JP2877222B2 - Renin monoclonal antibody and method for immunological measurement of active renin using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a renin monoclonal antibody and a method for immunologically measuring active renin using the same, and more particularly to a method for measuring renin which is indispensable for diagnosis of hypertensive disease. The present invention relates to a renin monoclonal antibody produced using recombinant human renin, and an immunoassay method for specifically measuring only active renin using the same.

[0002]

BACKGROUND OF THE INVENTION Renin is a proteolytic enzyme that degrades angiotensinogen and converts it to angiotensin I (ATI). ATI causes vascular smooth muscle to contract, resulting in an increase in blood pressure. For this reason, measurement of renin involved in ATI production has become an important test for understanding the pathology of hypertensive disease.

[0003] In particular, in the diagnosis of renal vascular hypertension, which is the main cause of secondary hypertension, the measurement of active renin in blood is important. It is also known that there is a rare but renin-producing tumor. In such a disease, the measurement of active renin in blood is also essential for the purpose of clarifying the nature of the tumor.

[0004] Renin has been measured for a long time,
The method of directly measuring renin was initially difficult. That is, in the initial measurement method for renin, a sample was injected into a non-anesthetized dog, and the average blood pressure was set to 30 mm within 2 minutes.
The renin activity when increasing Hg was defined as one Goldblatt Unit and the measurement was performed. Although this method is called a bioassay, it is not a practical method because it has poor measurement sensitivity and measurement reproducibility and cannot be repeatedly performed on the same animal.

[0005] Later, a practical measurement of renin was developed, but it was not a method of measuring renin itself. 196
In 9 years, Boyd et al. Conducted RIA on ATI produced by renin from angiotensinogen per unit time.
A method was developed in which the ATI production capacity was measured and the renin activity amount (ng / ml / hr) was substituted based on the ATI productivity.

[0006] In the above method, since angiotensinogen present in a sample (plasma) is used as an enzyme substrate for renin for the measurement of renin activity, it is necessary to carry out an immunoreaction after performing an enzymatic reaction in the measurement. In addition to the problem that extremely complicated operations such as quantifying the amount of ATI present in the sample from the beginning and subtracting it from the measurement result are necessary, fluctuations in the concentration of angiotensinogen, a substrate in blood, There is also the problem of being affected.

[0007] Furthermore, in the dysfunction of the liver that produces angiotensin, the renin substrate is reduced, and as a result, the renin activity value is underestimated. It is pointed out that it will be evaluated. In fact, in the measurement of renin activity, a phenomenon in which the apparent renin activity is increased by adding angiotensinogen from the outside to a sample considered to be deficient in angiotensinogen has been observed. In addition, this measurement system cannot directly measure a sample that does not contain angiotensinogen, such as amniotic fluid or an extract. In addition, there were concerns that this measurement system would cause measurement errors if control during the enzyme reaction was incomplete, and that the effects of proteases other than renin would be a problem.

On the other hand, a direct method for measuring renin was developed by Galen et al. In 1979, and it was possible to directly measure renin using renin purified from a renin-producing tumor and an antibody against the same.

However, it has been found that renin contains active renin and its precursor, inactive renin (prorenin), and it is impossible to discriminate between them by RIA using ordinary renin antiserum. It has been found. That is,
There are two types of renin present in the blood, active renin and inactive renin, of which active renin, which is thought to play a physiological role, accounts for about 10% or less of total renin, and active renin. In the measurement of the total renin, it was necessary to measure only the active renin only specifically and with high sensitivity.

For the above purpose, it is necessary to use an antibody having the property of specifically recognizing or binding to active renin, and at the same time having a high affinity for measuring a low concentration. Since a polyclonal antibody was used in the measurement, it was not possible to specifically measure only active renin.

As a method for measuring only active renin, a method for measuring active renin using a monoclonal antibody developed by Christoph et al. In 1985 that extremely strongly inhibits the enzyme activity of renin (JP-A-60-231624)
No.) is known. Although this method enabled specific measurement of active renin, this method used 5 × 1
The enzyme activity of renin was reduced to 50 even at a dilute antibody concentration of 0 ^-11 M.
% Of the antibody had to be used. An antibody having such properties is difficult to obtain by an ordinary monoclonal antibody preparation method, and is not a method that can be easily performed.

Also, Christoff et al. Have disclosed a sandwich assay for active renin using two types of antibodies as an example of the above assay, but both antibodies used in combination have strong activity. It is an antibody that has an enzyme inhibitory activity for type renin. In the above publication, the recognition sites of these antibodies are different, but antibodies having strong enzyme inhibitory activities are expected to be close to each other as recognition sites as antibodies. If you are measuring active renin accurately,
That is a very questionable one.

Further, as another measuring method, the present inventors developed a method for measuring renin using a renin inhibitor and a renin-specific antibody in 1987 (JP-A-63-217).
272). This method is also a method for specifically measuring active renin, and pepstatin A is used as a renin inhibitor.
Was used as an example, but the enzyme inhibitory activity of pepstatin A on renin was weak (1 × 10 ⁻⁶ M) and the measurement sensitivity was not always sufficient. Also, when pepstatin A was used as a renin inhibitor, it binds only active renin among total renin but also binds to other proteases, so it had to be combined with other renin-specific reagents. .

On the other hand, a kit for directly measuring the concentration of active renin in blood put into practical use by Pasteur is a truly desirable measurement system which is not affected by the substrate concentration.
Although the sample volume was as large as 250 μl, the sensitivity was still insufficient even in a large amount, and the measurement system was often lower than the sensitivity even in a normal person. For this reason, it has been pointed out that primary aldosteronism, which is a disease in which renin is significantly reduced, becomes insensitive in most cases.
This is because the blood concentration of active renin in normal humans is on the order of tens to picomolars, and in order to measure such renin theoretically, a monoclonal antibody with some high affinity was required. .

As described above, particularly in the measurement of active renin, substances or antibodies that recognize only active renin are important, as described above. Such an antibody must be an antibody that recognizes, particularly, at or near the enzyme active center among active renin molecules. This is because an antibody that recognizes a site far away from the active center of renin may recognize not only active renin but also inactive renin.

Although renin has a molecular weight of about 40,000, it can be easily understood that the enzyme active center is a very limited region. It is generally difficult to produce a monoclonal antibody specific to this limited region, and moreover, it has a high renin enzyme inhibitory activity and a high renin binding affinity that can accurately measure low concentrations of active renin. It was even more difficult to produce antibodies with them at the same time.

[0017]

Therefore, an antibody which simultaneously satisfies a high enzyme inhibitory activity and a high binding affinity can be prepared, or an antibody against active renin having the performance which can be obtained usually and another technique can be used. There has been a need to develop an accurate measurement system for active renin, and to provide a highly sensitive measurement system for active renin.

[0018]

Means for Solving the Problems The present inventors have made intensive studies to develop a method for measuring renin which has solved the drawbacks of the conventional method for measuring active renin, and as a result, it has been proposed to use recombinant human renin. It has been found that renin monoclonal antibodies produced by the hybridomas include those having high enzyme inhibitory activity and those capable of loading high radioactivity.

Further, the present inventors have found that by combining this monoclonal antibody with a monoclonal antibody which recognizes renin but does not have high enzyme inhibitory activity, it is possible to measure active renin with high accuracy. completed.

That is, an object of the present invention is to provide a renin monoclonal antibody produced by a hybridoma produced using recombinant human renin.
Another object of the present invention is to provide a hybridoma that produces the above-mentioned monoclonal antibody. Furthermore,
Another object of the present invention is to provide a method for measuring active renin using the above monoclonal antibody.

The monoclonal antibody against recombinant human renin of the present invention can be obtained by culturing a hybridoma obtained by fusing transformed cells with lymphocytes derived from spleen cells of an animal immunized with high-purity human activated renin. .

Highly active human renin for use in immunization can be obtained, for example, by using the gene sequence of human renin disclosed by Murakami et al. In 1983 (JP-A-60-47681). As animal spleen cells, mice immunized with recombinant human renin,
Splenocytes of rats, sheep and the like are used, and myeloma cells such as P3U1 are used as transformed cells.
Fusion of the transformed cells and lymphocytes derived from animal spleen cells can be performed by a known fusion method using electrofusion or polyethylene glycol. Among them, production of a hybridoma using spleen cells derived from a mouse is advantageous in that subsequent antibody collection is facilitated.

The culture of the hybridomas obtained as described above can also be performed, for example, by using a Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal bovine serum or a DMEM medium containing no fetal bovine serum. The method is performed in the following manner.

Further, as a method for collecting a monoclonal antibody from a hybridoma, a method of administering a fixed amount of the hybridoma to Balb / c or a nude mouse, preferably Balb / c mouse, collecting ascites fluid, and purifying the same is used. It is possible. In addition, a method of concentrating the culture supernatant of a cell culture device such as an Acucyst and purifying the same can also be used. In addition, the obtained antibody can be easily purified by a known purification method such as using a protein A column or an ion exchange column.

In the present invention, high-purity recombinant human renin is used as an immunizing antigen, so that a large number of monoclonal antibodies against a homogeneous antigen can be produced as compared with the case where a conventional extract is used. In experiments conducted by the present inventors, monoclonal antibodies covering dozens of clones were obtained, and all of them strongly bound to renin in the living body, that is, renin purified to a higher degree than human kidney. Degree of inhibition is 1 × 10
^At a concentration of ^-6 M, 1
Even at a concentration of × 10 ^-8 M, there was a wide range of antibodies having enzyme inhibitory activity.

[0027] Monoclonal antibodies having renin enzyme inhibitory activity are not necessarily strongly inhibited, and have high binding affinity. However, at a concentration of 5 × 10 ^-9 M, the inhibitory activity is insufficient. Was.

The monoclonal antibody obtained as described above can be bound to a solid phase according to a conventional method.
Labeling using enzymes or radioactive elements is also possible.
For example, examples of labeling of a monoclonal antibody include a method of labeling with a radioisotope such as ¹²⁵ I, ¹³¹ I and ¹²³ I using an oxidizing agent such as chloramine T, and an enzyme such as alkaline phosphatase and peroxidase by a hinge method or the like. Labeling method. The binding or labeling to these solid phases is carried out not only using the monoclonal antibody itself, but also by using known methods for producing Fab ′ or F
It is possible to indirectly immobilize or label those converted to (ab ') ₂ or modified with biotin, avidin or the like.

Among the obtained monoclonal antibodies,
It has been found that there are those which can load radioactivity about 5 times higher than normal radioactivity. For example, the monoclonal antibody 11-6 described in detail in Examples below.
Was an antibody capable of stably labeling ¹²⁵ I with a high specific activity of 25 μCi / μg.

Using the monoclonal antibody thus obtained, the amount of active renin contained in biological fluids such as serum, plasma, urine, ascites, pleural effusion, whey, amniotic fluid, spinal fluid, extract, and exudate is measured. In order to measure the amount of active renin with high sensitivity, a sandwich measurement method of active renin using the following two types of antibodies can be used. It is desirable to do.

When this sandwich immunoassay is carried out, if the substance to be measured is a substance having a relatively low molecular weight, a favorable reaction cannot be expected if the antibody recognition sites are close to each other. It is important to change the site recognized by the antibody.

In the present invention, in order to make the recognition sites of the two antibodies different from each other, a sandwich immunoassay is carried out by combining two types of antibodies that inhibit the enzyme activity of renin and antibodies that do not inhibit the enzyme activity. This gave good results. A sandwich immunoassay for renin
After reacting the solid-phased antibody specifically recognizing only renin (however, excluding the active site) with a sample (biological fluid) and washing, a labeled antibody recognizing the active site of renin is removed. It is performed by reacting. Further, it is preferable to react the immobilized monoclonal antibody and the labeled monoclonal antibody in one step in order to obtain high sensitivity.

More specifically, in the sandwich immunoassay, (a) at least one of the two antibodies used is a renin monoclonal antibody produced using recombinant human renin; While one inhibits the enzyme activity of active renin, the other antibody has a weak inhibition of the enzyme activity, so that active renin can be accurately sandwiched between the antibodies,
A highly sensitive measurement system was obtained.

As for the combination of different antibodies as in the above (b), dozens of monoclonal antibodies obtained by recombinant human renin are divided into several groups according to their properties, and the combination is performed by a monoclonal antibody combination test or a monoclonal antibody mutual test. It can be determined by examining an appropriate combination of antibodies from an inhibition test or the like. However, from the test results of the present inventors, the combination of monoclonal antibody 11-6 and an existing renin monoclonal antibody was 12-12 (Higaki et al, Acta endo
crinologica 120: 81-86).

In the above method, for example, 200 μl of a sample is placed in a tube, 100 μl of ¹²⁵ I-labeled renin monoclonal antibody and one renin antibody bead are added and mixed, and the mixture is stirred at room temperature for 1 to 24 hours, preferably 3 hours. After removing the reaction solution and washing three times with 2 ml of distilled water, the amount of radioactivity on the beads is measured, and the active renin concentration in the sample is determined from a standard curve obtained from the standard serum.

A major feature of the method of the present invention resides in that monoclonal antibodies against two types of active renin having different ability to inhibit the activity of active renin enzyme are used as described above. There is also a feature in improving the accuracy of the measured values.

That is, the immunoassay is generally carried out at ^10-9 to
This is a method for measuring a trace substance of about 10 ⁻¹² M, which is an extremely sensitive measurement method. For this reason, immunoassays need to measure a standard substance whose concentration has been strictly assayed together with the analyte, and accurately calculate the concentration contained in the test substance by comparison with the standard substance. Is the most important factor in the reliability of the measurements.

However, in the conventional methods for measuring renin, the preparation of a renin standard substance is required for biomaterials.
Therefore, it was inevitable that the concentration of renin present in the biological extract would change with each preparation. Another problem is that the physical properties of renin in the extract, which is considered to be the difference in antigenicity between the test substance and the standard substance, differ depending on the preparation, and the fundamental measurement principle of the immunological measurement method. Was a serious problem affecting

In the present invention, in order to ensure the reliability and reproducibility of the measured values in the immunoassay for renin,
The origin of the reference material was not determined from the biomaterial, but rather from recombinant human renin produced by a genetic engineering technique capable of stably supplying a constant amount of renin stably at all times. Since this recombinant human renin has high purity and high enzymatic activity of renin and is homogeneous without any change in antigenicity, the use of this as a standard substance makes the measurement reliability of the method of the present invention higher than before. Greatly improved.

Another factor supporting the method of the present invention is the presence of a monoclonal antibody which enables high radioactivity loading. That is, the specific radioactivity of the labeled antibody in the radioimmunoassay is 20 to 40 μCi / μg, desirably 23 to 35 μCi / μg, and as described above, the renin monoclonal antibody 11-6 contains ¹²⁵ I
Can be stably labeled with its specific activity of about 25 μCi / μg,
Sufficient activation intensity can be obtained as a radioimmunological assay. Moreover, the thus obtained renin monoclonal antibody 11 labeled with high specific activity
When -6 was used for a sandwich immunoassay, its maximum binding ability (B-BO) / T (%) stably maintained a binding ability of 70% or more immediately after production even after 8 weeks of production.

[0040]

According to the present invention, active renin in a body fluid is measured by a sandwich immunoassay method by combining monoclonal antibodies against human renin with those that inhibit active renin activity and those that do not. In addition, the use of a monoclonal antibody which can be loaded with high radioactivity as a monoclonal antibody and the use of recombinant reninin as a standard product enable highly sensitive and accurate measurement of active renin.

As a result, active renin which could not be measured without using a monoclonal antibody having an extremely high renin enzyme inhibitory activity can be measured with sufficient measurement sensitivity and at the lower limit of normality which could not be performed by the conventional measurement system. It has become possible to measure accurately.

[0042]

According to the method of the present invention, active human renin can be measured with high sensitivity even in a sample having a normal lower limit, which cannot be measured by the conventional human active renin measurement system. In addition, the reliability and reproducibility of measured values could be improved by using recombinant human renin as a standard substance. Therefore, according to the method of the present invention, it is possible to accurately evaluate the amount of active renin including normal subjects and diseases showing low renin activity, and it is advantageously used in diagnosis or treatment of diseases such as hypertensive diseases. It is possible.

[0043]

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

Example 1 Preparation of Renin Monoclonal Antibody: Recombinant human renin (6 μg) was added to Ba with complete Freund's adjuvant.
The mice were sensitized by administration to lb / c mice at three-week intervals. Three days after the final immunization, the spleen was collected and washed to obtain spleen cells. These spleen cells are treated with Pronase (from Sigma) for 1 minute, washed with an isotonic phosphate buffer solution (PBS), and then treated with an isotonic mannitol solution containing magnesium chloride and calcium chloride (pH
7.4) and mixed with mouse myeloma cells (P3U1) suspended in the same solution so as to have a cell ratio of 5: 1.

This mixed solution was placed in a parallel chamber (Shimadzu Corporation) of an electrofusion apparatus, and a DC pulse was applied at 420 V to fuse both cells. After the fusion, the cells were collected, washed with Dulbecco's modified minimum essential medium (DMEM), suspended in DMEM medium containing 10% fetal bovine serum, and dispensed into 24-well culture plates. From the next day, D containing hypoxanthine, thymidine and aminopterin
Hybridoma cells were selected from the MEM medium (HAT medium), and the culture supernatant of the growing hybridoma cells was used for screening.

6 μg of high-purity human renin purified from human kidney was reacted at room temperature for 20 seconds in the presence of 0.8 mCi of Na ¹²⁵ I and 20 μg of chloramine T. The reaction was stopped by adding 50 μg of sodium metabisulfite aqueous solution, and the reaction solution was separated by Sephadex G-
Purification was performed using a 50 column (1 × 30 cm, Pharmacia), and a 0.01 M phosphate buffer containing 0.5% BSA (p.
H7.4; hereinafter, diluted to) abbreviated as "buffer", ¹²⁵
I-labeled renin was obtained. The reaction rate was measured by paper chromatography using 75% methanol as a developing solution. As a result, the reaction rate was 60%, and the specific activity was 100 μCi / μg.

The two-antibody RIA method used for screening is based on the ¹²⁵ I-labeled renin 1 obtained above in 100 μl of culture supernatant.
After mixing with 00 μl (0.03 μCi) and reacting at 4 ° C. for 20 hours, 100 μl of a buffer containing 3% mouse normal serum was added.
1 ml of sheep antiserum dilution for mouse gamma globulin
Was added and left at room temperature for 30 minutes. After centrifugation at 3000 rpm for 30 minutes, the supernatant was removed, and the radioactivity of the precipitate was measured using a gamma counter (ARC-1000 manufactured by Aloka).

The positive wells in which the renin antibody activity of the supernatant was detected by the two-antibody RIA were cloned by the limiting dilution method to obtain hybridoma cells producing an antibody against human renin. The cells producing the antibody against human renin were intraperitoneally administered to Balb / c mice to which pristane had been administered one week beforehand, and after breeding for a certain period, ascites was collected.

The monoclonal antibody against human renin contained in the ascites was fractionated with ammonium sulfate, and then purified using an FPLC system (Pharmacia) with an ion exchange resin column.
Dozens of highly purified human renin monoclonal antibodies were obtained. These cells can also be cultured using a holofiber-type culture device, for example, Accucyst Jr, and a high-purity renin monoclonal antibody was similarly obtained from the culture supernatant obtained by the culture.

Example 2 Selection of Renin Monoclonal Antibody: (1) Grouping by Renin Enzyme Activity Inhibition Intensity 20 μl of Renin Monoclonal Antibody Obtained in Example 1 and Diluted Solution of Existing Renin Monoclonal Antibodies 12-12 Was treated with 5 mg of trypsin per 10 ml of plasma for 30 minutes at 37 ° C., and then activated human pooled plasma 200 supplemented with 10 mg of trypsin inhibitor.
μl and left at room temperature for 30 minutes.

After standing, the renin activity was measured using a renin activity measurement kit (renin rear beads II Dynabot). The activity inhibition was determined by dividing the result obtained by adding 20 μl of a diluted solution containing no human monoclonal antibody to the above-mentioned activated human pooled plasma by the renin activity measurement value (%).
Calculated as Table 1 shows the results.

[0052] (* Is a known monoclonal antibody)

(2) Combination test by IRMA Several monoclonal antibodies were selected based on the grouping of the above renin monoclonal antibodies and the results of the binding ability to ¹²⁵ I-labeled renin in Example 1. One of them was prepared by immobilizing 100 μl of the ¹²⁵ I-labeled renin monoclonal antibody prepared according to Example 8 and the antibody similarly prepared according to Example 8 on beads, and was subjected to the above-mentioned method. Human plasma, trypsin-treated plasma and 100 μl of serum before treatment were measured as samples. Table 2 shows the results.

[0054] (* Is a known monoclonal antibody)

The values in the first half of the results indicate the binding rate of plasma [(B−B0) / T (%)] before trypsin activation, and the latter figures indicate the binding rates after trypsin activation. Only the results obtained when the binding ratio before activation of trypsin is 2.0% or more are shown. As a measurement system using only activated renin, it is necessary to have a high activation binding rate and a relatively low plasma basal value. Therefore, the combination of 69-30 and 11-6 and the like constitute a system for measuring both active renin and inactive renin.

(3) Inhibition test between renin antibodies Anti-renin monoclonal antibody (100 μg) was placed in a test tube.
100 μl and 100 μl of the ¹²⁵ I-labeled renin prepared in Example 1 were taken and mixed. Further, beads immobilized with the renin monoclonal antibody shown above were added, and 6
After the reaction for 2 hours, the solid phase was washed twice with 2 ml of physiological saline, and the radioactivity of the solid phase was counted. As a control, 100 μl of a buffer solution containing no renin monoclonal antibody was used, and the count thus obtained was defined as 100%.

◎: when the radioactivity count of the solid phase is 98% or more of the control count, ○: 90 to 98%, Δ: 80 to 90%, and を: less than 80% The results are shown in Table 3 below as x.

[0058] (* Is a known monoclonal antibody)

From these results, it was found that monoclonal antibody 1
The combination of two types of monoclonal antibodies, 1-6 and existing renin monoclonal antibody 12-12, was judged to be optimal,
It was used for the subsequent active renin measurement system. The hybridoma A6 producing the monoclonal antibody 11-6
-11-6 was filed on September 21, 1994 with the FERM BP-4804
Deposited as

Example 3 Measurement of Affinity of Renin Monoclonal Antibody 11-6: The renin monoclonal antibody 11-6 was serially diluted with a buffer, and reacted with ¹²⁵ I-labeled renin by the two-antibody method RIA shown in Example 1. Binding rate [(B0-N) / T (%); bound count−nonspecific count / total count × 10
0)] was measured. The binding constant of the monoclonal antibody thus determined to renin was approximately 1 × 10 ⁻¹⁰ M.

Example 4 Measurement of renin activity inhibitory activity of renin monoclonal antibodies 11-6 and 12-12: High-purity renin monoclonal antibodies 11-6 and 12-12 were each subjected to 6.45 × 10 ^{-7 in a} buffer solution. Serial dilutions from M to 6.45 × 10 ⁻¹² M were made. 20 μl of each solution obtained by serial dilution,
10 minutes with 5 mg of trypsin per ml of plasma,
The mixture was treated at 37 ° C., added to 200 μl of activated human pooled plasma to which 10 mg of trypsin inhibitor had been added, allowed to stand at room temperature for 30 minutes, and then measured for renin activity using a renin activity measurement kit (Renin-Ria beads II Dynabot) did.

The activity inhibition was calculated by dividing the value obtained by adding 20 μl of the buffer solution containing no human monoclonal antibody to the above-mentioned activated human pooled plasma to the renin activity measurement value as the inhibition rate (%). As a result, the renin monoclonal antibody 11-6 inhibited the enzyme activity of renin by 50% or more at a concentration of 5 × 10 ^{−8 to} 5 × 10 ⁻⁹ M, but 5 × 10 ⁻⁸ M
^At a concentration of ^-10 M, the enzyme activity of renin was less than 50%. On the other hand, renin monoclonal antibody 12-12 is 5 ×
At a concentration of 10 ^-8 M, the enzyme activity of renin was less than 50%.

EXAMPLE 5 Binding of Renin Monoclonal Antibody to Horseradish Peroxidase: Binding of monoclonal antibody 11-6 to horseradish peroxidase (POD) was performed using activated maleimide and S-acetylmercaptosuccinimide. . 10 mg of the antibody was reacted at room temperature with S-acetylmercaptosuccinimide dissolved in dimethylformamide at a molar ratio of 1:10.

After the reaction, the acetyl group was removed using hydroxylamine hydrochloride to release the SH group. The reaction solution was purified using Sephadex G25, and the eluted fractions were collected, concentrated and stored at 4 ° C. POD (Boehringer) 1
0 mg was reacted with activated maleimide (Dojindo) at room temperature at a molar ratio of 1: 100. After the reaction, the reaction solution was purified using Sephadex G25, and the eluted fraction was collected and concentrated.

The thus prepared SH-introduced monoclonal antibody 11-6 and maleimide-introduced POD were mixed at a molar ratio of 1: 1 and reacted at 4 ° C. overnight. The next day, the reaction solution was further reacted at room temperature for 4 hours, and the reaction solution was purified by BioGel A-0.5m (Bio-Rad). The eluted fractions were collected and concentrated to obtain POD-bound renin monoclonal antibody 11-6.

Example 6 Enzyme-linked immunosorbent assay for human active renin (ELIS
A): 200 μl of the standard sample and 100 μl of the POD-conjugated renin monoclonal antibody diluent prepared in Example 5 were added to the reaction tray, and one bead having the renin monoclonal antibody 12-12 immobilized thereon was added. Rotated at room temperature for hours. After washing three times with 2 ml of purified water, the beads were transferred to a test tube, and the OPD solution 300 was added to the substrate.
The usual enzyme reaction using μl was performed at 25 ° C. for 1 hour, and the absorbance at 492 nm was measured. Table 4 shows the results.
Shown in

[0067]

Example 7 Reactivity of human activated renin ELISA to prorenin: Expression of a human prorenin-producing Chinese hamster oocyte by introducing an expression vector encoding human prorenin prepared by Murakami et al. Human prorenin was obtained from the culture supernatant and purified. The cross-reactivity of this purified human prorenin was measured by the sandwich EIA of human active renin shown in Example 6.

That is, human prorenin was added to a sandwich EIA
Was serially diluted using a standard substance diluent, and its reactivity was evaluated. At the same time, human prorenin was activated by trypsin (Type II-S Sigma) using the method of Murakami et al., And the reactivity of the sample to which the trypsin inhibitor was added was evaluated in the same manner using a sandwich ELISA system. Table 5 shows the results. From these results, it was confirmed that the measured value of human prorenin increased 10 times or more after activation of trypsin. This increase due to the activation showed a similar change in measured values in the renin activity measurement system. As a result of this renin activity measurement, it was confirmed that active human renin was mixed in the purified human prorenin by about several percent. The renin activity was in accordance with the method described in Example 4.

[0070]

Example 8 Method for Preparing Radioimmunological Assay for Active Renin: This assay uses an antibody (monoclonal antibody) that specifically recognizes the active site of human renin and recognizes other than the active site of human renin. This is a method for specifically measuring active renin with an antibody (monoclonal antibody). That is, a monoclonal antibody that specifically recognizes active renin is used as a label,
The other monoclonal antibody is bound to the bead sphere to form a solid phase, the renin standard and the sample are reacted simultaneously, and after washing, the amount of radioactivity bound to the solid phase antibody is measured to determine the active renin concentration in the sample. Things.

Therefore, in the measurement, it is necessary to produce the following reagents. (1) Production of ¹²⁵ I-labeled renin antibody As a labeling antibody, the renin monoclonal antibody 11-6 having high inhibitory activity shown in Example 2 was used. CIS Na
Take 0.99 mCi of ¹²⁵ I into a silicon-coated glass tube, add 42 μg of the above monoclonal renin antibody solution, and add 10 μg / 10 μl of chloramine T (20 mM-P
B, pH = 7.4), and after stirring for 60 seconds, sodium metabisulfite was added at 20 μg / 20 μl (20 mM
-PB, pH = 7.4) was added to stop the reaction.

Next, the reaction solution was added to 0.5 g / 5 ml of ion exchange resin (Amberlite IRA400T) (buffer solution 1), and unreacted ¹²⁵ I was adsorbed on the ion exchange resin to obtain ¹²⁵ I-labeled renin. The antibody was purified. The labeling ratio determined by paper chromatography using 95% methanol of the reaction solution as a developing solution was 96%.
%, Thus the specific activity was 25.7 μCi / μg. (900 μCi × 0.96 / 42 μg × 0.8;
However, 0.8 is the counting efficiency of the counter.)

The purity of the reaction solution after the purification was 99% or more. Purification reaction solution was Buffer 1
And make the final radioactivity concentration about 30 kBq (0.81 μCi).
/ Ml. (However, the amount used for the assay is about 2 kBq / 100 μl / tube.)

Composition of buffer 1; 0.05M-phosphate buffer 3% -BSA 0.9% -NaCl 0.1% -NaN ₃ 0.005% -normal mouse globulin red No. 2 trace pH = 6. 8

(2) Production of Renin Antibody Beads As the solid phase antibody, the renin monoclonal antibody 12-12 having low inhibitory activity shown in Example 2 is used. 1/4 inch diameter polystyrene beads with 5% scatter solution
After washing for 1 hour, renin monoclonal antibody 12-12 prepared at a concentration of 10 μg / ml was bound to 1 mM-phosphate buffer (pH = 5.7) (2 μg per bead),
0.2M-MgSO _4, 0.25M- phosphate buffer (0.5
% -BSA / 0.9% -NaCl / 0.1 % -NaN 3)
After the stabilization, the liquid is removed, and the beads to which the antibody is bound are dried at a constant temperature and a low humidity (25 ° C./15%) to produce the beads.

(3) Production of standard serum for measuring active renin (standard renin serum) This product is produced by adding recombinant human renin antigen to normal horse serum. First, commercially available normal horse serum (SE105L)
C, manufactured by Equitech) and heat-treated (56 ° C./30 minutes) so as to have the same reactivity as human serum.
M-PMSF (phenylmethylsulfonyl fluoride)
/ Isopropanol, 0.2% -EDTA2Na, 0.1
After adding% -NaN ₃ , the mixture is filtered through a 0.2 μm filter to prepare a base serum.

Next, the recombinant human renin antigen (2
50 ng / ml) to the base serum to give 5, 20,
Prepare standard renin serum at a concentration of 100, 500 pg / ml. The prepared standard renin serum is stored frozen at −20 ° C. or lower or freeze-dried before use. In addition, the concentration of the recombinant human renin antigen was tested using a renin international standard (68/356) supplied from WHO.
In this case, the conversion factor between the active unit and the weight unit is 1p
g = 6 × 10 ⁻⁶ GU.

Example 9 Measurement of active renin concentration in a healthy subject: 200 μl of standard renin serum at each concentration, 100 μl of ¹²⁵ I-labeled renin antibody
l, one renin antibody bead was placed in a tube, mixed, and reacted by stirring at room temperature for 3 hours. The reaction solution was removed, and 2 ml of distilled water was added.
After washing three times with, the amount of radioactivity on the beads was measured, and a standard curve of active renin shown in FIG. 1 was obtained.

Next, the amount of radioactivity on the beads was measured in the same manner as described above for 536 healthy subjects, and the active renin concentration in each sample was determined from the standard curve of active renin. The results are shown in FIG. 2. This measurement method was highly sensitive, and was able to accurately measure not only the high active renin concentration but also the lower limit of healthy subjects.

Example 10 Correlation with renin activity: The correlation between this assay and renin activity was determined for 364 disease specimens. For the measurement of renin activity, renin rear beads II (manufactured by Dynabot) was used, and the measurement method was according to the instruction manual of the kit. Disease specimens include:
Includes essential hypertension, renal vascular hypertension, chronic renal failure, primary aldosteronism, renin producing tumor, pheochromocytoma, and other diseases. The results are shown in FIG. 3, where Y = 7.03
A positive correlation was observed at X + 1.45 (r = 0.944). (Y: active renin concentration: this measurement method, X: renin activity: renin / rear beads II)

[Brief description of the drawings]

FIG. 1 is a drawing showing a standard curve of the method of the present invention.

FIG. 2 is a drawing showing the distribution of active renin concentration in the serum of a healthy subject.

FIG. 3 is a drawing showing the correlation between the method of the present invention and renin activity.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12N 15/02 C12N 5/00 B C12P 21/08 15/00 C (C12P 21/08 C12R 1:91) (58) (Int.Cl. ⁶ , DB name) C07K 16/40 C12N 5/10 C01N 33/53 C01N 33/577 C01N 33/60 C12N 15/02 C12P 21/08 BIOSIS (DIALOG) WPI (DIALOG)

Claims (12)

(57) [Claims] (1) At an antibody concentration of 5 × 10 ⁻⁹ M,
Inhibits nin enzyme activity by more than 50%,
Load more than 20μCi / μg specific radioactivity and stably
Renin monoclonal antibodies that can be used . 2. The renin monoclonal antibody according to claim 1, designated as renin monoclonal antibody 11-6. 3. The renin monoclonal antibody according to claim 1, which is labeled with an enzyme or a radioisotope. 4. A renin enzyme prepared using recombinant human renin and having an antibody concentration of 5 × 10 ⁻⁹ M.
Inhibits the activity by 50% or more,
A laser that can be loaded with specific activity of at least Ci / μg
A hybridoma that produces a nin monoclonal antibody . 5. The hybridoma according to claim 4, wherein the hybridoma is obtained by fusing transformed cells with spleen cells derived from a non-human animal immunized with recombinant human renin. 6. The hybridoma according to claim 4, wherein the hybridoma is designated as hybridoma A6-11-6. 7. A method for immunologically measuring an active renin protein by using two or more kinds of monoclonal antibodies recognizing sterically different sites on an active renin protein and sandwiching active renin in a biological fluid between these antibodies. (A) at least one of these antibodies is 5 × 10
^-9 Te antibody concentration odor M, 50% or more renin activity
20 μCi / μg or more by radioactive iodine
Specific activity was allowed load is stable renin monoclonal antibodies that can be used is a compound that inhibits one enzyme activity of active renin (b) an antibody, other antibody is weak inhibition of the enzyme activity A method comprising: 8. The antibody that inhibits the enzyme activity of active renin is a renin monoclonal antibody 11-6 .
The method described in the section . 9. An antibody that inhibits the enzymatic activity of active renin is labeled antibody The method of claim 7 or paragraph 8 Claims weak antibody of enzymatic activity of the active renin is immobilized antibody. 10. The method according to claim 9, wherein the labeled antibody is labeled with radioactive iodine at a specific radioactivity of 20 to 40 μCi / μg. 11. The method of any of claims of claims paragraph 7 to paragraph 10 using a high-purity active renin as a standard. 12. The biological fluid is serum, plasma, urine, ascites, pleural effusion, whey, amniotic fluid, spinal fluid, extract or Ms. any of the claims paragraph 7 to paragraph 11 is either exudates The method described in the section.