JPH08285852A - Reagent for measuring human prorenin and measuring method using the same - Google Patents

Abstract

PURPOSE: To measure the quantity of human prorenin directly by combining an antibody for specifically recognizing the profragment part of human prorenin with an antibody for recognizing human renin. CONSTITUTION: The reagent for measuring human prorenin is prepared by combining an antibody for specifically recognizing the profraqment part of human prorenin with an antibody for recognizing human renin. More specifically, a profraqment antibody of anti-human prorenin for specifically recognizing an enzyme kinetically inactive human prorenin is combined with an antibody for recognizing the structure of human renin at other than the active part. With such arrangement, human prorenin can be measured directly. The antibody for specifically recognizing an enzyme kinetically inactive human prorenin is prepared from the serum of house rabbit. With such method, quantity of human prorenin can be measured with high sensitivity through simple operation as compared with an indirect measuring method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel reagent for immunologically measuring human prorenin and a method for measuring human prorenin using the same.

[0002]

2. Description of the Related Art Diabetic patients often tend to be complicated by vascular disorders such as arteriosclerosis as the duration of sickness increases, which causes an increase in mortality. by the way,
When this diabetic patient is complicated by angiopathies, the human prorenin level in the blood increases depending on the severity of the disease, and when the treatment is successful, the elevated human prorenin level in the blood decreases. The value is used as a marker of diabetic angiopathy ["The New England Journal of Medicine (N. En.
g. J. Med. ) ", Volume 312, 1412-14
17 pages (1985), "Higashijo Medical Journal", Volume 60,
Pages 342-350 (1990), “Clinical
Investigator (Clin. Investi
g. ) ”, Vol. 71, pp. 3-6 (1993)].

Therefore, some methods for measuring the prorenin level in human plasma have been proposed so far.
Since human plasma contains active renin as an enzyme protein and human prorenin, which is a precursor of active renin and is enzymatically inactive, human prorenin in plasma is partially activated in advance by cold acid. , After converting to renin using trypsin, the total renin amount is determined by the enzymatic mechanical method as the renin active amount or as the active renin amount by the immunological method, and the renin amount is separately determined by the enzymatic mechanical method or the immunological method. The indirect means of calculating the amount of human prorenin as the difference between the former and the latter had to be taken.

As a result of the recent discovery of a method capable of converting inactive human prorenin into an active form by a renin inhibitor,
Using a monoclonal antibody that specifically recognizes renin and a monoclonal antibody that recognizes both human prorenin and renin, the amount of total renin was measured immunologically, and the amount of human prorenin was determined from the difference between the amount of total renin and the amount of active renin. A method for calculating was developed [“Journal of Hypertens.”, Volume 11, Addendum 5, pages 240 to 241 (1993)]. This method has the advantage of being able to prevent the excessive degradation of human prorenin and renin by trypsin, but it utilizes the conversion of human prorenin to active renin by trypsin, and the total amount of renin and the amount of renin It is still an indirect method of separately measuring and and calculating the amount of human prorenin from the difference.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a method for directly measuring the amount of human prorenin instead of the conventional method for indirectly determining the amount of human prorenin. The purpose of this is to detect rapidly and accurately.

[0006]

Means for Solving the Problems The present inventors have found that if an anti-human prorenin profragment antibody that specifically recognizes enzymatically inactive human prorenin is obtained, this anti-human prorenin profragment antibody can be obtained. Focusing on the fact that human prorenin can be directly measured by using it in combination with an antibody that recognizes a structure other than the active site of human renin, as a result of various studies, an enzymatically inactive human prorenin profragment was identified. An antibody that specifically recognizes has been successfully prepared from rabbit serum, and the present invention has been completed based on this.

That is, the present invention provides a reagent for measuring human prorenin, which comprises a combination of an antibody specifically recognizing a profragment site of human prorenin and an antibody recognizing human renin.

Next, a method for preparing an antibody specifically recognizing the profragment site of human prorenin and an antibody recognizing human renin in the present invention will be described. First,
Human renal-derived prorenin cDNA is introduced into an expression vector (eg PSVDPR _n PA33) and incorporated into Chinese hamster ovary cells (hereinafter abbreviated as CHO cells). This is described, for example, in Poorman R.
A. ) Method [“Protein, Structure, Function, Genetics (St
ruture, Function, Genetic
s) ", Volume 1, pp. 135-145 (198)
6)].

Then, the CHO cells thus obtained were cultured in Dulbecco's modified Eagle medium (containing 10% fetal calf serum), and when the CHO cells were sufficiently grown, they were replaced with serum-free medium to remove prorenin. Obtain the containing culture supernatant.

The culture supernatant thus obtained is optionally centrifuged and then concentrated by salting out or by fractional precipitation using polyethylene glycol. The concentrate is further fractionated by CM-cellulose chromatography and then gel filtered to obtain purified prorenin.

It has been confirmed that this purified prorenin is the same as the prorenin in human plasma [American Journal of Physiology (Am. J.
Physiol. ), Vol. 258, E451-E45
Page 9 (1990)]. The amount of this prorenin is
Prorenin is treated with trypsin to be converted into renin, which can be used to digest sheep angiotensinogen, and the amount of angiotensin I produced can be measured and calculated.

Purification of renin can be carried out, for example, by adding trypsin (end point concentration: 100 μg / ml), reacting at 24 ° C. for 2 hours, and then performing affinity chromatography using a pepstatin-aminohexyl sepharose 4B column.

Next, the purified human renin obtained as described above is used as it is as an immunogen, and human prorenin is subjected to solid phase peptide synthesis at its profragment site (1-43 amino acid residues) to give a maleimide compound. A human renin antibody and a human prorenin antibody are prepared by using a substance bound to a carrier protein such as bovine serum albumin, ovalbumin, and hemocyanin of keyhole limpet using various crosslinking agents as an immunizing antigen.

That is, each immunizing antigen is mixed well with Freund's complete adjuvant and administered to a mature rabbit (body weight: about 2.5 kg) for immunization. This immunization is performed every two weeks, and a small amount of blood is collected from the marginal ear vein after the 5th time.
For example, the antibody titer is examined by the Octaloni method. And
When the antibody titer rises sufficiently, whole blood is collected to obtain antiserum. The antiserum is then treated with salting out and DEAE-cellulose chromatography to prepare IgG.

Further, if necessary, F (ab ') ₂ is prepared by using pepsin digestion, affinity chromatography with gel filtration protein A, etc.
Anti-human renin Fab 'and anti-human prorenin Fab' can be obtained by reduction with mercaptoethylamine or the like.

Preparation of Fab ′ from such IgG is carried out by a known method such as “J. Immunoassay”, Vol. 4, 20.
It can be performed by the method described on pages 9 to 327 (1983).

As a combination of antibodies in the case of measuring human prorenin using the immunoassay reagent thus prepared, when the human renin antibody is immobilized, the profragment site of human prorenin is specific. When an antibody that specifically recognizes the profragment site of human prorenin is immobilized, the human renin antibody may be the enzyme-labeled antibody.

Regarding these antibodies, since a relatively large amount of antibody is generally required for the immobilized antibody, it is preferable to use a monoclonal antibody that can stably obtain a large amount of antibody, but if desired, an anti-antibody may be used. It is also possible to use a polyclonal antibody that is serum.

The enzyme-labeled antibody may be either a monoclonal antibody or a polyclonal antibody, as long as it recognizes a site different from the site recognized by the immobilized antibody. At this time, as the labeling enzyme, alkaline phosphatase, β-D-galactosidase, peroxidase, glucooxidase and the like can be used. In the present invention, horseradish oxidase is preferably used.

As the cross-linking agent, N, N'-o-phenylene dimaleimide, 4- (N-maleimidomethyl) is used.
Cyclohexanoic acid / N-succinimide ester, 3-
(2-Pyridyldithio) propionic acid / N-succinimide ester, 4,4′-dithiodipyridine, and other known crosslinking agents can be used.

The reaction of these cross-linking agents with the labeling enzyme and the antibody can be carried out according to a known method. In some cases, the antibody is a fragment thereof, such as F
Ab ′, Fab and F (ab ′) ₂ may be used.

If the enzyme-labeled product obtained by using the above-mentioned cross-linking agent is purified by affinity chromatography or the like, an immunoassay system with higher sensitivity becomes possible. The purified enzyme-labeled product is added with thimerosal or glycerin as a stabilizer, or freeze-dried and stored in a cool and dark place.

The combination of the thus-prepared antibody recognizing the pro-fragment (1-43 amino acid residue) site of human prorenin and the antibody recognizing human renin does not react at all with the human renin preparation. However, it reacts specifically and quantitatively with human prorenin preparation.

FIG. 1 is a graph showing the action specificity of the measuring reagent of the present invention for human prorenin (A) and human renin (B). As can be seen, the absorbance is remarkably increased with the decrease of human prorenin. In the case of human renin, while it decreased, there was no change. Also, in FIG.
It is a graph showing the dose dependency of the measurement reagent for human prorenin at this time, but this graph is in good agreement with the standard curve, and it is possible to measure human prorenin within the concentration range of 10 to 1000 pg / ml. I understand.

For comparison, FIG. 3 is a graph showing the results when a combination of anti-human renin rabbit serum and enzyme-labeled anti-human renin rabbit serum was reacted with human prorenin preparation and human renin preparation. However, as is clear from this, since this reacts with both the human prorenin preparation (A) and the human renin preparation (B), it is unsuitable as a reagent for directly measuring human prorenin.

Next, a specific method for measuring human prorenin using the reagent of the present invention will be described. Immobilization of the antibody prepared as described above is appropriately diluted with a sodium carbonate buffer (pH 9.6) and adsorbed on a 96-well ELISA plate. After that, the liquid in the well is removed, and in order to prevent nonspecific adsorption of the measurement sample and the enzyme-labeled antibody to the well, a phosphate buffer solution (hereinafter abbreviated as PBS) containing ovalbumin, bovine serum albumin, milk casein, etc. is used. Fill wells and store at 4 ° C until used for measurements.

Then, the measurement sample is diluted with an appropriate amount of a surfactant, for example, PBS containing 0.05% Tween 20 and
Collect in wells. After standing at 25 ° C for 2 hours,
Wash wells with PBS containing 0.05% Tween 20. Next, an enzyme-labeled antibody appropriately diluted with the above PBS is added. After standing under the same conditions as the sample solution, washing with the above buffer solution again, adding the color former and H ₂ O ₂ and reacting at 37 ° C for 30 minutes, then adding 2M sulfuric acid to stop the reaction and measure the absorbance To do.

At this time, if the antibody is labeled with peroxidase, ABTS [2,2'-azinobis (3-ethylbenzothiazoline) -6-sulfonic acid], TMZ (tetramethylbenzidine), OPD can be used as a color former. (O
-Phenylenediamine), TMBZ (3,3 ', 5,
Known reagents such as 5'-tetramethylbenzidine) can be used.

The reaction conditions between the solidified antibody and the measurement reagent and the reaction condition between the measurement sample and the labeled antibody are 25 ° C. for 2 hours or 37 ° C. for 1 hour. However, it is necessary to be careful when performing under these conditions, because the background becomes high and the sensitivity tends to decrease accordingly.

Instead of the enzyme-labeled antibody, a radioactive compound such as ¹²⁵ I is labeled on the antibody by a known method,
It is also possible to measure human prorenin using a conventional radioimmunoassay and a similar method such as a fluorescent immunoassay.

[0031]

EXAMPLES The present invention will be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

Reference Example 1 [Preparation of human prorenin profragment (1-43 amino acid residue) antibody] 8 mg of hemocyanin in 0.1 M PBS (pH 7.0).
Dissolve in 0.5 ml, and add N- (γ-maleimide butyrooxysuccinimide (hereinafter abbreviated as GMBS)) in dimethylformaldehyde solution (15 mg / ml) 50
μl is added, and the mixture is reacted at room temperature for 3 hours. After the reaction, the reaction solution was passed through a Sephadex G-25 column to remove unreacted G
Remove MBS. The reaction product was added to 0.1M PBS (p
Elution with H6.0) gives 2 ml of eluate. Next, to the above-mentioned eluate, 4 mg of a prorenin profragment (1-43 amino acid residue) synthetic peptide without a protecting group was added, and the mixture was reacted at room temperature for 3 hours to give a prorenin profragment synthetic peptide hemocyanin conjugate (immune antigen). obtain. This solution is freeze-dried until the animal is immunized -20
Store at ℃. The immune antigen is adjusted to a protein concentration of 1 mg / ml with physiological saline. An immunizing antigen (1 mg as protein amount) is mixed well with an equal amount of Freund's complete adjuvant, and the whole amount is injected subcutaneously into several sites in a New Zealand White rabbit (body weight: about 2.5 kg).
Thereafter, the amount of the immunizing antigen is half the initial amount, and immunization is similarly performed 5 to 6 times at 2-week intervals. After the final immunization, whole blood is collected by a conventional method to obtain antiserum.

Reference Example 2 (Preparation of human renin antibody) Purified human renin (0.1 mg) was mixed well with 1 ml of Freund's complete adjuvant, and a total amount of it was subcutaneously applied to several New Zealand white rabbits (body weight: about 2.5 kg). Inject separately. Thereafter, the same immunization is performed 5 to 6 times at 2 week intervals. After the final immunization, whole blood is collected by a conventional method to obtain antiserum.

Reference Example 3 (Preparation of Enzyme-Labeled Human Prorenin Profragment Antibody) 1 ml of the human prorenin profragment antibody obtained in Reference Example 1 was introduced into a Protein A Sepharose 4B column and treated by a conventional method to prepare IgG fraction. Get the picture. IgG above
Pepsin (0.2 mg) was added to the fraction, and the mixture was reacted with 0.1 M sodium acetate buffer (pH 4.5) at 37 ° C. for 48 hours, followed by gel filtration using 0.1 M PBS (pH 7.0) to obtain F (ab ′). _Two fractions (4 mg / ml) are obtained.
The F (ab ') ₂ fraction then contained 0.5 mM EDTA.
100 μl of a solution containing 0.5 mM 2 mercaptomethylamine dissolved in 1 M PBS (pH 6.0) was added to the mixture at 37 ° C. and 9
After 0 minutes of reaction, treatment with Sephadex G-25 column chromatography gives a solution containing 2 mg of Fab 'fraction of the anti-human prorenin profragment antibody.

Then, 2 ml of horseradish-derived peroxidase (hereinafter abbreviated as POD) was added to 0.3 ml of 0.1 M PBS.
70 mM N-succinimidyl-in a solution of g
4- (N-maleimidomethyl) -cyclohexane-1-
30 μl of dimethylformamide solution containing carboxylic acid was added and reacted at 30 ° C. for 60 minutes, and the unreacted activator was separated by Sephadex G-25 column using 0.1 M PBS (pH 6.0) and activated. Get POD. This activated POD solution was adjusted to pH 7.0 to 7.5, the above antibody Fab 'fraction (2 mg) solution was added, and the mixture was reacted at 4 ° C for 18 hours to obtain a POD prorenin antibody Fab' conjugate.

This POD prorenin antibody Fab ′ conjugate (POD-labeled prorenin antibody) was treated with Ultrogel ACA4.
Purify using 4 columns. The POD-labeled prorenin antibody is stored at -20 ° C until used for measurement.

Reference Example 4 (Preparation of Enzyme-Labeled Human Renin Antibody) The human renin antibody obtained in Reference Example 2 was used in place of the human prorenin profragment antibody in Reference Example 3, and the POD renin antibody Fab conjugate (POD labeled) was similarly used. Renin antibody).

Example 100 μl of the anti-renin antibody obtained in Reference Example 2 (diluted 3,000 times with carbonate buffer (pH 9.6)) was dispensed into each well of a Nunc 96-well microplate at 4 ° C. Let stand for 16 hours. Remove antibody solution, 0.05% Tween 2
After washing the plate 3 times with PBS containing 0 (pH 7.4) (Tween PBS), PBS containing 0.1% bovine serum albumin (BSA) (pH 7.4) (BSA / PB)
S) is added and the mixture is allowed to stand at 4 ° C. for 16 hours or longer. BSA / P
Discard the BS, wash with Tween PBS and store at 4 ° C. A human prorenin preparation is appropriately diluted with BSA / PBS, 100 μl of each is added to the antibody-treated microplate, and the mixture is reacted at room temperature for 2 hours. After the reaction, the plate was washed 5 times with 300 μl of Tween PBS, 100 μl of POD-labeled prorenin antibody (diluted 300 times with BSA / PBS) was added to each well and reacted at room temperature for 2 hours, and Tween P
Wash 5 times with 300 μl of BS. Next, a coloring liquid (3,
150 μl of 0.1 M acetate buffer, pH 5.5 containing 5.5 mM of 3 ′, 5,5′-tetramethylbenzidine is injected into each well. After standing at 37 ° C. for 5 minutes, 50 μl of hydrogen peroxide solution (10 μl of H ₂ O ₂ diluted to 30 μl with distilled water)
Is added and the mixture is reacted at 37 ° C. for 20 minutes. 100 μl of 2M H ₂ SO ₄ is added as a reaction stop solution, and the absorbance at OD 450 mμ is measured with a plate reader.

Next, the same operation is performed using the POD-labeled renin antibody obtained in Reference Example 4 instead of the POD-labeled prorenin antibody and the Renin sample instead of the PR sample. The results are shown in FIG.

As can be seen from FIG. 1, in the direct measurement using the reagent of the present invention, a dose-response curve was shown according to the dilution series of the prorenin preparation, whereas no reaction was observed with the renin preparation. Was not shown.

On the other hand, the immunological measurement of renin shows a dose-response curve according to the dilution series of the prorenin preparation and the renin preparation as shown in FIG. 3, and recognizes the common site of prorenin and renin. It indicates that

Next, regarding the measurement range and the sensitivity, a linear relationship is obtained in the range of 10 pg / ml to 1,000 pg / ml as shown in FIG. 2, and it is possible to measure in the practical sensitivity and concentration range. Was shown.

[0043]

INDUSTRIAL APPLICABILITY The present invention is a human prorenin assay reagent comprising a combination of an antibody that recognizes a human prorenin profragment site and an antibody that recognizes human renin. It is possible to measure, and the amount of human prorenin can be measured with high sensitivity as compared with the conventional indirect measuring method, which is easy to operate. Therefore, it is suitable as a diagnostic agent for various complications of diabetes.

[Brief description of drawings]

FIG. 1 is a graph showing the specificity of the action of the measuring reagent of the present invention.

FIG. 2 is a graph showing the dose dependence of the measurement reagent of the present invention.

FIG. 3 is a graph showing the non-specificity of conventionally known anti-human renin rabbit serum.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yuichi Ishida 1947 Higashi-Owa, Washimiya-cho, Kita-Katsushika-gun Saitama 8-205 West Heights 8-205 (72) Inventor, Yasuhiko Hatano 95-25, Mukoengaru, Engaru-cho, Monbetsu-gun, Hokkaido

Claims (5)

[Claims] 1. A reagent for measuring human prorenin, which comprises a combination of an antibody specifically recognizing a profragment site of human prorenin and an antibody recognizing human renin. 2. The reagent for measuring human prorenin according to claim 1, wherein the profragment site of human prorenin is amino acid residues 1 to 43. 3. An immobilized antibody that specifically recognizes the pro-fragment site of human prorenin is added to a measurement sample and an enzyme-labeled antibody that recognizes human renin, and the mixture is reacted. Then, the amount of change in human prorenin is measured and prepared in advance. A method for measuring human prorenin, which is used to determine the amount of human prorenin in comparison with the prepared standard line. 4. An immobilized antibody that recognizes human renin is added with a measurement sample and an enzyme-labeled antibody that specifically recognizes the profragment site of human prorenin, and the mixture is reacted. Then, the amount of change in human prorenin is measured and prepared in advance. The method for measuring human prorenin to determine the amount of human prorenin in comparison with the standard line. 5. The method according to claim 3, wherein the enzyme-labeled antibody is developed.
Or the method for measuring human prorenin according to 4 above.