JPH08160046A - Measuring method for activity of angiotensin conversion enzyme(ace) - Google Patents

Abstract

PURPOSE: To measure only the activity of an angiotensin conversion enzyme(ACE) in a specimen with high accuracy by a method wherein an anti-ACE antibody immobilized on an inactive carrier and the ACE in the specimen are made to react with each other and foreign substances are removed by a washing operation. CONSTITUTION: A bead made of polyethylene is preferable for an inactive carrier whereon an antibody is immobilized, and blood, a serum, urine or the like can be cited for a liquid specimen containing ACE. After refined human ACE being an antigen is mixed in Freund's complete adjuvant or the like, a rabbit, for instance, is immunized against it and a rabbit anti-human ACE antibody is prepared from an antiserum thereof by refinement. This antibody is immobilized on the inactive carrier. Then, the carrier whereon the antibody is immobilized is put in a solution of a phosphoric acid buffer and the specimen containing the ACE and an antigen-antibody reaction is brought about. After the reaction, foreign substances are removed by a washing operation and the activity of the ACE in the specimen is measured. According to this constitution, an effect of noise due to the foreign substances in the specimen is eliminated and measurement of the activity at a low concentration level becomes possible.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring angiotensin converting enzyme (ACE) activity, and more particularly to a method for measuring ACE activity at a low level.

[0002]

BACKGROUND ART As is well known, ACE acts on angiotensin-I in the body to release C-terminal dipeptide, that is, L-histidyl-L-leucine, to thereby increase blood pressure. It is an enzyme that produces -II. This enzyme plays an important role in vivo in association with the renin-angiotensin system or the quinine-kallikrein system. By measuring the level of this enzyme in blood, ACE such as sarcoidosis is used as an index. Diagnosis of diseases and accurate understanding of pathological conditions are possible.

Therefore, it is physiologically or clinically meaningful to measure the above-mentioned ACE activity. Conventionally, the measuring method is 1) a method using a radioisotope 2) a method using fluorescence 3) liquid chromatography 4) A buffer solution containing p-hydroxyhypryl-L-histidyl-L-leucine, hippurase, p-hydroxybenzoic acid hydroxylase, nicotinamide adenine-dinucleotide phosphate reduced form proposed by Kasahara et al. Is added to a solution containing ACE, the mixture is reacted at a constant temperature for a predetermined time, and then the decrease in absorbance is measured.

The ACE activity measuring method proposed by Kasahara et al. Is widely used because it can be measured quickly with a simpler device than other methods, but it was measured without separating ACE from the test object. , Coexisting in the subject
The influence of the E-like active substance, the activity-inhibiting substance and the reaction intermediate product was exerted, and the ACE activity was measured including these noises. In particular, the reproducibility at the low value level and the difference between the measured value and the theoretical value in the addition recovery test were large.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to eliminate the effects of the above-mentioned noises and measure only the activity of ACE in a subject with high accuracy. In particular, the aim is to improve reproducibility at low concentration levels.

[0006]

The above object of the present invention has been achieved by the following constitution.

1) When measuring ACE activity in a sample, anti-ACE antibody immobilized on an insoluble carrier and AC in the sample
A method for measuring the activity of ACE, which comprises reacting E and removing contaminants by a washing operation, and then measuring the enzyme activity.

2) After reacting ACE with an anti-ACE antibody in a sample, they are reacted with an antibody against the anti-ACE antibody immobilized on an insoluble carrier, and a contaminant is removed by a washing operation to separate the enzyme. A method for measuring ACE activity, which comprises performing activity measurement.

3) The ACE described in 1 or 2 above, wherein the anti-ACE antibody is a monoclonal antibody.
Activity measurement method.

In the present invention, purified human AC which is an antigen
E (Protogen) is Freund's complete adjuvant
(Manufactured by Difco), Libi adjuvant (manufactured by Ribi), BC
Animals are immunized after mixing with G, an adjuvant such as aluminum hydroxide. The animals to immunize are rabbits,
Examples include goats, mice such as Balb / c and NZB, rats, Armenian hamsters, and sheep. The dose of the immunogen is about 100 μg / mouse for rabbits and about 50 μg / mouse for booster immunization, and this is intradermally in the back skin at intervals of 1 to 4 weeks, preferably 2 to 10 times at intervals of 2 weeks. Administer. In the case of mice, the immunogenic dose is 25 to 50 μg / mouse, which is injected intraperitoneally or subcutaneously. When Freund's complete adjuvant is used for the first immunization, Freund's incomplete adjuvant and the immunogenic conjugate are mixed in the second and subsequent booster immunizations, and the mixture is administered about 2 to 10 times every 2 weeks.

After the initial immunization, booster immunization is performed every 2 weeks, and the target antibody titer in the obtained antiserum is measured. It is preferable to measure by an enzyme immunoassay as shown below.

In the present invention, examples of the body fluid or sample containing ACE include blood, serum, plasma, semen, spinal fluid, urine and the like. Examples of the insoluble carrier for immobilizing the antibody include beads and gels made of polyethylene, chitin, chitosan, ABS resin, magnetic, etc., but polyethylene beads are preferably used.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0014]

Examples Example 1 1) Preparation of antibody 100 μg of purified human ACE (Protogen) as an antigen
Was dissolved in phosphate buffer (pH 7.2) and injected subcutaneously into rabbits (New Zealand White) with Freund's complete adjuvant. From the second time onward, 50 μg of the antigen was similarly injected together with incomplete adjuvant. After continuing this every 2-3 weeks, rabbit antisera were collected 4-6 months later. For the antiserum, the immunoglobulin fraction was purified by a conventional method using a 50% ammonium sulfate salting-out method and a protein A column.

2) Preparation of antibody-immobilized beads Carbon beads buffer (pH 5.0) containing polyethylene anti-human ACE antibody or goat anti-rabbit immunoglobulin (Nordic) on polyethylene beads (Sekisui Chemical Co., Ltd.). 20μ in
The dissolved solution was added to g / ml, and the mixture was allowed to stand at 4 ° C for 24 hours. After removing the antibody solution by suction, heat treatment (56 ℃, 30
The obtained phosphate buffer containing 1 w / v% bovine serum albumin was added, and the mixture was allowed to stand at 37 ° C. for 15 hours.

3) Antigen-antibody reaction a. Reaction 1 100 μl of phosphate buffer (pH 7.2) and 200 μl of sample are dispensed into the wells of the reaction tray, and the beads to which the rabbit anti-human ACE antibody is immobilized are placed therein. After sealing the upper part of the well with a seal, it is left at 37 ° C. for 2 hours for antigen-antibody reaction. After the reaction, remove the seal, and wash 3 times with 1 ml of phosphate buffer.

B. Reaction 2 Rabbit anti-human ACE antibody is diluted in phosphate buffer (pH 7.2) to a final concentration of 3 μg / ml. Dispense 100 μl of this antibody solution and 200 μl of sample into the wells of a reaction tray that had been blocked with 1 w / v% BSA solution in advance, seal with a seal, and allow to react at 37 ° C. for 1.5 hours. The beads to which the goat anti-rabbit immunoglobulin antibody has been immobilized are added thereto, and the mixture is sealed again and further reacted at 37 ° C. for 1.5 hours. After the reaction, the same washing operation is performed except for the seal.

4) Enzyme activity measurement An enzyme activity was measured by a modified method using a Kasahara method measurement reagent kit (Fujirebio Inc.). The measuring method is performed according to the operating method of this kit, except that the beads reacted with the sample are used instead of the sample, and the reaction condition between the beads and the substrate solution is 37 ° C. for 60 minutes.

The activity value of ACE was determined by the following equation.

(AB) / 12000 × 1/60 × 10 ⁶ (unit: nmol / min) A: Absorbance of sample B: Absorbance of sample blank or reagent blank 5) Sample measurement: Addition recovery test 3 Concentration The ACE activity of the pooled sera (samples A to C) and the five kinds of human sera (samples 1 to 5) and a 1: 1 mixture thereof as shown in the following table were measured by the above-mentioned method and Kasahara method. The measurement was performed 3 times, and the measured value was obtained from the average. The theoretical value is the average of the measured values of the individual samples of pooled serum and human serum, and the recovery rate was calculated from the following formula.

Recovery rate = (measured value−theoretical value) / theoretical value × 100 (%) The obtained results are shown in Table 1.

[0022]

[Table 1]

From the results shown in Table 1, it is clear that the difference between the theoretical value and the measured value is small in the two measuring systems in this patent, as compared with the existing enzyme activity measuring method. It is considered that the cause of the divergence of the current method is that the enzyme-like substance existing as a contaminant in the test substance and the enzyme activity-inhibiting substance are affected, and thus the accurate quantitative determination of the enzyme amount is lacking. On the other hand, according to the method disclosed in this patent, a good recovery rate was obtained because accurate measurement can be performed irrespective of the change in the amount of contaminants due to the mixing of the sample.

[0024]

According to the present invention, the conventional Kasahara method and Cushman method are used.
There is no effect of coexisting substances in the sample, which is seen in the method, etc., and ACE in the sample can be easily separated by using an antibody, and noise due to contaminants can be measured by measuring the activity of the ACE. It was possible to measure activity at low concentration levels without any influence.

Claims (3)

[Claims] 1. An angiotensin converting enzyme (A) in a sample
CE) In measuring the activity, the anti-ACE antibody immobilized on the insoluble carrier is reacted with ACE in the sample, and the contaminants are removed by washing operation, and then the enzyme activity is measured. . 2. After reacting ACE with an anti-ACE antibody in a sample, the ACE is reacted with an antibody against the anti-ACE antibody immobilized on an insoluble carrier, and a contaminant is removed by a washing operation to separate the enzyme. A method for measuring ACE activity, which comprises performing activity measurement. 3. The ACE according to claim 1 or 2, wherein the anti-ACE antibody is a monoclonal antibody.
Activity measurement method.