also a major problem.

The present invention has been made to overcome the problems of the conventional quantitative methods described above, and its purpose is to provide a method for immunologically quantifying the amount of procollagen C-terminal in a sample such as a body fluid;
and to provide a foot turtle kit for use therewith.

[Means for solving problems]

To summarize the present invention, the first invention of the present invention is the preparation of a monoclonal antibody that specifically reacts with procollagen C wood tips.

The second invention of the present invention relates to a kit for quantitative determination of procollagen C bed edge as a use of the first invention, and includes the following (a), (
b), a procollagen C bed edge determination kit containing C9 as an essential component.

(a) Anti-glocollagen O-terminal antibody-bound solid phase substance (b) Enzyme m-fiber anti-procollagen 0-terminal antibody C'9
b) Substrate specific for The present invention is a method for quantifying procollagen C terminus in which an anti-procollagen C terminal antibody-bound solid phase substance and an enzyme-based anti-glocollagen C terminus antibody are simultaneously applied to the sample.

IiM antibodies! During preparation, the procollagen C bed end, which is used as an antigen, was prepared using the method of Hoffman et al. [HlP, Hoff-r 7
(H, P, Hoffman) Proceedings of the National Academy of Sciences x y
x U, 8. A, (Proc, Natl, A
cad, sci.

U, 8. ), Volume 75 m4304-4! S08
(1976)], it can be obtained from culture P solution of human fibroblasts. Fibroblasts are usually cultured in a serum-free medium for 24 hours, and the culture fluid is collected. The tongue fraction containing procollagen is precipitated with ammonium sulfate, and the procollagen C-terminus is purified by #I treatment and column chromatography as in the examples illustrated below. Separation of procollagen C terminus al! ! This makes it possible to produce specific antibodies. Antiserum against this purified antigen can be produced using an already established method! Niji can be prepared. That is, an experimental animal such as a rabbit can be immunized with several 1 ml of the sheathed glocollagen C husks, and after an appropriate period of time, the serum can be extracted from the animal.

The presence of specific antibodies in the antiserum can be tested by techniques such as #prime immunoassay (KL Engineering 8A J, Western Protein Ink assay).

However, in order to obtain specific antibodies using this method, the antigen used for immunization must be a single protein in terms of purity. In addition, in order to immunize large experimental animals such as rabbits and goats, large amounts of antigen must be administered. It is necessary to make 4. Therefore, it has been extremely difficult to obtain specific antibodies using conventional methods.

The present inventors developed the Köhler and Milstein method (K6hler, Mi) in order to solve various problems in preparing Eri anti-procollagen C-terminal antibodies using conventional methods.
latein, Nature (Natur-rθ), 25th
6, p. 476 (1975)). By using this method, there is no need to prepare a large amount of antigen,
Moreover, there is no need to make the antigen absolutely pure.

This is because the liver cells of an experimental animal immunized with human E type procollagen are fused with myeloma cells, and cloned cells that produce specific antibodies are selected under in vitro conditions. In particular, mice are often used as experimental animals. For example, a Ba1b10 mouse is intraperitoneally immunized with human type E procollagen extracted from a fibroblast culture solution, a booster immunization is performed 4 weeks later, and the mouse liver is removed 3 days later. Fuse lung cells and mouse myeloma cells with the action of polyethylene glycol, and then use the conventional method! Culture in a 1196-well plate. The supernatant of each culture solution was collected, and cells producing specific antibodies were selected using the ELI method, and further cloning was performed using the limiting dilution method to obtain anti-procollagen C monoclonal antibody producing cells (hybridoma). get.

These hybridomas secrete monoclonal antibodies into the culture medium.

Antibody fractions can be obtained from the supernatant by culturing No. hybridoma using a commercially available serum-free medium or the like by performing operations such as ammonium sulfate salting out. In addition, No. 1 hybridoma was cultured in the body of a syngeneic mouse, and the same procedure was performed using the animal's body fluid.
Antibody fractions can also be obtained.

These anti-P4 collagens. The terminal antibody is used in the quantitative kit that forms part of the present invention as an antibody-bound solid phase material immobilized on an insolubilized carrier or as an enzyme-labeled antibody as a conjugate with an enzyme.

The antibody used in the present invention may be either an antiserum or a monoclonal antibody, but monoclonal antibodies are particularly suitable. For example, two different anti-procollagen C-terminal monoclonal antibodies can be used as the antibodies used in (a) above.

In this case, both of them show tropism toward the glocollagen O hemis, but in particular, those showing tropism against distinct epitopes of the procollagen C husks are used. Furthermore, these antibodies are not limited to being used as immunoglobulins; F'(a) obtained by treating these antibodies with pepsin
It can also be used as a fragment such as b')2sFab''. When these fragments are used, non-specific adsorption can be prevented.

That is, the method for quantifying Procolla-def C-terminus according to the present invention includes: a) using anti-procollagen as the specimen; The terminal antibody or antibody fragment-bound solid phase is reacted with the enzyme-labeled anti-procollagen C wood end antibody or antibody fragment simultaneously in one step, and the glocollagen O-terminus in the sample is in phase with the antibody or antibody fragment and the enzyme-labeled antibody. or by forming a sandwich with an antibody fragment, b) reacting the enzyme on the resulting sandwich with a substrate specific to the enzyme, and measuring the enzyme activity.

As the insolubilizing carrier used in the antibody-binding solid phase material, those having the ability to bind antibodies can be used, such as polystyrene, polypropylene, polyvinyl chloride,
Alternatively, polycarbonate microplates, beads, sticks, test tubes, etc. may be used.

Further, the #-labeled antibody is produced by enzyme labeling by methods well known to those skilled in the art, such as the maleimide method and the pyridyl disulfide method. Examples of enzymes used for enzyme labeling include alkaline phosphatase, β-D-galactosidase, glucose oxidase, acetylcholinesterase, lactate dehydrogenase, glucoamylase, tyrosinase, horseradish peroxidase, lactoperoxidase, etc. Among them, alkaline phosphatase is preferred. .

The substrate may be any substrate specific to the enzyme used for enzyme labeling. For example, when the enzyme is alkaline phosphatase, p-nitrophenyl phosphate may be used as a substrate, and the concentration of p-nitrophenol produced by the enzymatic reaction may be measured as absorbance at a wavelength of 420 nm.

From the enzyme activity thus determined, the procollagen C-terminal cap can be calculated and quantified.

As described above, the present invention relates to procollagen in a specimen. We propose a standard method of fixing ψ in the measurement of the terminal end,
Liver'&i! It is used for diagnosing tissue fibrosis such as cancer.

〔Example〕

Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

In addition, FIG. 1 and FIG. 2 show the relationship between the absorbance at 420 nm (vertical axis) and Procortough 0 terminal m degree (ng/d, horizontal axis) for the first line used in the stationary method of the present invention. This is a graph shown in .

Example 1 (1) Isolation of procollagen 0-terminal antigen For extraction of procollagen 0-terminal, human fetal lung-derived fibroblasts (
MR-90) is cultured at 57°C in Dulbecco's modified Eagle (DMFtM) medium containing 10-tallow serum. When the cells have grown to an appropriate density, 50 mW/
1 Ascorbine 6N 20 mW/l L -7'
Replace with serum-free DMKM medium supplemented with Lorin,
After culturing for 24 hours, the culture supernatant was collected. Add 25mM ethylenediaminetetraacetate A (EDTA) as a proteolytic enzyme inhibitor to this culture supernatant.
mM N-ethylmaleimide (NKNJ, 1mM
Phenylmethylhonyl fluoride (PMBF),
After adding 1 mM p-aminobenzamide hydrochloric acid, the protein was salted out and precipitated with ammonium sulfate (176 mf7 ml). The precipitate was treated with 2M urea, 2.5M
MF! α1M Tris-HCl containing DT
H7,8) DI the dissolved K! fAIli Sephacel column and the same buffer 150- and [L2MNaO
Elution was performed using a gradient of the same buffer solution 15011d containing T, and the eluted peaks were collected at 05M at 19°C. We confirmed that the human E type procollagen thus obtained and S-produced Bacteria-Yumei collagenase (manufactured by Sigma,
America J was mixed at a ratio of 50:1 and incubated at 4°C for 20 hours to decompose only the collagen portion in the human ear procollagen molecule. Procollagen from this breakdown product. Final purification of the ends was performed using Bio-Gel A-
1.5 m (/(Iorad Co., Ltd. - America i column chromatography), and a peak corresponding to a molecular weight of about 100,000 was collected as N procollagen 0 Song end fraction.

(2) Procollagen. Preparation of antiserum against terminal purified pulled collagen 0 terminal antigen 15mf in saline α
5sdK was dissolved, and an equal amount of 70% India adjuvant was added thereto, emulsified, and then subcutaneously injected into rabbits. 2
Weekly tl! ! The same amount of antigen was emulsified with incomplete 70 India adjuvant and subcutaneously injected 4 times to final immunization! After 10 days, the whole blood was collected, left at room temperature for 60 minutes, and then centrifuged to remove anti-globin collagen.
Antiserum containing terminal antibodies was obtained. The specificity and titer of the antibody can be determined by one skilled in the art using methods well known in the art, namely B.
It was confirmed by the L Technique 88 method and the Western blotting method.

13+ Preparation of monoclonal antibody against procollagen C-terminus Purified human type I glycollage 750 μm was dissolved in saline α1.
- and emulsified by adding Totsutsukuda's complete 70 indium adjuvant, and injected intraperitoneally into Bal'b/c mice. Four weeks later, 50 μ2 of the antigen alone was injected intraperitoneally into the mice. Three days later, from the lungs removed from the mouse,
Liver cells were obtained and mouse myeloma cells (8P210
) and cells at a ratio of 10:1 and left for 1 minute in the presence of 50% polyethylene glycol and 20% dimethyl sulfoxide to perform cell fusion. Serum-free DME: M
After diluting by adding t@di, remove the supernatant by centrifugation, suspend the cells in DMBM medium containing 10 times tallow serum, and prepare 2 x 10'm cells in a 96-well microtiter plate. It was established as a public corporation. then 1
~ Replace half of the medium with HAT medium every 5 days,
After ~20 days, collect the culture supernatant from the well in which the fused cells (hybridoma) have grown, and check for the presence or absence of antibody production using FfL.
Using the Engineering 8A method, etc., five hybridoma strains producing antibodies against the C-terminus of procollagen were selected.

These hybridomas were cloned twice using the limiting dilution method, and PO5-5 and PO3- were cloned as the hybridoma clones that produced the highest antibody titer.
Acquired 2 shares of 7.

It was confirmed by Western blotting that the monoclonal antibodies produced by these hybridomas specifically reacted with procollagen C wood ends. To obtain large amounts of these monoclonal antibodies, approximately 2 x 10 ml of Ba1b10 mice were intraperitoneally administered. Ejaculate the individual's Ibridoma,
An ascites M field was created, and after 10 days, ascites was collected, and an anti-procollagen O-end monoclonal antibody was obtained.

(4) Binding of antibody to unsaponifiable carrier The antiserum obtained in (2) was subjected to ammonium sulfate salting out according to a conventional method.
Anti-procollagen C Songtian antibody was detected by FiAW cellulose column chromatography. The obtained antibody was prepared using the method of Ishikawa et al. [E.

J Ishikawa a Scandinavian Journal of Immunology (5can, y
- Engineering mmunoL), p. 8, p. 43 (1978
)) was treated with ↓9pepsin, and antibody fragment P (ab
') Must have.

This antibody fragment was dissolved in 10mM sodium carbonate AH.
The antibody fragments were dissolved in a buffer solution (pHa5) to a concentration of 100 fit/-, and a polystyrene ball (1 * manufactured by Suikaji Co., Ltd., particle size: 4 x 2) was immersed in this solution, and the antibody fragments were incubated at 4°C for 18 hours. Fixed to the ball. This is 1%
Bovine serum albumin, 11196) laNm was stored in Tris-buffered saline (TB8) at 4°C.

(5) Alkaline phosphatase labeled anti-procollagen C
Preparation of bed end monoclonal antibody (3) From ascites containing anti-procollagen 0 Song end monoclonal antibody (PO8-73), anti-procollagen C bed end monoclonal antibody (PO8-73
was purified to obtain a monoclonal antibody fragment (Fab') according to the method of Ishikawa et al.

Monoclonal antibody thus obtained (PO8-7)
Using N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (manufactured by Dibenkencal Co., Ltd., Sokuto), the fragment was prepared using the method of Ishikawa et al. (Enzyme immunoassay, 2nd edition, p. 92, 1982). Year 12
4C (Igaku Shoin, published on May 15, 2013) was then bound to alkaline phosphatase (Boehringer, West Germany).

(6) Collect 50 μt of the sample containing procollagen C terminus and side leg procollagen 0 terminus into a test tube.
11.1% of the enzyme-recognizing antibody fragment prepared in 5)
A solution diluted to 1 f/- with TBS containing bovine serum albumin was immediately added to [L6-] and incubated for 30 minutes at room temperature. The polystyrene balls were then separated and washed four times with 2 gLt of TBS containing 01% Tween 20 (Ushii Chemical Co., Ltd., Bettei), and p-nitrophenyl phosphate (Wako Pure Chemical Industries, Ltd.) as a substrate. Table 1. Add CL6d to a solution of Osaka J dissolved in α05M Tris-HCl buffer (pH 9, 7, 2mM MgO, proprietary) to 10mM, and incubate at room temperature for 15 minutes. was added to stop the reaction, and the absorbance at a wavelength of 420 nm was measured.A calibration curve obtained from the measurement results is shown in FIG.

Example 2 (1) Procollagen. Isolation of terminal mass was carried out according to Example 1.

(2) Preparation of anti-Glocolaken C-terminal monoclonal antibody The procedure of Example 1 was followed.

(3) Binding of anti-procollagen C-terminal monoclonal antibody to insolubilized carrier The same procedure as in Example 1 (4) was carried out. That is, monoclonal antibody (PO5-53) was prepared from ascites containing monoclonal antibody (PO5-5) by fpI, and Eri antibody fragment F(ab)s was obtained by pepsin treatment. This was added to 10 mM sodium carbonate buffer (pHa5). 10
A polystyrene ball was manufactured by incubating a polystyrene ball in this solution at 4° C. for 18 hours.

(4) Preparation of enzyme-labeled anti-procollagen 0-terminal monoclonal antibody The procedure of Example 1 was followed.

(5) Measurement of procollagen C-terminus The measurement was carried out according to Example 1. The calibration curve obtained from the measurement results is shown in Figure 2.

〔Effect of the invention〕

As explained above in Part 1, the present invention provides procollagen. It became possible to quickly place the foot on the thigh with a high sensitivity of 5 of/- in one hour.

[Brief explanation of drawings]

Figures 1 and 2 show test 1i1 used in the foot placement method of the present invention.
11i! , absorbance at 420 nm and glocollagen O
This is a graph showing the relationship with Emperor Kobata.

Claims (1)

[Claims] 1. Human type I procollagen. A monoclonal antibody characterized by being specific to a terminal peptide. 2. The following (a), (b) and (c) (a) Anti-human type I procollagen C-terminal peptide antibody-bound solid phase (b) Enzyme-labeled anti-human type I procollagen C-terminal peptide antibody (c) A kit for quantifying human type I procollagen C-terminal peptide, which is characterized by containing an enzyme (b)-specific substrate as an essential component. 3. The quantitative kit according to claim 2, wherein the antibody is a monoclonal antibody specific for human type I procollagen C-terminal peptide. 4. When quantifying human type I procollagen C-terminal peptide in a sample using enzyme immunoassay, anti-human type I procollagen C-terminal peptide antibody-bound solid phase and enzyme-labeled anti-human type I procollagen are added to the sample. A method for quantifying human type I procollagen C-terminal peptide, which is characterized by simultaneously using a C-terminal peptide antibody. 5. The quantitative method according to claim 4, wherein the antibody is a monoclonal antibody specific for human type I procollagen C-terminal peptide.