JPH04324357A - Measurement method of collagen - Google Patents

Abstract

PURPOSE:To inspect measurement of collagen in an object at a high sensitivity with a high reproducibility by performing a heating treatment thereof at a specified temperature in the presence of an anion surfactant. CONSTITUTION:An object to be inspected undergeoes a heating treatment at 39-60 deg.C, preferably at 40-50 deg.C for about 0.5 hour or more, preferably for about 1-5 hour with the presence of an anion surfactant such as sodium lauryl sulfate (SDS) or sodium lauryl benzene sulfonate (LBS) with the optimum concentration of 0.005-0.5% or preferably 0.04-0.15% and then, a part of the object treated is added to an antibody solidifying well. Thereafter, a measurement is performed by a normal sandwiching method. Also available is a method of thermal treatment at 39-60 deg.C which is carried out during an immunological reaction. This enables measurement of collagen with high reproducibility while allowing rise in a collagen measured value in blood. The measurement of IV type collagen in the blood facilitates the diagnosis of a liver disease, especially liver fibrosis.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a method for measuring collagen,
In particular, it relates to improvements in methods for measuring collagen using immune reactions.

0002

2. Description of the Related Art Measuring various collagen-related substances present in blood is known to be effective in diagnosing liver diseases. Furthermore, recently, its clinical significance and usefulness as a marker of liver fibrosis have been pointed out. Among these, blood human type III collagen N-terminal peptide, human type IV collagen N-terminal peptide 7-S domain, and human type IV collagen C-terminal peptide NC1 domain were measured by radioimmunoassay using each polyclonal antibody. [Rohde et al., European Journal of Clinical Investigation (Eur. J. Clin. Invest.
．． ), 9, 451-459, 1979, Hogemann
Gemann et al., Clinical Chimica Acta (Cl
in. Chim. Acta. ), 144, 1-10, 1
984, Schuppan et al., Journal of Clinical Investigation (J.
Clin. Invest. ), 78, 244-248,
1986)]. Regarding blood human type IV collagen, Japanese Patent Application Laid-open Nos. 63-63971 and 63-78067
No. 63-246396 and JP-A-2-1553
Among these, JP-A-63-78067 and JP-A-63-246396 mainly describe the characteristics of the antibodies used in the present invention. Unexamined Japanese Patent Publication 1986
-63971 and JP-A-2-1553 are about measurement methods, the former is about enzyme immunoassay using both monoclonal and polyclonal antibodies, and the latter is about enzyme immunoassay using monoclonal antibodies. It is listed. As described above, measurement of type IV collagen has recently attracted attention.

0003

[Problems to be Solved by the Invention] However, when performing measurements using the monoclonal antibodies described in JP-A-63-78067 and JP-A-63-246396, and the method described in JP-A-2-1553, When measured using conventional enzyme-linked immunosorbent assays, their reproducibility is poor. As a solution to this problem, the present applicant and others have filed a patent application for a method of heat treatment (processing the specimen at 39-60°C in advance or heat-treating it during the immune reaction). The development of a reproducible measurement method is expected. The present invention was made in view of the problems of the prior art, and its purpose is to achieve good reproducibility,
Moreover, it is an object of the present invention to provide a method that can measure collagen in a specimen with high sensitivity.

0004

[Means for Solving the Problem] As a result of intensive studies by the present inventors to achieve the above object, it was possible to reproduce the problem by heat-treating the specimen in the presence of an anionic surfactant such as sodium lauryl sulfate (SDS). The present inventors have discovered that it is possible to measure human type IV collagen, which has a unique characteristic, and have completed the present invention.

That is, the present invention provides a method for measuring collagen in a specimen by a sandwich method using a monoclonal antibody.
The present invention provides a method for measuring collagen, which is characterized by heat treatment at 0°C.

Specifically, the method of the present invention includes (1
) In the presence of an anionic surfactant such as SDS, a sample is preheated at 39 to 60°C, a portion of the treated sample is added to an antibody-immobilized well, and then measured using a normal EIA method; 2) There is a method in which the immune reaction is carried out at 39 to 60°C in the presence of an anionic surfactant such as SDS.
A method of performing an immune reaction in one step, that is, an EIA method in which an immune reaction is performed in one step of adding an enzyme-labeled antibody solution and a sample to an antibody-immobilized well (However, in this case, the surfactant deactivates the enzyme-labeled antibody. Therefore, it is desirable to make the concentration of the surfactant (hereinafter referred to as weight %) as low as possible, for example, about 0.005 to 0.03%), and a two-step method, namely , an EIA method in which an immunoreaction is performed in two steps: first, a sample is added to an antibody-immobilized well and reacted for a certain period of time, then after washing, an enzyme-labeled antibody is added and reacted (in this case, the first immunoreaction is performed using an anionic surfactant). carried out at a temperature of 39 to 60 °C in the presence of
The second reaction is carried out in a conventional manner).

[0007] In the method of the present invention, the reaction time for heat-treating the specimen in the presence of an anionic surfactant is 0.5 hours or more, preferably 1 to 5 hours. In addition, buffers to be used are preferably near neutral and have a high buffering capacity for immunoreactions, such as Good's buffers such as Hepes buffer, phosphate buffers, and the like. For enzymatic reactions, select a buffer suitable for the enzyme used. For example, in the case of peroxidase, the substrate is A
In the case of BTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], citrate buffer is suitable.Also, the pH should be 5 to 9, preferably medium during immune reaction. The optimum pH of the enzyme used during enzyme reaction
Preferably nearby.

Examples of anionic surfactants include sodium lauryl sulfate (SDS), sodium laurylbenzenesulfonate (LBS), lithium lauryl sulfate (LDS), etc., and their optimum concentration is SD
S and LBS are 0.005-0.5%, preferably
It is 0.04-0.15%. The temperature of heat treatment is 39-6
The temperature range is 0°C, particularly preferably 40 to 50°C.

[0009] The monoclonal antibodies used include embodiments in which IgG itself is used, or the specific binding portion F(ab')2 or Fab' itself of these antibodies is used. In addition, "collagen" in the present invention
is preferably human type IV collagen. In addition, "type IV collagen" in this specification refers to human basement membrane-related collagen, and also includes a vascular basement membrane collagen complex, which is a collagen complex.

0010

[Examples] Next, the present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to these examples. Preparation of reagent 1 [Preparation of biotinylated antibody (biotin-Fab')] (a) Preparation of Fab': Monoclonal antibody against type IV collagen (see JP-A-63-246396, (
JK199 (provided by Shiseido Co., Ltd., 10 mg/2 ml) was dialyzed against 0.1 M acetate buffer (pH 4.2), and pepsin solution (Boehringer Mannheim, 20 mg/2 ml; 0.1 M acetic acid) was added to the solution. A buffer solution (50 microliters) was added, and the mixture was reacted at 37°C for 60 minutes. Centrifuge the reaction solution and transfer the supernatant to Sephacryl S.
-200 column (diameter 1.5 cm x 50 cm, eluent: 0.1 M phosphate buffer pH 6.0) and F(a
b') Two fractions were obtained. Dithiothreitol was added to this F(ab')2 fraction at a concentration of 20 mmol/liter, and the mixture was reacted at 37°C for 1 hour. The reaction solution was centrifuged, and the supernatant was transferred to a Sephacryl S-200 column (diameter 1.
5 cm x 50 cm, eluent: 0.1 M phosphate buffer (pH 6.0) to obtain a Fab' fraction.

(b) Biotinylated antibody (biotin-Fab
) Preparation: Analytical Biochemistry (A
nal. Biochem. ), 149, 529-536
(1985), 3-(N-maleimidobutyryl)-biocytin (MBB) was synthesized and Fab'
(1.66mg/1.3ml) to MBB solution (
0.5mg/ml; 0.1M phosphate buffer, p
H6.0, 0.5 ml) was added, and the mixture was allowed to react at room temperature for 2 hours. The reaction solution was centrifuged, and the supernatant was applied to a Sephadex G-25 column (1.5 cm in diameter x 30 cm, eluent: 0.1 M phosphate buffer pH 6.0) to obtain biotin-Fab'.

Preparation of reagent 2 [Preparation of solid phase] (a) Preparation of biotin-BSA: BSA (manufactured by Oriental, F-V
, 30 mg) in 0.1 M phosphate buffer (pH 7.0, 0
．． NHS-SS-biotin solution (manufactured by Pierce, 16.8 mg/ml; 0.1 M phosphate buffer, pH 7.0, 1 ml) was added thereto, and the mixture was reacted at 30°C for 1 hour. I let it happen. The reaction solution was centrifuged, and the supernatant was transferred to a Sephadex G-25 column (diameter 1.
5cm x 30cm, eluent: 50mM carbonate buffer p
H9.6) to obtain a biotin-BSA fraction.

(b) Preparation of solid phase: Biotin-BSA obtained in the above step (a) was dissolved in 50 mM carbonate buffer (pH 9.6).
The biotin-BSA concentration was adjusted to 10 μg/ml. This solution (150 microliters) was dispensed into the wells of a plate (manufactured by Nunc, Immunoplate I, 96 wells) and reacted at room temperature for 1 hour to immobilize it. After washing the wells several times with physiological saline, a block solution (0.5% BSA, 300 microliters) was dispensed into the wells and allowed to stand at room temperature for 1 hour. Then, after washing the wells several times with physiological saline, an avidin solution (manufactured by Vector, Avidin D, 50 μg/ml; 0.1 M phosphate buffer, pH 7.0, 150 microliters) was dispensed into the wells. The reaction was allowed to proceed at room temperature for 2 hours. After washing the wells several times with physiological saline, biotin-Fab' solution prepared in reagent preparation 1 [1.6 μg/ml (10
mM phosphate buffer, pH 7.0, 0.15M NaCl
, 0.5% BSA), 150 microliters] and reacted overnight (18 to 22 hours) at room temperature to obtain a solid phase.

Preparation of reagent 3 [Enzyme-labeled antibody (POD-F
ab') Preparation] (a) Preparation of Fab': Monoclonal antibody against type IV collagen (see JP-A No. 63-246396, (
Fab' was obtained using reagent JK132 (provided by Shiseido Co., Ltd.) and following the same procedure as in Reagent Preparation 1(a) (however, the pepsin digestion time was 30 minutes). (b) P.O.D.
Preparation of (peroxidase)-maleimide: POD (manufactured by Boehringer Mannheim, 6 mg) was dissolved in 0.1 M phosphate buffer (pH 7.0, 0.8 ml), and N-(γ-maleimidobutyryl Oxy)succinimide (manufactured by Dojindo Chemical Co., Ltd.) solution (80 mg/ml N,N-dimethylformamide, 50 microliters) was added, and the mixture was reacted at 30°C for 1 hour. The reaction solution was centrifuged, and the supernatant was applied to a Sephadex G-25 column (diameter 1.5 cm x 30 cm, eluent: 0.1 M phosphate buffer pH 6.0) to obtain a POD-maleimide fraction. (d) Preparation of POD-Fab': POD-maleimide (1 mg) was added to Fab' (1 mg) and reacted in a cold place for 20 hours. The reaction solution was centrifuged, and the supernatant was transferred to a Sephacryl S-200 column (1.5 cm in diameter x 50
cm, eluate: 0.1M phosphate buffer pH 6.5) to obtain a POD-Fab' fraction.

Example 1 [Quantification of type IV collagen: SD
Effect of S] Type IV collagen (manufactured by Sigma) was dissolved in human serum that does not contain type IV collagen, and the collagen concentration was 0 to 100.
A sample of 0 ng/ml was prepared. Preparation of reagent 2
Put 20 microliters of the sample into the well prepared in
SDS addition buffer [10mM HEPES, pH 7.0
, 0.1% BSA, 0.15M NaCl, SDS concentration 0-5%; 100 microliters] and heated at 45°C for 2 hours. After the reaction, the reaction solution was removed by suction, a washing solution (physiological saline) was added, and the washing solution was removed by suction. This washing operation was repeated four more times. After washing, reagent preparation 3
Enzyme-labeled antibody solution [POD-Fab' (approximately 20
IU) in 10mM HEPES, pH 7.0, 0.1%
BSA, 0.15 M NaCl) was added to bring the total volume to 10 ml, 150 microliters] and heated at 37° C. for 1 hour. After the reaction, wash the wells in the same manner as above, add substrate solution [ABTS 1 mg/ml, 0.01% hydrogen peroxide, 0.1M citrate buffer pH]
4.2; 150 microliters] and incubate at 37°C.
After reacting for 0 minutes, the absorbance of this liquid was measured at 415 nm (main wavelength) and 650 nm (secondary wavelength). The results are shown in Figures 1 and 2. SDS concentration 0.005-0.5%
, preferably at 0.04 to 0.15%.

Example 2 [Quantification of type IV collagen: LB
Effect of S] Type IV collagen (manufactured by Sigma) was dissolved in human serum that does not contain type IV collagen, and the collagen concentration was 0 to 100.
A sample of 0 ng/ml was prepared. Hereinafter, the same method as in Example 1 was used. However, LBS was added instead of SDS. The concentration added was 0-0.5%. The results are shown in Figure 3. By adding LBS, the measured value of collagen in the serum increased, and the effect was recognized from 0.005%, and even 0.5% was effective.

Example 3 [Quantification of type IV collagen: LD
Effect of S] Type IV collagen (manufactured by Sigma) was dissolved in human serum that does not contain type IV collagen, and the collagen concentration was 0 to 100.
A sample of 0 ng/ml was prepared. Hereinafter, the same method as in Example 1 was used. However, instead of SDS, 0.1% LDS was added and compared with that without the addition.

The results are shown in Table 1. Table 1 shows the collagen values of the specimens with and without LDS addition (0.1%) at the same time as the heat treatment.

[Table 1] As is clear from Table 1, the addition of LDS clearly increased the measured value and had an effect of addition. Above, Example 1
Methods 3 to 3 are methods in which an anionic surfactant is added during the immune reaction and heat treatment is performed at the same time, and the immune reaction is performed in two steps.

Example 4 [Quantification of type IV collagen: Examination of the effect of adding SDS by pre-processing the specimen] Type IV collagen (manufactured by Sigma) was dissolved in human serum that does not contain type IV collagen, and the collagen concentration was 0. ~1000ng/
A milliliter sample was prepared. Prepare the sample from 39 to 60
After applying a temperature of 0.degree. C. and simultaneously treating with an anionic surfactant, a portion of the treated sample was added to the antibody-immobilized well, and a method was then investigated in which measurement was performed using a conventional EIA method. The method is to put 40 microliters of sample and SDS addition buffer (
After adding 200 microliters of SDS (with an SDS concentration of 0.1%) and reacting at 45°C for 1.5 hours, 120 microliters of this solution was placed in the well prepared in reagent preparation 2.
It was heated at 37°C for 2 hours. After the reaction, the reaction solution was removed by suction, a washing solution (physiological saline) was added, and the washing solution was removed by suction. Thereafter, the same operations as in Example 1 were performed. As a control,
A similar method without the addition of SDS and a method in which heat treatment was performed during the immune reaction without the addition of SDS were performed.

The results are shown in Table 2. Table 2 shows that S
When DS was added and subjected to immune reaction after heat treatment, and S
The collagen values are shown when an immune reaction is performed after heat treatment without adding DS.

[Table 2] Even with the method of pre-treating the sample, addition of SDS clearly increased the measured value and had an effect of addition. Next, we investigated a two-step method for carrying out the immune reaction by adding an anionic surfactant during the immune reaction and simultaneously performing heat treatment.

Example 5 [Quantification of Type IV Collagen: Effect of Temperature] Type IV collagen (manufactured by Sigma) was dissolved in human serum that does not contain type IV collagen, and the collagen concentration was 0 to 100.
A sample of 0 ng/ml was prepared. Hereinafter, the same method as in Example 1 was used. However, the SDS concentration is 0.
1%, and the temperature of the first reaction was 37-60°C. The results are shown in Figures 4 and 5. For those without SDS additives, 40~
Purified collagen reacted sufficiently at 56°C, but although the reactivity of collagen in serum increased slightly, it was insufficient. The one with SDS added had some effect at 37°C, but by raising the temperature to 40 to 56°C, the reactivity of collagen in serum in particular reached equilibrium.

Example 6 [Quantification of Type IV Collagen: Calibration Curve] Type IV collagen (manufactured by Sciuma) was dissolved in human serum that does not contain type IV collagen, and the collagen concentration was 0 to 100.
A sample of 0 ng/ml was prepared. Hereinafter, the same method as in Example 1 was used. However, the SDS concentration is 0.
1%, and the temperature of the first reaction was 45°C. The results are shown in FIG. At 1000ng/ml, the absorbance is approximately 1.
It was 00.

Example 7 [Quantification of Type IV Collagen: Confirmation of Reproducibility] Using 30 serum samples, measurements were performed on the first and second days to examine reproducibility. The results are shown in FIG. The reproducibility on the 1st and 2nd day (shown by the correlation of 30 cases) is 30 cases, and the correlation coefficient r =
0.994, the regression equation y=0.89X+20.5,
It was confirmed that this method is reproducible.

Example 8 [Quantification of Type IV Collagen: Blood Concentration Distribution by Disease] Blood collagen was measured in 14 normal cases, 44 cases of chronic hepatitis, and 34 cases of liver cirrhosis, and the distribution by disease was investigated. The method was the same as in Example 6. Also, for comparison, heat treatment (45
We also investigated the method using only ℃). The results are shown in Figures 8 and 9.
Shown below. In both methods, the level of type IV collagen increased with the progression of liver fibrosis such as liver cirrhosis. However, S.D.
The addition of S better reflected the progress of fibrosis such as liver cirrhosis. As shown in Figure 8, by measuring blood type IV collagen using the method according to the present invention, reproducible values can be obtained, which is useful for diagnosing and predicting liver diseases, especially liver fibrosis. Therefore, the present invention has significant utility.

[0025]

Effects of the Invention According to the method for measuring collagen according to the present invention, collagen can be measured reproducibly by heat-treating the specimen at 39 to 60°C in the presence of an anionic surfactant such as SDS. and can increase the measured value of collagen in the blood. Furthermore, by measuring type IV collagen in blood using this measurement method, diagnosis of liver diseases, particularly liver fibrosis, can be facilitated.

[Brief explanation of drawings]

FIG. 1 is a graph showing the reactivity of antibodies and collagen with changes in SDS concentration (0 to 2%) when SDS was added to the specimen at the same time as heat treatment.

FIG. 2 is a graph showing the reactivity of antibodies and collagen with changes in SDS concentration (0 to 0.1%) when SDS was added to the sample at the same time as heat treatment.

FIG. 3 is a graph showing the reactivity of antibodies and collagen with changes in LBS concentration (0 to 0.5%) when LBS was added to the specimen at the same time as heat treatment.

[Figure 4] When the specimen is heat-treated without SDS addition,
It is a graph showing the reactivity of antibodies and collagen with changes in temperature.

FIG. 5 is a graph showing the reactivity of antibodies and collagen with changes in temperature when a sample is heat-treated with the addition of SDS (0.1%).

FIG. 6 is a calibration curve diagram of collagen.

FIG. 7 is a correlation diagram of 30 specimens with day-to-day reproducibility in collagen measurement.

FIG. 8 is a diagram showing the relationship between collagen measurements and diseases when SDS (0.1%) was added at the same time as heat treatment.

FIG. 9 is a diagram showing the relationship between collagen measurement values and diseases after heat treatment (45° C.).

Claims (2)

[Claims] 1. A method for measuring collagen in a specimen by a sandwich method using a monoclonal antibody, which method comprises heating at 39 to 60°C in the presence of an anionic surfactant. . 2. The measuring method according to claim 1, wherein the anionic surfactant is sodium lauryl sulfate, sodium laurylbenzenesulfonate or lithium lauryl sulfate.