JPH03215747A - Enzyme immunological measurement for elastase 1 and reagent for measurement - Google Patents

Abstract

PURPOSE:To easily measure the elastase 1 in a specimen to be inspected by conjugating this elastase 1 with a solid phase, separating the elastase 1, measuring the activity of a labeling enzyme, and determining the elastase 1 in the specimen to be inspected by a standard curve obtd. by using the standard elastase 1 of a known concn. CONSTITUTION:Monoclonal antibodies which recognize two kinds of different antigenic determinants to the elastase 1 are used. One thereof is conjugated with the solid phase and the other is made to recognize the enzyme and to conjugate the elastase 1 in the specimen to be inspected with the solid phase; thereafter, the activity of the labeling enzyme on the solid phase is measured. The quantity of the elastase 1 in the specimen to be inspected is then determined by the standard curve formed by making the similar operation with the standard elastase 1 of the known concn. Peroxidase, etc., which are usually used as the labeling enzyme are used as the labeling enzyme of the labeled antibodies. Polystyrene beads, etc., are usable as the solid phase carrier to be used for the solid-phased anti-elastase 1 monoclonal antibodies.

Description

[Detailed description of the invention] ^. INDUSTRIAL APPLICATION FIELD The present invention relates to an enzyme immunoassay method and reagent for measuring pancreatic exocrine proteinase elastase 1 present in body fluids, which is useful for diagnosing pancreatic diseases.

B. Conventional technology There have been attempts for a long time to measure enzymes released into the blood from the pancreas and use them to diagnose pancreatic diseases, and the measurement of serum and urinary amylase, serum lipase, etc. is said to have high diagnostic significance. ing. However, these enzymes are also produced by organs other than the pancreas, and are present even in cases other than pancreatic disease, so they lack specificity as a screening test for pancreatic disease, so the development of other biochemical diagnostic methods is desired. Ta.

Elastase is the only proteolytic enzyme that can degrade connective tissue elastin, and there are two types (elastase), 2) that differ physicochemically, enzymatically, and immunologically (Toshiro Oyama: Medical Science). History 103:697 (1
977>).

Activity measurement methods using elastin or synthetic substrates have been attempted to measure elastase I. However, measurement in blood has been considered difficult because the activity is inhibited by protease inhibitors present in large amounts. For these reasons, an attempt was made to measure elastase 1 by an immunological method, and a radioimmunological assay (RfA method) was developed by Oyama et al. (Japanese Patent Publication No. 59-251113).

Elastase 1 in the blood is a protease inhibitor 1
Although it has no immunological activity in the form of a complex bound to α2-macroglobulin, it has immunological activity in the form of a complex bound to α1-antitryprine. In immunological methods using labeled antigens, it has been difficult to accurately measure blood elastase I because some or most of the antigenicity of the labeled antigen disappears due to binding to protease inhibitors in the blood. However, R
In the IA method, the labeled antigen is synthesized with D, which is a synthetic inhibitor.
The labeled antigen was treated with FP (diisobrobyl fluorophosphate) to prevent it from binding to protease inhibitors in the blood. DFP treatment does not change the antigenic activity of elastase I, making immunoassay possible.

However, in the EIA system using a polyclonal antibody, it was found that the DFP-treated elastase I was different in antigenicity from the original elastase I, and therefore did not show an accurate value of elastase I. Furthermore, since the RIA method uses radioactive substances, there are safety issues, and since polyclonal antibodies are used, there are differences in binding properties depending on the antibody manufacturing port, so manufacturing control is required. There was a problem with the surface. Therefore, the development of another measurement method has been desired. Later, in order to solve the latter problem among the shortcomings of RIA, there was a report on the RIA method in which polyclonal antibodies were changed to monoclonal antibodies.
-73152) It does not solve the fundamental drawback of the RIA method (the use of radioactive substances).

C. Problems to be Solved by the Invention As mentioned above, the conventional method for measuring elastase I uses a radioactive substance and has problems in terms of safety. Furthermore, differences in binding properties occur depending on the manufacturing method, and there are also problems in terms of manufacturing control for supplying stable reagents.

The purpose of the present invention is to provide a measurement method that does not use radioactive substances and can be easily performed in general clinical laboratories without special equipment, and a stable measurement reagent that does not cause differences in binding properties depending on the manufacturing loft. . Furthermore, DFP is available as a standard product.
Alternatively, a complex of elastase l and α1-antitrypsin or a complex of proelastase l and α1-antitrypsin can be used instead of elastase l converted into PMSF (7enylmethylsulfonyl fluoride), and the blood concentration can be adjusted. The goal is to ensure that it is accurately reflected.

D. Means and Effects for Solving the Problem This discovery was made using two monoclonal antibodies against the complex of elastase 1 and α,-antitribucin or the complex of proelastase 1 and α,1-antitribucin in body fluids. The present invention provides a method for enzyme immunoassay of elastase 1 and a reagent for the measurement. The present inventors have obtained two monoclonal antibodies that recognize the complex of elastase 1 and α1-antitrib/n and the complex of proelastase 1 and α1-antitrib/n, which have excellent sensitivity and high precision. We succeeded in establishing a method for measuring elastase I in body fluids and at the same time ensuring a stable supply of antibodies.

In the measurement of elastase 1 according to the present invention, when DFP-treated or PMSF-treated elastase 1 is used, almost no immune activity is shown, but elastase 1 and α1-
complex with antitrib7 or proelastase l
When a complex of α1-antitribun and α1-antitribune was used, both complexes showed comparable immunoreactivity. This is DF
This shows that the antigenicity of elastase 1 is changed by P treatment and PMS F treatment.

On the other hand, it is said that elastase I exists in the blood as a complex of elastase I and α2-macroglobulin or α1-antitribunin, but it has also been confirmed that proelastase I exists in pancreatic juice. Furthermore, in the RrA method using polyclonal antibodies, elastase 1 and α1-
The reactivity with the complex with antitribucin or the complex with proelastase I and α1-antitrypun was low.The reactivity with the serum sample was similarly low. Therefore, it is thought that there is a problem in using DFP-treated or PMS F-treated elastase, which changes the antigen immunoactivity, as the standard elastase. This paper proposes the validity of using a complex between proelastase I and α1-antitrypun, whose existence has been confirmed by the present inventors.

The measurement of elastase l according to the present invention involves elastase l
Using a monoclonal antibody that targets two different antigenic determinants for U2, one bound to a solid phase and the other labeled to the enzyme, elastase l in the sample is bound to the solid phase, and then The activity of the labeled enzyme on the solid phase is measured, and the amount of elastase in the sample is determined using a standard curve prepared in the same manner using a standard elastase with a known concentration instead of the sample. be.

A monoclonal antibody against elastase 1 is prepared by immunizing a mammal, such as a mouse, with elastase 1, and then producing antibody-producing cells extracted from the mouse. For example, it can be obtained by the Kohler-Mllstein cell fusion method, in which lung cells are fused with myeloma cells, and cells that produce the desired monoclonal antibody are cloned from the resulting fused cells (hybridoma).

Labeling enzymes for labeled antibodies include peroxidase and alkaline phosphatase. Commonly used labeling enzymes such as β-galactonase can be used. Labeling of antibodies with these enzymes can be carried out by known methods such as the glutaraldehyde method and the periodate oxidation method.

As the solid phase carrier used for the immobilized anti-elastase 1 monoclonal antibody, known carriers such as polystyrene beads can be used.

The measurement reagent kit used in the present invention includes: 1) Standard elastase 1 consisting of a complex of elastase 1 and α1-antitribucin or a complex of proelastase 1 and α1-antitribucin 2) Solid-phase anti-elastase 1 monoclonal antibody 3) Contains an enzyme-labeled anti-elastase 1 monoclonal antibody. Each immobilized or enzyme-labeled anti-elastase 1 monoclonal antibody recognizes a different antigenic determinant.

E. Example (+) Preparation of anti-elastase I monoclonal antibody Purified elastase IfI solution o. B
It was administered intraperitoneally to ALB/C mice at 3 week intervals. 2
After ~3 months, 0.1 ml of elastase 1 solution (containing 70 μg of elastase 1) was injected from the tail vein. 3EI
Afterwards, the lungs were removed, and 3 x 10' of tile cells and 3 x 10 mouse myeloma cells (×63^gL 6.5.3 strains) were collected.
Cell fusion was performed in the presence of 50% PEG4000i. 0.0 ml each in a 96-well culture dish. Dispense 1ml each,
After culturing in HAT medium for 24 hours, antibodies in the culture soil were assayed. Antibody-producing hybridomas were cloned by limiting dilution method to obtain antibody-producing strains. The obtained hybridoma 1xlo'+11 was pre-injected with 1.0 ml of 2J, 10.14-tetramethylbentadecane.
Administered into the peritoneal cavity of B/C mice, and 14 days later, 5 ml of ascites was administered.
An anti-elastase 1 monoclonal antibody was obtained from each mouse. The two antibodies obtained as above react with the complex of human esterase 1 and α1-antitrypsin or the complex of proelastase 1 and α■-antitrypsin, and are suitable for enzyme immunoassay. It was possible.

(2) Labeling of anti-elastase l antibody with bellookindase 40 mg of bellookindase was dissolved in a 1.25% glutaraldehyde solution, and gel filtration was performed using Sepade, Kusu G-25. The velocindase fraction was collected, anti-elastase 1 antibody solution (20 mg of antibody) and 0.4 ml of IM carbonate buffer (PH9.5) were added, and the mixture was allowed to stand overnight. After standing overnight, gel filtration was performed using a Sephacryl S-300HR force ram to obtain peroxidase mark E[. An elastase 1 antibody solution was obtained.

(3) Preparation of immobilized anti-elastase l antibody After washing polystyrene beads with Scanoto 20X-PF, add purified water and wash with anti-elastase l antibody solution (antibody 100
mg) and left at 4°C overnight, the polystyrene beads were washed with phosphate buffer (PH 7.0) containing 0.1% bovine serum albumin to obtain immobilized anti-elastase 1 antibody. .

(4) Purification of proelastase 1 20 ml of unactivated human pancreatic juice was developed on anti-human elastase 1 antibody-conjugated Sepharose 4B equilibrated with 0.1 M Tris-MCI buffer (PH 8.0), and after removing the portion that passed through, 0.2M green-MCI buffer (0.5
A proelastase l fraction was obtained by elution with M (containing sodium chloride, pH 2.8). This fraction was mixed with O. Cephacryl S-20 equilibrated with 1M acetate buffer (pH 5.5)
Purified proelastase developed into 0HH; o. 2mg
I got it. As a result of measuring the activity of this purified product using succinyl-alanyl-alanyl-alanyl-P-nitroanilide as a substrate, no activity was observed, but expression of activity was observed for the first time with the addition of tribun (derived from bovine). or,
This refined product is SDSI! It was aerophoretically homogeneous.

(5) Preparation of complex of α,-antitrypsin and proelastase 1 α1-antitrypsin 0.5 ml (3.16 m
g/m 1 ) and proelastase 1; 20 μ+(
200 μg/m I) and heated at 37°C.
A complex of α-vine antitribucin and proelastase 1 can be prepared by allowing the mixture to stand at 4°C for 3 hours and then overnight at 4°C. This preparation is used as Cefacryl-SIO.
As a result of developing on OHR column, proelastase 1 and 9
It was revealed that 5% or more was bound to α8 antitrypun.

(6) Measurement of blood elastase l by enzyme immunoassay (system using a complex of elastase 1 and α1-antitribucin as a standard) Camphor elastase l (
Add 0.3 ml of Beruoxidase standard anti-elastase 1 antibody solution and 1 piece of polystyrene peas (immobilized anti-elastase 1 antibody) to 50 μl of test serum or test serum, and incubate at 37°C. After standing for 30 minutes, the polystyrene beads were soaked in OmM phosphate buffer containing 0.15M sodium chloride (pH 7.0).
) After washing twice with 2 ml, the polystyrene beads were transferred to another tube and treated with 3% 0-phenylenediamine and 0.0 ml.
After adding 0.5 ml of citric acid buffer containing 2% hydrogen peroxide and leaving it at room temperature for 15 minutes, add 2 ml of 1.6N sulfuric acid.
was added to stop the reaction, and the absorbance at a wavelength of 492 nm was measured. Standard elastase l (elastase l and α,
The elastase concentration in the test serum was calculated from a standard curve prepared using the antitribun complex.

As a result of measuring standard elastase l (complex of proelastase 1 and α1-antitribucin) using proelastase as a sample, the displayed concentration of elastase l and the elastase l concentration calculated by this measurement method matched.

(7) Measuring method of blood elastase 1 by enzyme immunoassay (system using a complex of proelastase 1 and α,-antitrypsin as a standard) Standard elastase 1 (proelastase 1 and α,-antitrypsin) complex) or 50 μl of test serum, 0.3 ml of peroxidase standard anti-elastase 1 antibody solution and polystyrene beads (immobilized anti-elastase 1 antibody).
After adding 1 piece and standing at 379C for 30 minutes, the polystyrene beads were placed in a lo
After washing twice with 2 ml of mM phosphate buffer (pH 7.0), the polystyrene beads were transferred to another test tube and treated with 3% 0-
Add 0.5 ml of citric acid buffer containing 7-enylene diamine and 0.02% hydrogen peroxide, let stand at room temperature for 15 minutes, and then add 2 ml of 1.6 N sulfuric acid to stop the reaction.
Absorbance at a wavelength of 492 nm was measured.

The amount of elastase I in the test serum was calculated from a standard curve prepared with standard elastase I (a complex of proelastase ■ and α-antitrib/n).

(8) Composition of Elastase 1 (system using standard elastase 1 and α1-antitrypun complex) The composition of Elastase 1 as determined by enzyme immunoassay is shown below.

1) Standard erasure
1 (Erastase 1
and α1-antitrypone complex) Ong/m12》5ng/ml3)
20ng/ml4)
50ng/ml5)
100ng/m+6)
200ng/m+7) buffer (
(contains animal serum) 8) Buffer solution 9) Beruoki/Dase standard antibody 1G) Washing solution 11) Immobilized antibody 12) O-phenylenediamine 13) Citrate buffer containing 0.02% hydrogen peroxide + 4 )
1. Composition of 6 N sulfuric acid (9) elastase 1 (system using a complex of proelastase 1 and α1-antitributane as a standard product) The composition of elastase 1 obtained by enzyme immunoassay is shown below.

1) ml elastase l (proelastase l and α1-
Antitrypin complex) Ong/m 1 2) No/
5
ng/ml3)
20ng/ml4)
50ng/m+5)
100ng/ml6)
200ng/ml 7) Buffer (contains animal serum) 8) Buffer solution 9> Verokindase camphor antibody 10) Washing solution 11) Immobilized antibody 12) 0-phenylenediamine + 3) Citric acid containing 0.02% hydrogen peroxide Acid buffer 14)
1. 6 N sulfuric acid F. Effects of the Invention Since elastase I is an enzyme specific to the pancreas, it is possible to diagnose various pancreatic diseases, and is particularly useful for screening pancreatic cancer, diagnosing acute pancreatitis, and monitoring its progress.

[Brief explanation of drawings]

FIG. 1 is a standard curve for measuring elastase I by enzyme immunoassay.

Claims (6)

[Claims] (1) Elastase 1 in the specimen is bound to the solid phase using a solid-phase anti-elastase 1 monoclonal antibody and an enzyme-labeled anti-elastase 1 monoclonal antibody, and elastase 1 is separated from the specimen, and The activity of the labeled enzyme bound to the phase is measured. Enzyme immunoassay of elastase 1, which consists of determining the amount of elastase 1 in the specimen from a standard curve obtained by performing the same procedure using standard elastase 1 of known concentration instead of the specimen. Method. (2) Standard elastase 1 is elastase 1 and α_1-
Claim 1, which is a complex with antitrypsin
The method described in section. (3) Standard elastase 1 is proelastase 1 and α_
1. The method according to claim 1, which is a complex with 1-antitrypsin. (4) The monoclonal antibody according to claim 1, which is obtained by fusing mouse antibody-producing cells obtained by immunizing a mouse with elastase 1 and mouse myeloma cells by a cell fusion method. (5) an enzyme-labeled antibody obtained by purifying an anti-elastase 1 antibody obtained using purified human elastase 1 as an antigen and covalently bonding the anti-elastase 1 antibody to an enzyme;
An anti-elastase 1 antibody-bound carrier in which an anti-elastase 1 antibody is bound to an insoluble carrier by physical adsorption, and a standard elastase consisting of a complex of elastase 1 and α_1-antitrypsin. Reagent for measuring elastase 1 in a sample. (6) Standard elastase 1 is proelastase 1 and α_
The reagent for elastase 1 measurement according to claim 5, which comprises a complex with 1-antitrypsin.