JP2617299B2 - Immunoassay method for elastase 1 - Google Patents

Description

The present invention relates to a method for immunologically measuring pancreatic exocrine protease elastase 1 contained in biological fluid, which is useful for diagnosing pancreatic disease.

It is said that pancreatic diseases are still difficult to diagnose even with the progress of various examination methods. The reason is that the pancreas is located deepest in the abdominal cavity, so it is difficult to grasp the existence or nature of the disease by classical diagnostic methods such as palpation, visual inspection, auscultation, and X-ray examination. Are similar to other gastrointestinal disorders, and there is no convenient and reliable test method.

Biochemical diagnosis of pancreatic disease has long been made by measuring amylase, but (1) in chronic pancreatitis, blood amylase rarely shows high values except in extreme periods, and urinary amylase measurement is also difficult. Not very practical and not suitable for diagnosing chronic pancreatitis. (2) In acute pancreatitis, blood and urine amylase show high levels, but the period of high levels after onset is short, and diagnosis cannot be made if measurement time is missed , (3) Amylase is also produced from organs other than the pancreas, and blood amylase levels are abnormal in non-pancreatic diseases. Therefore, measurement of blood amylase lacks specificity as a screening test method for pancreatic disease and is insufficient. Therefore, development of a new biochemical diagnostic method for pancreatic disease has been desired.

Elastase is a general term for pancreatic exocrine protease that specifically hydrolyzes elastic fiber elastin of connective tissue,
There are two types of these, which are physicochemically, enzymatically, and antigenically different from each other. Acidically charged elastase is elastase 1 and basic charged elastase is elastase 2 I'm calling

The determination of the amount of elastase 1 was first attempted by an enzymatic method, but was not successful mainly for the following reasons. In other words, the sensitivity is insufficient, and since a large amount of protease inhibitors are present in the blood, when the active enzyme enters the blood, these inhibitors are instantaneously bound and the enzyme activity is lost. Furthermore, the enzyme activity assay using a substrate is affected by enzymes other than elastase, and thus lacks specificity. For these reasons, an attempt was next made to measure the enzyme by an immunological method. The presence or absence of the immunological activity is different from the presence or absence of the enzymatic activity, and the elastase inhibitor α ₂
The complex bound to macroglobulin has no immunological activity, whereas the complex bound to α ₁ -antitrypsin has immunological activity. However, even with an immunoassay using a labeled antigen, the elastase inhibitor in the blood sample binds to the labeled antigen, so that part or most of the antigenicity of the labeled antigen is lost, immunological activity is inhibited, and blood elastase is inhibited. An accurate quantification of 1 was not possible. On the other hand, Oyama changed the labeled antigen to antigen elastase 1
Pretreated with DFP (diisopropylfluorophosphate), a synthetic inhibitor of E., and the labeled antigen was masked with DFP to prevent the elastase inhibitor in the blood from binding to the labeled antigen (Japanese Patent Publication No. 59-2518).
3). Since the binding of DFP to elastase 1 itself did not change the antigen activity of elastase 1, it became possible to immunologically measure the elastase 1 concentration in the body fluid.

However, Oyama's method was not satisfactory as a method for measuring elastase 1 in body fluids. This is because conventional antibodies recognize various antigenic determinants of elastase 1, and the binding portion between elastase 1 and α ₁ -antitrypsin (FIG. 1, C), or a close portion of the binding portion (FIG. 1, C).
There are also antibodies that recognize 1, D). Therefore, when α ₁ -antitrypsin is bound to elastase 1 in a conventional anti-elastase 1 antibody, it cannot react at all or contains an antibody with extremely low reactivity, so that its titer is low. There was a problem with sensitivity. The elastase 1 by the antibodies of manufacturing Rotsuto, particularly alpha ₁ - since caused a large difference in binding of the antibody to complex with anti-trypsin and elastase 1, is a variation in the measurement of elastase 1 occurs, large in terms of manufacturing control Was an obstacle.

In a disease in which α ₁ -antitrypsin increases, such as pancreatic cancer, α ₁ -antitrypsin and elastase 1 to which α ₁ -antitrypsin is bound are compared with elastase 1 as an index. It is considered that when only the elastase 1 to which ₁ -antitrypsin is bound is used as an index, the rate of increase from the normal value is emphasized and expressed, so that it is easier to find an abnormality. Therefore, elastase 1 and α ₁ -Development of a monoclonal antibody that recognizes a binding site with antitrypsin (Fig. 1, B) has been awaited.

The present invention relates to elastase 1 and elastase 1 and α ₁ −
It is intended to provide a method and a reagent for measuring elastase 1 in a body fluid using a monoclonal antibody against a complex with antitrypsin. The present inventors have obtained a monoclonal antibody recognizing an antigenic determinant (FIG. 1, A) at a site distant from the binding portion between elastase 1 and α ₁ -antitrypsin, and bound to α ₁ -antitrypsin. Establishes a highly accurate method for measuring elastase 1 in body fluids, which shows almost the same reactivity with elastase 1 as free elastase 1, and ensures a stable supply of antibodies. It was done. Furthermore, the present inventors succeeded in obtaining a monoclonal antibody recognizing the binding site between elastase 1 and α ₁ -antitrypsin (FIG. 1, B), and specifically measure α ₁ -antitrypsin-bound elastase 1. As a result, an excellent measurement system was established particularly as a diagnostic method for cancer.

In the measurement of elastase 1 according to the present invention, the above-mentioned monoclonal antibody against elastase 1 is subjected to a competitive reaction between elastase 1 in a test sample and labeled elastase 1 to which α ₁ -antitrypsin is bound, followed by anti-elastase 1 Labeled elastase 1 bound to the monoclonal antibody and labeled elastase 1 not bound are separated by an appropriate method (B / F separation), and the activity of one or both labeling agents in the fractions is measured. Instead of the sample, the amount of elastase 1 to be measured is determined from a standard curve prepared by performing the same operation on standard elastase 1 of known concentration.

A monoclonal antibody against elastase 1 is obtained by immunizing a mammal, for example, a mouse, with elastase 1, and fusing myeloma cells, Epstein-Barr virus (EBV), or the like to antibody-producing cells, for example, spleen cells isolated from the mouse, The cells can be obtained by the Khler-Milstein cell fusion method, which confers permanent growth ability and clones a cell producing the desired monoclonal antibody from the obtained fused cells. Elastase 1 and alpha ₁ - junction of the antitrypsin (Figure 1, B) a monoclonal antibody that recognizes the alpha ₁ - It is possible to obtain by immunizing antitrypsin binding elastase 1, alpha ₁ - Anti Since trypsin has no species specificity, when a mouse is immunized with elastase 1, α ₁ -antitrypsin in the mouse blood binds to elastase 1, and therefore, the binding site between elastase 1 and α ₁ -antitrypsin (FIG. 1) , B) to produce monoclonal antibodies.

As the labeling agent for labeled elastase 1, those usually used as labeling agents such as radioisotopes, enzymes, fluorescent substances and luminescent substances can be used. Labeling of elastase 1 with these labeling agents can be performed by a known method, for example, a chloramine-T method or a periodate method.

B / F separation can be performed by a known method such as the PEG method, the two-antibody method, and the solid phase method.

As a test sample, urine, pancreatic juice, pleural effusion, ascites, and the like can be used in addition to blood.

alpha ₁ - as an anti-elastase 1 monoclonal antibody in order antitrypsin to specifically measure only elastase 1 bound, elastase 1 and alpha ₁ - junction of the antitrypsin monoclonal antibody that recognizes (Figure 1, B) When used, it is needless to say that labeled elastase 1 to which α ₁ -antitrypsin is bound must be used.

The kit kit for measurement used in the present invention comprises (i) a buffer containing α ₁ -antitrypsin, (ii) labeled elastase 1 (iii) standard elastase 1 (iv) anti-elastase 1 monoclonal antibody. Although the anti-elastase 1 monoclonal antibody recognizes a single antigenic determinant, a mixture of these can also be used.

Furthermore, elastase 1 in a test sample can be measured by the San Deutsch method using two or more types of monoclonal antibodies that recognize different sites of the antigenic determinant of elastase 1. That is, after the elastase 1 in the test sample is bound to the anti-elastase 1 monoclonal antibody bound to the solid phase, the anti-elastase 1 has a recognition site different from that of the anti-elastase 1 monoclonal antibody bound to the solid phase. 1 monoclonal antibody is labeled with a labeling agent, and a labeled anti-elastase 1 monoclonal antibody is reacted therewith to form a San Deutsche antigen-antibody complex of (immobilized antibody)-(elastase 1)-(labeled antibody). The activity of the labeling agent on the antigen-antibody complex or of the labeling agent in the solution is measured, and the activity of the labeling agent to be measured is determined by a standard curve prepared by the same operation using standard elastase 1 with a known concentration instead of the test sample. The purpose is to determine the amount of elastase 1 in the test sample. As the solid phase, those commonly used as the solid phase such as glass, polystyrene, nylon, and filter paper can be used. The shape of the solid phase may be a rod shape, a plate shape, a tube shape, a bead shape, or the like. The kit for measurement used in the San Deutsch method includes (i) immobilized anti-elastase 1 monoclonal antibody, (ii) labeled anti-elastase 1 monoclonal antibody, and (iii) standard elastase 1.

A binding site between elastase 1 and α ₁ -antitrypsin is attached to either the immobilized antibody or the labeled antibody (FIG. 1,
By using a monoclonal antibody that recognizes B), elastase 1 bound to α ₁ -antitrypsin can be specifically measured.

Since elastase 1 is an enzyme specific to the pancreas, it can be applied to diagnosis of various pancreatic diseases. In particular, it is useful for diagnosis of acute pancreatitis, follow-up thereof, and screening for pancreatic cancer.

Next, the present invention will be described by way of examples. The essence of the present invention is that an anti-elastase 1 monoclonal antibody or elastase 1 which shows almost the same cross-reactivity as elastase 1 even in a complex in which α ₁ -antitrypsin is bound to elastase 1 and alpha ₁ - examples in the binding portion there of on the measuring method and the antibody elastase 1 monoclonal antibody elastase 1 in bodily fluids, which comprises using a monoclonal antibody that recognizes and below the anti-trypsin It is not limited.

Example 1 Preparation of Anti-Elastase 1 Monoclonal Antibody 0.1 ml of Freund's complete adjuvant was added to 0.1 ml of purified elastase 1 solution (containing 25 μg) to prepare an emulsion. 0.2ml of this emulsion is BALB /
C mice were intraperitoneally administered. One month later, 0.2 ml of elastase 1 solution (containing 50 μg) was intravenously injected into the tail vein. Three days later, the mice were sacrificed, spleen was removed, and 1 × 10 ⁸ spleen cells and 2 × 10 ⁷ mouse myeloma cells (P3U1 strain) were subjected to cell fusion in the presence of 50% PEG. 0.1 ml each is seeded on 10 96-well plates, and HAT
After culturing in a medium for 2 weeks, an antibody assay in the culture supernatant was performed. Cloning was performed from the antibody-positive wells by the limiting dilution method, and antibody-producing strains were selected. The obtained monoclonal antibody producing strain 1 × 10 ⁷ BALB /
C mice were inoculated into the abdominal cavity, and ascites was collected 2 weeks later.

Example 2 Labeling of elastase 1 with ¹²⁵ I About 300 μCi of Na ¹²⁵ I was collected, and a phosphate buffer was added to adjust the pH.
Was adjusted to 7.5. Add 150 μg of elastase, stir, add 25 μl of chloramine T (1 mg / ml), shake for 30 seconds, and add 100 mg of sodium metabisulfite (1 mg / ml).
μ was added and the reaction was stopped by adding a labeled reaction solution Sephadex G-25 column (1 × 30 cm), and eluted with phosphate buffer (pH 7.5), to remove free ¹²⁵ I ¹²⁵ I-labeled elastase 1 was recovered.

Example 3 Determination of Elastase 1 in Blood by Competition Method 100 μl of ¹²⁵ I-labeled elastase, 100 μl of anti-elastase 1 monoclonal antibody and 100 μl of bovine γ-globulin (10 mg /
ml), and the mixture was incubated overnight at room temperature. 1 ml of a 20% PEG solution was added, and the mixture was stirred and mixed, followed by centrifugation at 3000 rpm for 10 minutes. After removing the decantation of the supernatant, the radioactivity of the remaining precipitate was measured.

The elastase 1 concentration in the test serum was determined from a standard curve (FIG. 2) created from the values obtained by measuring the elastase 1 standard solution.

Example 4 Preparation of Immobilized Anti-Elastase 1 Antibody Ammonium sulfate powder was added and dissolved in ascites fluid containing anti-elastase 1 monoclonal antibody so as to be 40% saturated.
The precipitated precipitate is collected by centrifugation, and then the phosphate buffer (pH
7.5) to give a γ-globulin solution. 2 μg / 0.3 ml of γ-globulin solution was added per polystyrene beads, and shaken at room temperature for 24 hours. Unreacted γ-globulin was washed away with physiological saline to prepare antibody-adsorbed beads.

Example 5 Labeling of Anti-Elastase 1 Monoclonal Antibody with ¹²⁵ I Ammonium sulfate powder was added and dissolved in ascites fluid containing anti-elastase 1 monoclonal antibody so as to be 40% saturated.
The precipitated precipitate is collected by centrifugation, and then the phosphate buffer (pH
The solution was dissolved in 7.5) to a concentration of 1 mg / ml to obtain a γ-globulin solution.

Take about 300 μCi of Na ¹²⁵ I, add phosphate buffer and add
Was adjusted to 7.5. After adding 1 ml of the above γ-globulin solution and stirring, 25 μl of chloramine T (1 mg / ml) is added, and after a shaking reaction for 30 seconds, 100 μl of sodium metabisulfite (1 mg / ml) is added.
Was added to stop the reaction. The labeling reaction solution was added to a Sephadex G-25 column (1 × 30 cm), eluted with a phosphate buffer (pH 7.5), and free ¹²⁵ I was removed to recover a ¹²⁵ I-labeled elastase 1 monoclonal antibody.

Example 6 Quantification of Blood Elastase 1 by San Deutsch Method One immobilized antibody bead was added to 100 μl of elastase 1 standard solution or test serum, and the mixture was incubated at room temperature with shaking for 2 hours. After washing the beads three times with physiological saline, ¹²⁵ I-labeled anti-elastase 1 monoclonal antibody solution
200 μl was added, and the mixture was incubated for 2 hours while shaking at room temperature. After washing the beads three times with physiological saline, the radioactivity of the beads was measured.

The elastase 1 in the test serum was determined by a standard curve (FIG. 3) prepared from the values obtained by measuring the elastase 1 standard solution.
The concentration was determined.

[Brief description of the drawings]

FIG. 1 schematically depicts the antigenic determinant site of elastase 1 bound to α ₁ -antitrypsin. FIG. 2 is a standard curve of blood elastase 1 measurement by the competitive method, and FIG. 3 is a standard curve of blood elastase 1 measurement by the San Deutsch method.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kiyoshi Sekiguchi 624-2 Toyoshiki, Kashiwa-shi, Chiba Prefecture (72) Inventor Public Road 2-18-13 Nishiyama, Kashiwa-shi, Chiba Prefecture (56) References Tokiko Sho59- 25183 (JP, B2) Ishikawa, Kawai, Miyai, Ed., Enzyme Immunoassay (Second Edition) (1982-12-15) 41-44

Claims (8)

(57) [Claims] 1. Elastase 1 in a test sample to be measured
And a labeled elastase 1 to which α ₁ -antitrypsin has been bound is allowed to undergo a competitive reaction with an anti-elastase 1 monoclonal antibody, and then labeled elastase 1 not bound to labeled elastase 1 bound to the anti-elastase 1 monoclonal antibody , The activity of one or both labeling agents in those fractions is measured, and the activity to be measured is determined by a standard curve prepared by the same operation using a standard elastase 1 of known concentration instead of the test sample. An immunoassay for elastase 1 comprising determining the amount of elastase 1 in a test sample. 2. The method according to claim 1, wherein the anti-elastase 1 monoclonal antibody recognizes an antigenic determinant located at a site distant from a binding site between elastase 1 and α ₁ -antitrypsin. 3. The method according to claim 1, wherein one or a mixture of two or more monoclonal antibodies is used as the anti-elastase 1 monoclonal antibody. 4. The method according to claim 1, wherein the anti-elastase 1 monoclonal antibody recognizes a binding site between elastase 1 and α ₁ -antitrypsin. 5. The method according to claim 1, wherein the labeling agent is a radioisotope, an enzyme, a fluorescent substance or a luminescent substance. 6. The method according to claim 1, wherein the separation of the labeled elastase 1 not bound to the labeled elastase 1 bound to the anti-elastase 1 monoclonal antibody is carried out by a PEG method, a two-antibody method or a solid phase method. 7. An anti-elastase 1 monoclonal antibody bound to a solid phase, to which elastase 1 in a test sample is bound, and then the anti-elastase 1 monoclonal antibody bound to the solid phase has a different recognition site. And reacting the labeled anti-elastase 1 monoclonal antibody obtained by labeling the anti-elastase 1 monoclonal antibody with a labeling agent to form a sandwich-type antigen-antibody complex of (immobilized antibody)-(elastase 1)-(labeled antibody). A standard curve formed by measuring the activity of the labeling agent on the antigen-antibody complex and / or the labeling agent in the solution and separately operating in the same manner for standard elastase 1 of known concentration instead of the test sample A method for immunologically measuring elastase 1 comprising determining the amount of elastase 1 in a test sample to be measured by the method. 8. The method according to claim 7, wherein the labeling agent is a radioisotope, an enzyme, a fluorescent substance or a luminescent substance.