JP3271159B2 - Renin active substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel renin active substance which expresses an enzymatic activity, that is, a renin activity, in which a peptide-specific antibody capable of forming a conjugate by binding to a human prorenin profragment peptide binds to human prorenin. It is about.

[0002]

BACKGROUND OF THE INVENTION Prorenin is mainly found in the kidney.
It is produced as a precursor of ripe renin, that is, a profragment having a 43 amino acid sequence bound to the N-terminal of the ripe renin, and released into the blood. This prorenin is present in the blood in an amount about 10 times that of the ripe renin, but does not show any enzymatic activity itself, so that the profragment portion is cleaved with a proteolytic enzyme such as trypsin or pepsin to obtain the ripe renin. Conversion to renin has been performed for a long time. As a relatively new method, a method using no enzyme has also been proposed. For example, a method of activating without changing the primary structure in an acidic or low-temperature state [“Natur”
e) ", Volume 288, Pages 702-705 (198
0), "Journal of Biological Chemistry (J. Biol. Chem.)", Vol. 262, pp. 2472-2577 (1987), "Clinical Chemistry (Clin. Chem.), Vol. 37,
1811-1819 (1991) "," Journal of Biological Chemistry (J. Bi)
ol. Chem. ) ", Vol. 267, No. 11753-1
1759 (1992)], a low-molecular-weight renin inhibitor is bound to the enzyme active site hidden in the deep groove of the three-dimensional structure of prorenin to form an open form, and an antibody that recognizes the vicinity of the active site of ripe renin is bound. Method of enabling [Journal of Biological Chemistry (J. Bi
ol. Chem. ) ", Vol. 267, No. 22837-2
2842 page (1992)]. In addition, there have been many reports that anti-peptide antibodies at the middle and C-terminal of prorenin profragment bind to prorenin to form a conjugate.
This was not known to have enzymatic activity.

[0003]

SUMMARY OF THE INVENTION The object of the present invention is to provide a novel renin active substance using an anti-peptide antibody capable of recognizing a specific amino acid sequence in a human prorenin profragment. .

[0004]

Means for Solving the Problems The present inventors have firstly developed a peptide having an amino acid sequence composed of amino acids from the first leucine to the fifteenth arginine at the N-terminal of human prorenin profragment. Provided a renin-active substance comprising a conjugate of an antibody specifically recognizing as an antigen and human prorenin (Japanese Patent Application Laid-Open No. 10-27).
No. 9600), and as a result of further study, it has an arbitrary amino acid sequence in the amino acid sequence composed of 33 amino acids from the 11th isoleucine to the 43rd arginine in the human prorenin profragment. An antibody that specifically recognizes a peptide also binds to human prorenin to become a substance exhibiting renin activity, and the antibody contains 11 to 11th leucine to 11th isoleucine in the human prorenin profragment.
The conjugate of human prorenin and an equimolar mixed antibody to which an anti-peptide antibody specifically recognizing an amino acid sequence composed of amino acids is compared with the conjugate of each anti-peptide antibody alone and human prorenin. The present inventors have found that the activity is improved, and have accomplished the present invention based on this finding.

[0005] That is, the present invention provides an amino acid sequence composed of 33 amino acids from the 11th isoleucine to the 43rd arginine in a human prorenin profragment having an amino acid sequence composed of 43 amino acids. A conjugate of at least one anti-peptide antibody specifically recognizing an amino acid sequence composed of 15 or more amino acids at any position in human prorenin, or an amino acid sequence composed of 43 amino acids Amino acid sequence consisting of 15 or more amino acids at any position in the amino acid sequence consisting of 33 amino acids from the 11th isoleucine to the 43rd arginine in a human prorenin profragment having At least one anti-peptide antibody that specifically recognizes A renin active substance comprising a conjugate of human prorenin and a mixed antibody with an anti-peptide antibody that specifically recognizes an amino acid sequence composed of 11 amino acids from the first leucine to the eleventh isoleucine in the fragment Is provided.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a renin active substance comprising a conjugate of an anti-peptide antibody and human prorenin,
This anti-peptide antibody is composed of 15 or more amino acids at any position in the amino acid sequence composed of 33 amino acids from the 11th isoleucine to the 43rd arginine in the human prorenin profragment. It is characterized in that it specifically recognizes a peptide having a modified amino acid sequence. Then, the anti-peptide antibody and 11 to 11th isoleucine from the first leucine to the eleventh isoleucine in the human prorenin profragment are used.
When an equimolar mixed antibody with an anti-peptide antibody specifically recognizing an amino acid sequence composed of two amino acids is used, renin activity is significantly increased. This human prorenin profragment is a substance having the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing.

[0007] The renin active substance of the present invention comprises a peptide consisting of 16 amino acids from the 11th isoleucine to the 26th arginine (hereinafter referred to as M peptide) and a C-terminal portion of the prorenin profragment. That is, a peptide having a cysteine residue added to the C-terminal of each peptide of a peptide consisting of 15 amino acids from the 27th glycine to the 41st methionine (hereinafter referred to as C peptide) is synthesized, and these are used. For example, it can be prepared by the following method. Each of these peptides is bound to a carrier protein such as bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, etc. using a cross-linking agent such as a maleimide compound to prepare immunizing antigens, and then each immunizing antigen is treated with Freund's. Immunization was performed by subcutaneously administering to adult rabbits at intervals of 2 weeks mixed with complete adjuvant, and a small amount of blood was collected from the peripheral vein of the ear after the 5th time, and the antibody titer was examined. When the antibody titer was sufficiently increased Perform whole blood collection to obtain antiserum. Next, the antiserum is salted out and purified by affinity chromatography using an affinity gel to which a predetermined synthetic peptide is bound.

[0008] From the preparation of the immunizing antigen to the affinity purification of the antibody, known methods (for example, published by Shujunsha on September 10, 1994, Shinobu Okai, Kunio Tsujimura, Akiki Inagaki, "Anti-Peptide Antibody Experiment Protocol", 48 page).

Next, the anti-peptide antibody thus affinity-purified was added to a human prorenin solution diluted with a physiological saline solution containing bovine serum, and added at a temperature of 4 ° C.
The conjugate can be prepared by reacting for up to 24 hours. Next, a sheep-angiotensinogen solution was added to this reaction solution as a human renin substrate, and the solution was added at 37 ° C. for 60 hours.
After reacting for 1 minute, the reaction is stopped by cooling on ice, and angiotensin I is produced in the reaction solution. Therefore, this conjugate is a substance that expresses enzymatic activity, that is, renin activity.

The renin active substance thus obtained was subjected to Western blotting and protein-protein interaction measurement using surface plasmon resonance to confirm that the purified antibody specifically bound to human prorenin. did. Next, each conjugate was allowed to react with sheep angiotensinogen as a renin substrate, and the renin activity was compared using trypsin-treated prorenin as a positive control and normal rabbit serum instead of the antibody as a negative control. As a result, the antibody conjugate showed renin activity equivalent to that of the positive control, whereas the negative control showed almost no renin activity. The renin active substance was found to be completely different from the open-type substance produced by the low molecular renin inhibitor.

FIG. 1 shows the results of Western blot analysis using human prorenin and ripe renin obtained by treating prorenin with trypsin. As can be seen, spots where the respective antibodies were bound were observed at the migration position for prorenin (lanes 1 and 3), whereas no spot was observed for the fully matured renin (lanes 2 and 4). 2 and 3
Is a method in which human prorenin is passed at a constant flow rate through a flow cell in which an antibody purified by affinity chromatography is chemically bound using a protein-protein interaction measuring apparatus (Pharmacia Biotech, Biacore 100) to change the surface plasmon resonance. By measuring the amount, the results of examining the binding ability between the anti-peptide antibody and prorenin were measured, and the horizontal axis was the reaction time, and the vertical axis was RU (Resonance)
(Units). FIG. 2 shows an anti-peptide antibody (hereinafter referred to as anti-M) which specifically recognizes an amino acid sequence composed of 16 amino acids from the 11th isoleucine to the 26th arginine of the profragment.
FIG. 3 shows an anti-peptide antibody (hereinafter referred to as an anti-C antibody) that specifically recognizes an amino acid sequence composed of 15 amino acids from glycine 27 to methionine 41 of the profragment. The solid line in each figure is for prorenin, and the chain line is for control buffer. As can be seen from these figures, binding of each anti-peptide antibody to prorenin is observed, whereas binding is not observed in the control buffer.

Next, FIG. 4 shows that a conjugate obtained by reacting an affinity peptide-purified anti-peptide antibody with human prorenin is reacted with sheep angiotensinogen as a renin substrate, and is reacted with an enzyme, ie, a renin active substance. FIG. 4 is a graph showing the results obtained by quantifying the amount of angiotensin I to be measured by enzyme immunoassay and determining renin activity. From this figure, the renin activity of ripe renin obtained by treating prorenin with trypsin and the anti-M peptide antibody were used as positive controls. Alternatively, the renin activity of the conjugate of the anti-C peptide antibody and human prorenin shows almost the same activity, whereas the system using normal rabbit serum instead of the anti-peptide antibody as a negative control shows almost renin activity. I understand that there is no.

Thus, an anti-M peptide antibody having an intermediate portion of the prorenin profragment, ie, the amino acid sequence from the 11th to the 26th, and a C-terminal portion, ie, the 27th amino acid sequence.
The anti-C peptide antibody having the 4th to 41st amino acid sequences specifically binds to human prorenin, and the formed conjugate expresses renin activity. It can be seen that they are converted to different renin active substances.

FIG. 5 shows an anti-M peptide antibody or an anti-C peptide antibody and an amino acid sequence composed of 11 amino acids from the first leucine to the eleventh isoleucine in the human prorenin profragment. A conjugate formed by combining an equi-molar mixture of an anti-peptide antibody that specifically recognizes (hereinafter referred to as an anti-N-peptide antibody) with human prorenin and each anti-peptide antibody alone are combined with human prorenin. Figure 3 is a graph comparing renin activity for bound conjugates with the trypsin activation level of prorenin used as 100%. From this figure, the renin activity was 1.22 times higher in the mixture of the anti-M peptide antibody and the anti-N peptide antibody than in the anti-M peptide antibody alone, and higher in the mixture of the anti-C peptide antibody and the anti-N peptide antibody. It can be seen that both are 1.52 times higher than the peptide antibody alone. In particular, the combination of an anti-C peptide antibody and an anti-N peptide antibody showed 128%, far exceeding the conventionally known case of prorenin trypsin activation.

[0015]

Next, the present invention will be described in more detail by way of examples.

Example 1 (1) Preparation of prorenin profragment peptide immunizing antigen A cysteine residue was added to the C-terminal side of a peptide having the amino acid sequence of SEQ ID NO: 2 according to the solid phase method commonly used for peptide synthesis. A peptide having 17 amino acid residues was synthesized, separated and purified by high performance liquid chromatography. On the other hand, 16 mg of hemocyanin as a carrier protein was added to a 0.1 M phosphate buffer (pH 7.0).
2) The solution was dissolved in 1 ml, and 100 μl of a solution of N- (γ-maleimidobutyroxy) succinimide in dimethylformamide (15 mg / ml) was added to the resulting solution, followed by reaction at room temperature for 3 hours. This reaction mixture was separated with Sephadex G-
25 columns (Pharmacia Bio Inc., 15 mmφ × 3
00mm) and gel filtration to remove unreacted N- (γ-maleimidobutyroxy) succinimide.
The reaction product was eluted using a 0.1 M phosphate buffer (pH 6.0). Next, 10 mg of the above purified peptide was added to this eluate and reacted at room temperature for 3 hours to obtain a peptide hemocyanin conjugate, that is, an immunizing antigen. The pro-fragment intermediate peptide immunizing antigen thus obtained is stored at -80 ° C until use.

(2) Preparation of Anti-Peptide Antibody The pro-fragment intermediate peptide immunizing antigen obtained in (1) was adjusted to a protein concentration of 1 mg / ml with physiological saline, and thoroughly mixed with an equal volume of complete Freund's adjuvant. , A New Zealand white rabbit (weight about 2.
5 kg) was injected subcutaneously in several places. Since then
The first half of the immunizing antigen was injected subcutaneously at two-week intervals, and this was repeated until a sufficient increase in antibody titer was confirmed. Thereafter, whole blood was collected by a conventional method to obtain an antiserum.

(3) Preparation of Affinity Chromatography Purified Antibody 1 mg of the purified peptide obtained in the above (1) was mixed with 3 ml of amine-bonded gel (manufactured by Bio-Rad Co., trade name: Affigel 102).
Was chemically bonded using N- (γ-maleimidobutyroxy) succinimide to prepare 3 ml of an affinity chromatography column. Then, the antiserum 1 obtained in (2)
0 ml and 0.1 M-phosphate buffer (pH 7.0) were mixed and fractionated and precipitated using 50% ammonium sulfate.
After collecting the IgG fraction, this was solubilized by adding 10 ml of a 0.1 M phosphate buffer, and the same 0.1 M-phosphate buffer was added.
Dialysis was performed at 4 ° C. for 15 hours using 2000 ml of a phosphate buffer. Next, the whole amount of the dialyzed IgG solution was passed through the affinity column (2 ml) to completely remove the non-adsorbed fraction, and the antibody was eluted with a 0.1 M glycine hydrochloride solution (pH 2.5). The solution was added for neutralization. The purified anti-M peptide antibody thus obtained is stored frozen until use.

(4) Production of a renin active substance A known method [Journal of Hypertensions (J. Hypertens.), Vol. 4, 388-3]
90 (1986)], a human renal-derived prorenin cDNA was introduced into an expression vector and incorporated into Chinese hamster ovary cells (hereinafter abbreviated as CHO cells). The cells were cultured in Eagle's medium, and when the CHO cells had grown sufficiently, the medium was replaced with a serum-free medium to obtain a culture supernatant containing recombinant human prorenin. The CHO culture supernatant thus obtained was dialyzed against a phosphate buffered saline containing 5 mM-EDTA, and purified to prepare human prorenin.

Next, about 8 ng of this human prorenin was electrophoresed on an SDS-polyacrylamide gel (gel concentration: 7.5%) according to a conventional method, and then transferred onto a nitrocellulose membrane. ) Was allowed to react with the purified fragment intermediate peptide affinity antibody obtained in the above step and allowed to bind. The reaction product was colored using an ABC kit (manufactured by Vector Laboratory) and analyzed to confirm that the antibody was bound at the position where human prorenin migrated. Further, in a flow cell (manufactured by Pharmacia Biotech) in which the affinity chromatography-purified antibody obtained in (3) was amine-coupled, 8 nM human prorenin was added.
The binding state between human prorenin and the affinity chromatography-purified antibody was determined by flowing a phosphate buffer in which the solution was dissolved at a flow rate of 10 μl / min and observing the change in surface plasmon resonance using a protein-protein interaction measuring device. Upon examination, it was confirmed that both formed a conjugate.

Example 2 A peptide having 16 amino acid residues obtained by adding a cysteine residue to the C-terminal side of a peptide having the amino acid sequence of SEQ ID NO: 3 in accordance with the solid phase method commonly used for peptide synthesis was synthesized. Then, a peptide hemocyanin conjugate was prepared in the same manner as in Example 1 and used as an immunizing antigen. Next, after preparing an antiserum from this immunizing antigen in the same manner as in Example 1,
Treatment with an affinity chromatography column prepared using the purified peptide gave a purified anti-C peptide antibody.
Next, human prorenin obtained in the same manner as in Example 1 was reacted with the above purified antibody to produce a renin active substance.

Example 3 A peptide having 12 amino acid residues obtained by adding a cysteine residue to the C-terminal side of the N peptide in the amino acid sequence of SEQ ID NO: 4 in accordance with the solid phase method commonly used for peptide synthesis was prepared. The peptide was synthesized, and a peptide hemocyanin conjugate was prepared in the same manner as in Example 1 and used as an immunizing antigen. Next, an antiserum was prepared from the immunized antigen in the same manner as in Example 1, and then treated with an affinity chromatography column prepared using a purified peptide to obtain a purified anti-N peptide antibody. Next, 50 μl of the 0.6 μM human prorenin solution, 50 μl of the 0.6 μM antibody solution of the purified anti-M peptide antibody obtained in Example 1 or the purified anti-C peptide antibody obtained in Example 2 and the above were obtained. 0.6 μ of purified anti-N peptide antibody
A renin active substance was produced by reacting each mixed antibody with human prorenin by adding 50 μl of M antibody solution and reacting at 4 ° C. for 20 hours.

Reference Example 1 (1) Preparation of enzyme-labeled angiotensin I 5 mg of horseradish peroxidase (manufactured by Boehringer Mannheim) was dissolved in a 0.2 M phosphate buffer, and 1 ml of a 2.5% glutaraldehyde aqueous solution was added to the obtained solution. After the addition, the unreacted glutaraldehyde was removed by gel filtration. Next, to this solution was added 130 μl of a solution prepared by dissolving 1 mg of synthetic angiotensin I (manufactured by Peptide Laboratories) in 1 ml of purified water, followed by a reaction. The reaction was stopped by adding a 0.2M-lysine aqueous solution. Gel filtration was again performed to remove unreacted angiotensin I.
5 ml were obtained.

(2) Preparation of angiotensin I antibody 3.6 mg of synthetic angiotensin I was dissolved in 0.2 M phosphate buffer, and N- (γ-maleimidobutyroxy)
A solution prepared by dissolving 3.6 mg of succinimide in tetrahydrofuran was added dropwise, and the mixture was reacted at 30 ° C. for 30 minutes. Then, ethyl alcohol and diethyl ether were added thereto.
The crystals were allowed to stand at −80 ° C. for 10 minutes to precipitate crystals.
The crystals were washed twice with diethyl ether to obtain a complex of angiotensin I and N- (γ-maleimidobutyroxy) succinimide. Separately, 10 mg of bovine serum albumin was dissolved in 2 ml of 0.2 M tris-HCl buffer (pH 8.6) containing 8 M urea, and 15 μmol of dithiothreitol (manufactured by Sigma) was added, followed by reaction at 37 ° C. for 1 hour. 3 ml of 10% trichloroacetic acid was added for precipitation, and the precipitate was washed three times with distilled water to obtain reduced bovine serum albumin. Then, angiotensin I
Conjugate with N- (γ-maleimidobutyroxy) succinimide and reduced bovine serum albumin containing urea 6M.
It was dissolved in 1.5 ml of a 2M aqueous solution of sodium ethylenediaminetetraacetate and reacted at 25 ° C. for 2 hours. After the reaction, the mixture was dialyzed to obtain an angiotensin I-bovine serum albumin conjugate (immune antigen). This solution is stored at -80 ° C until immunization of the animal.

(3) Angiotensin I antibody Angiotensin I-bovine serum albumin conjugate (immune antigen) was treated in the same manner as in Example 2 to obtain an antiserum.

(4) Angiotensin I antibody plate A solution obtained by diluting the angiotensin I antiserum 5000-fold with a 0.05 M sodium bicarbonate solution (pH 9.6).
00 μl of 96-well microplate (Greiner)
, And allowed to stand at 4 ° C. for 16 hours to immobilize on a microplate. Further, 200 μl of a phosphate buffer containing 1% bovine serum albumin was added to the angiotensin I antibody-immobilized plate, and the plate was allowed to stand at 4 ° C. for 16 hours.

(5) Renin activity of the conjugate 50 μl of a 0.6 μM purified anti-peptide antibody solution was added to 50 μl of a 0.6 nM human prorenin solution, and the mixture was reacted at 4 ° C. for 20 hours to form a conjugate. 100 μl of an angiotensinogen reagent was added to this reaction solution,
For 60 minutes and transferred to an ice bath to stop the reaction. Next, 100 μl of the enzyme-labeled angiotensin I solution was added and mixed, and 100 μl of the mixture was transferred to the angiotensin I antibody plate and shaken gently at 25 ° C. for 2 hours. Further, 200 µl of a coloring reagent was added, and the mixture was reacted at 25 ° C for 15 minutes to form a color.
Was added to stop the reaction, and the absorbance at 450 nm was measured using a colorimeter. The same operation was carried out using 100 μl of ripe renin as a positive control. In addition, as a negative control, the same operation was performed by adding the same amount of normal rabbit serum to human prorenin solution instead of the anti-M or C peptide antibody. As a result, human prorenin expressed renin activity when forming a conjugate with an anti-M peptide antibody or an anti-C peptide antibody, and showed almost the same renin activity as the ripe renin as a positive control, whereas it showed a negative control as a negative control. Of human prorenin showed almost no renin activity. From the above, it can be seen that human prorenin was converted to a conjugate expressing renin activity when bound to an anti-M peptide antibody or an anti-C peptide antibody.

[0028]

The conjugate of human prorenin obtained by the present invention and its anti-pro fragment peptide antibody is a novel renin active substance expressing renin activity. It can be easily put into practical use as a method for measuring prorenin in body fluids using a measurement reagent.

[0029]

[Sequence List] SEQUENCE LISTING <110> Tokiwa Chemical Industries Co., Ltd; Ishida, Yuichi <120> Renin-active substance <130> PTKK99-001 <150> JP10-291124 <151> 1998-10-13 <160> 4 <210> 1 <211> 43 <212> PRT <213> Artificial Sequence <400> 1 Leu Pro Thr Asp Thr Thr Thr Phe Lys Arg Ile Phe Leu Lys Arg Met 1 5 10 15 Pro Ser Ile Arg Glu Ser Leu Lys Glu Arg Gly Val Asp Met Ala Arg 20 25 30 Leu Gly Pro Glu Trp Ser Gln Pro Met Lys Arg 35 40 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <400> 2 Ile Phe Leu Lys Arg Met Pro Ser Ile Arg Glu Ser Leu Lys Glu Arg 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <400> 3 Gly Val Asp Met Ala Arg Leu Gly Pro Glu Trp Ser Gln Pro Met 1 5 10 15 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <400> 4 Leu Pro Thr Asp Thr Thr Thr Phe Lys Arg Ile 1 5 10

[Brief description of the drawings]

FIG. 1 is a pattern showing the results of Western blot analysis of human prorenin and ripe renin.

FIG. 2 is a graph showing the binding ability of an anti-M peptide antibody to human prorenin.

FIG. 3 is a graph showing the binding ability of an anti-C peptide antibody to human prorenin.

FIG. 4 is a graph showing the renin activity of a conjugate of an anti-peptide antibody and human prorenin.

FIG. 5 is a graph showing the relative renin activity of a conjugate of an anti-peptide antibody mixed antibody or a single anti-peptide antibody and human prorenin.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // C12N 5/10 C12N 5/00 B 15/09 15/00 A (72) Inventor Yuichi Ishida Higashi-Owawa, Washinomiya-cho, Kitatsukatsuka-gun, Saitama 1947 West Heights 8-205 (56) Reference JP-A-3-87200 (JP, A) JP-A 8-285852 (JP, A) JP-A 10-279600 (JP, A) Canadian Patent Specification No. 2234204 J.I. Immunol. Methods (1995), Vol. 184, no. 1,91-100 Biochem. Biophys. Res. Commun. (1985), Vol. 132, No. 3, p. 1038-1045 J.P. Biochemistry (1989), Vol. 106, no. 3, p. 430-435 Biomedical. Reseach (1999), Vol. 20, No. 2, p. 117-121 Biosci. Biotechnol l. Biochem. (1999), Vol. 63, No. 3, p. 550-554 (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 7/00 C07K 19/00 C07K 16/40 C12N 9/64 G01N 33/53 C12N 5/00 C12N 15/09 BIOSIS (DIALOG ) MEDLINE (STN) WPI (DIALOG) JICST file (JOIS)

Claims (5)

(57) [Claims] 1. An eleventh amino acid sequence of a human prorenin profragment having an amino acid sequence composed of 43 amino acids.
An anti-peptide antibody that specifically recognizes an amino acid sequence consisting of 33 amino acids from the isoleucine to the 43rd arginine, which retains the ability to activate human prorenin by binding to human prorenin A conjugate of one kind of antibody and human prorenin. 2. The method according to claim 1, wherein the amino acid sequence of the peptide used as the immunogen for the preparation of the anti-peptide antibody is from the 11th isoleucine to the 11th isoleucine in the human prorenin profragment having an amino acid sequence composed of 43 amino acids.
An amino acid sequence composed of 16 amino acids up to the 6th arginine or an amino acid sequence composed of 15 amino acids from the 27th glycine to the 41st methionine in the human prorenin profragment. Item 7. The conjugate according to item 1. 3. A human prorenin profragment having an amino acid sequence composed of 43 amino acids, comprising 11 amino acids from the first leucine to the eleventh isoleucine.
Claims 1. An anti-peptide antibody prepared using a peptide consisting of an amino acid sequence composed of 10 amino acids as an immunogen, wherein the antibody retains the ability to activate human prorenin by binding to human prorenin. Or a mixed antibody with at least one of two anti-peptide antibodies;
Conjugate with human prorenin. 4. A reagent for measuring human prorenin, comprising the conjugate according to claim 1 as a measurement target. 5. A method for measuring human prorenin, comprising using the conjugate according to claim 1 as an object to be measured.