New monoclonal antibodies directed against the propart segment of human prorenin as a tool for the exploration of prorenin conformation

JOURNAL OF

MMMJ~GGICAL ELSEVIER

Journal

of Immunological

Methods

184 (1995) 91-100

New monoclonal antibodies directed against the propart segment of human prorenin as a tool for the exploration of prorenin conformation Pascale Cohen a, Dominique Simon a,*, Gabriel Badouaille “, Jean-Claude Jean-Michel Portefaix ‘, Bernard Pau ’

Mani

h ,

’ Sanofi Recherche, 371 rue du Professeur Blayac, 34184 Montpellier Cedex 4. Frunce h UMR CNRS 9921. Faculte’ de Pharmacie, 15 at:enue Ch. Flahault, 34060 Montpellier Cedex I. France Received

20 October

1994; revised 5 January

1995: accepted

24 March

1995

Abstract Six monoclonal

antibodies

with a peptide corresponding

(MAbs)

directed

against human prorenin

by immunizing BALB/c mice These new MAbs were screened for their

were produced

to the sequence ( - 17 to + 9) of prorenin.

ability to first bind to the immobilized peptide and then to prorenin previously captured by an anti-total renin MAb. The specificity of the MAbs was confirmed by the total lack of binding to active renin. Using BIAcore ’ technology, equilibrium affinity constants of the MAbs were determined and ranged from 3.2 X 10’ to 5.7 X 10L’ I/mol. Immunoradiometric assays (IRMA) for prorenin were performed using the anti-total renin MAb and the anti-prorenin MAbs. The best results were obtained when an anti-prorenin MAb was immobilized and the anti-total renin MAb was used as tracer in a one-step procedure. Moreover, the signal was significantly increased by the presence of the renin inhibitor SR 43845 suggesting that the inhibitor-induced conformational change of prorenin could be detected by the MAbs. Keywords:

BIAcore

technology;

Monoclonal

antibody;

1. Introduction Renin is an aspartic proteinase (EC 3.4.23.15) which is synthetized in the form of a precursor, prorenin. Prorenin is enzymatically inactive but

Abbreviations: MAb, monoclonal antibody; IRMA, immunoradiometric assay; IEMA, immunoenzymometric assay. * Corresponding author. Tel: 33/67.10.65.26; Fax: 33/67.10.67.67. ’ BIAcore and Bialogue are registered trade marks. 0022.1759/95/$09.50 Q 1995 Elsevier SSDI 0022-1759(95)00079-8

Science

Prorenin;

Immunoradiometric

assay

the removal of an N-terminal prosegment transforms prorenin into active renin. The function of this prosegment is however, unclear at present. In human plasma, 90% of the total renin present is ‘inactive renin’ consisting of a mixture of various prorenin derivatives N-terminally truncated Kim et al., 1985; Gaillard et al., 1986). The most widely used method for the determination of prorenin is an indirect procedure which requires the prior conversion of prorenin to active renin. Prorenin can be hydrolyzed to give active renin

B.V. All rights reserved

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of Immunological Methods 184 (1995) 91-100

using enzymes such as trypsin (Sealey et al., 1979) which cleaves the prosegment from the polypeptide chain. Prorenin can also be activated by exposure to low pH (Leckie and McGhee, 1980) or low temperature (Laragh, 1992) and, by renin inhibitors as recently shown (Derkx et al., 19921. In the latter case, activation seems to be due to a conformational change permitting access of the renin substrate to the active site of the enzyme. Several polyclonal (Hirose et al., 1985; Kim et al., 1985; Price-Jones et al., 1993) and monoclonal antibodies (Gaillard et a!., 1986; Ishizuka et al., 1989) against prorenin have been described. In the present study, we describe the production and characterization of new monoclonal antibodies directed against a portion of the prorenin prosegment which specifically recognizes prorenin and, we report on the effect of their binding as well as that of a renin inhibitor on the conformation of the prorenin molecule.

2. Materials

and methods

2.1. Prorenin and renin sources

Human recombinant prorenin was kindly furnished by Sanofi Recherche, Labege, France. International active human renin standard (lot 68/356) was kindly provided by the National Institute for Biological Standards and ControI (Blanche Lane, Hertfordshire, UK). 2.2. Preparation of F(ab’),

antibodies

The human anti-total renin monoclonal antibody used (3E8) was described by Galen et al. (1984). This MAb recognizes both prorenin and renin. Samples of antibody 3E8 were dialyzed against sodium formate buffer, pH 2.8, followed by dialysis against sodium acetate buffer, pH 4.2. The F(ab’1, fragments were then prepared by digestion with pepsin (1 Kg/ml) for 5 h at 37°C (Rousseaux et al., 1983). 2.3. Preparation of the SP91681-OVA immunogen Hirose et al. (1985) determined that the prosegment contains 43 residues by sequential Ed-

man degradation of 35S Met-radiolabelled human prorenin. These 43 residues are numbered negatively from the activation site ( - 43 to - 1) in the present study. The peptide (- 17 to + 9) with the following sequence was synthesized: G-V-D-MA-R-L-G-P-E-W-S-Q-P-M-K-R-L-T-L-G-N-T-TS-S. A linker composed of GGY residues was added at the C-terminus to permit coupling and iodination. Pure peptide (> 80%) as established by amino acid analysis was obtained by preparative reversed phase high performance liquid chromatography (HPLC). This peptide was called SP91681. SP91681 was coupled to ovalbumin (OVA) by the bisdiazobenzidine condensation procedure. The coupling efficiency was 23 mol peptide/mol OVA. Synthesis, purification and coupling were performed by Neosystem (Strasbourg, France). 2.4. Preparation of monoclonal antibodies (MAbs) BALB/c mice were immunized using two different procedures. One group of mice received two simultaneous injections of 5 Kg prorenin given intraperitoneally and subcutaneously (in complete Freund’s adjuvant). A third injection of 10 pg prorenin was given subcutaneously in incomplete Freund’s adjuvant. A second group of mice was immunized by three subcutaneous injections of 200 pg immunogen (SP91681 coupled to OVA) emulsified in complete Freund’s adjuvant followed by one injection (s.c.1 of 10 pg prorenin in incomplete Freund’s adjuvant. The booster injection was 100 ,ug of SP91681-OVA given intraperitoneally 3 days before fusion. Animals were cared for in accordance with Sanofi Recherche institutional guidelines. For fusion, spleen cells were fused with a myeloma P3-X63-Ag8-653 line according to the method described by Di Pauli and Raschke (1978) and modified by Galen et al. (1984). Anti-prorenin antibodies in the mouse serum, culture supernatants, ascites or purified IgG fractions were detected by their binding to peptide SP91681 and prorenin and by the absence of binding to active renin. Antibody-producing hybridomas were subcloned and then frozen in liquid nitrogen. Ascites were produced following intraperitoneal injection

P. Cohen et al. /Journal

of Immunological Methods I84 (1995) 91-100

of cloned hybridoma cells into y-irradiated (350 rads) and pristane-treated BALB/c mice. Monoclonal antibodies were then purified from ascites by affinity chromatography on protein A-Sepharose according to the method of Ey et al. (1978). 2.5. Enzyme immunoassays Indirect solid phase enzyme immunoassay of peptide SP91681 SP91681 (10 pg/ml) in phosphate-buffered so-

lution (PBS), pH 7.4, was adsorbed onto polyvinyl 96-well microtiter plates (ImmunoPlate Maxisorp, Nunc, Roskilde, Denmark) by incubating 100 ~1 of peptide overnight at 4” C. After washing (PBS), SP91681-coated wells were saturated for 30 min at 37” C with PBS containing 4% fetal calf serum (FCS). Test antibody, in culture supernatant, ascites or purified IgG fraction was incubated (100 pi/well) for 3 h at room temperature in PBS-4% FCS in the peptide-coated wells. After washing, bound antibody was detected by adding a peroxidase-conjugated anti-mouse Ig (Silenus Laboratories, Hawthorn, Australia). Controls were performed by replacing the test antibody by the anti-prorenin ( - 19 to - 7) MAb 4Cl (Gaillard et al., 1986; Simon et al., 1992a). To determine the antibody isotype in culture supernatants, the peroxidase-conjugated anti-mouse Ig was replaced by a peroxidase-conjugated anti-mouse y heavy chain (Jackson Immunoresearch Laboratories, Interchim, Montluqon, France) to detect IgG antibodies and by a peroxidase-conjugated anti-mouse p heavy chain (Sigma, St. Louis, MO, USA) to detect IgM antibodies.

93

then added (100 pi/well) and the plate was incubated at room temperature for 3 h in PBS-4%~ FCS. After washing, the test antibody preparation was added and incubated for 18 h at 4” C. The wells were then washed and prorenin which had been recognized by the second monoclonal antibody was detected by adding the peroxidaseconjugated anti-mouse y heavy chain antibody. The active renin enzyme immunoassay was carried out under the same conditions by replacing prorenin by active renin (50 ng/ml in PBS-4% FCS). Controls were performed by replacing the test MAb by the anti-prorenin MAb 4Cl in the prorenin enzyme immunoassay or by the anti-active renin MAb 4Gl described by Galen et al. (1984) in the active renin enzyme immunoassay. To detect IgM antibodies in culture supernatants, the peroxidase-conjugated anti-mouse y heavy chain was replaced by the peroxidase-conjugated anti-mouse F heavy chain. 2.6. Liquid-phase radioimmunoassay CR1541of peptide SP91681 SP91681 was iodinated according to the chloramine T method described by Greenwood et al. (1963); 1 mCi of Na”‘I (Amersham, Buckinghamshire, UK) was allowed to react with 1 pg peptide. Free iodine was separated from the iodinated peptide on an Amprep column (phenyl pH, RPN 1914, Amersham). The specific activity of the iodinated peptide was approximately 0.4 mCi/pg, which is equivalent to 0.66 mol of iodine per mol of peptide. The RIA test was performed according to the procedure described by Simon et al. (1992b).

Enzyme immunoassays of prorenin and of active renin

2.7. Biosensor measurement of antibody actirity

To detect anti-prorenin or anti-active renin antibodies in culture supernatants, ascites, or purified IgG fractions, a two-site immunoassay was performed. The F(ab’), fragment of the anti-total renin MAb 3E8 was coated (100 Fl/well) onto polyvinyl microtiter plates in PBS (10 pg/ml) overnight at 4°C. After washing (PBS), F(ab’), 3E8-coated wells were saturated with PBS-4% FCS for 30 min at 37” C. Prorenin (50 ng/ml) was

Equipment and reagents

The running and dilution buffer in all experiments was 0.1 M imidazole HCl, pH 7.4. Regeneration of the sensor chip was performed using 15 ~1 of 100 mM HCI. Preparation of the sensor surfaces

Rabbit anti-mouse y heavy chain (RAM-Gl, Pharmacia) in acetate buffer (pH 5.0) was immo-

P. Cohen et al. /Journal

94

of Immunological Methods 184 (199.5) 91-100

bilized by coupling dextran activated with 100 mM N-ethyl-N’-(3-dimethylaminopropyll carbodiimide hydrochloride/400 mM N-hydroxy succinimide. Anti-protein MAbs were bound to RAM-G1 and the binding of prorenin to each MAb was monitored. Binding kinetics were studied in channels with low concentration coat, whereas characterization of the binding of the MAbs to prorenin and the effects of a renin inhibitor, SR 43845 (Nisato et al., 19871, were performed in channels with a high concentration coat. Evaluation of antibody kinetic constants

Kinetic runs were performed using six different ligand concentrations. The antibody bound CR,) to the antigen as well as the reaction rate (dR,/dt) were computed by the BIAcore software. Kinetic rate constants (k,, and k,,) and the apparent equilibrium affinity constants (K, = k,,/koff) were determined using Bialogue Kinetics Evaluation sofhvare. 2.8. Immunoradiometric

assay (IRMA) forprorenin

Immobilization of the Mb 500 ~1 of an antibody solution at 20 pg/ml

in PBS were incubated in Maxisorp immunotubes (StarTube, Nunc) overnight at room temperature.

then incubated with shaking for 5 h at room temperature. Finally, the tubes were washed three times with 0.05 M Tris-HCl, pH 7.4, containing 0.1% Tween 20 (buffer 0, and the bound radioactivity was counted in a y counter. Two step-procedure assay.

The antibody-coated tubes were rinsed and saturated as previously described. The prorenin (300 ~1, 0.2-20 ng/ml) in buffer A was then added and incubated with shaking for 2 h at room temperature. After rinsing with buffer C, the labelled antibody (300 ~1; 200 000 counts/min) in buffer B was added and incubated with shaking for 3 h at room temperature. The tubes were then washed three times and counted as previously described. 2.9. Immunoenzymometric prorenin

assay

(IEM)

of

Conjugates.

The anti-total renin MAb 3E8 and the antiprorenin MAb 13Gll were coupled to horseradish peroxidase (Boehringer Mannheim, Mannheim, Germany) by the sodium periodate method described by Nakane and Kawaoi (1974). The conjugates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Iodination of the second I&lb

The tracer antibody was lz51 labelled by the chloramine T method at room temperature as previously described (Larue et al., 1991). The specific activity of iodinated antibody was approximately 19 pCi/Fg, which is equivalent to 1.3 mol of iodine per mol of immunoglobulin. One step-procedure assay

The antibody-coated tubes were rinsed with PBS and saturated for 30 min at room temperature with PBS containing 0.3% bovine serum albumin (Sigma). After saturation, prorenin (300 ~1, 0.2-20 ng/ml) in 0.05 M K/K,PO,, pH 7.4, containing 0.15% casein (buffer A), and the labelled antibody (100 ~1; 200000 counts/min) in 0.05 M Tris-HCl, pH 7.4, containing 10% horse serum (buffer B), was added. The tubes were

One-step procedure

The assay used immunotubes coated with MAb 13Gl1, saturated and rinsed as described above for the IRMA. Prorenin (200 ~1, 20-200 pg/ml) in FCS and the peroxidase-conjugated MAb 3E8 (200 ~11 in 0.1 M imidazole . HCl, pH 7.4, were added. The tubes were incubated with shaking for 18 h at 4°C. Finally, the tubes were washed four times with 0.1 M imidazole 9HCl, pH 7.4, containing 0.1% Tween 20. Peroxidase substrate, 400 ~1 of a solution of one tablet of o-phenylene diamine (Sigma) dissolved in 10 ml of citrate buffer 0.1 M, pH 5, was then added to each tube. After incubation for 15 min in the dark, 400 ~1 of 4 N H,SO, was added and the absorbance was measured at 492 nm in a microELISA autoreader.

P. Cohen et al. /Journal

of Immunological Methods 184 (1995) 91-100

Two-step procedure.

This assay was performed using immunotubes coated with MA\, 3E8. After saturation and rinsing, prorenin (400 ~1, 20-200 pg/ml) in 0.1 M imidazole . HCl, pH 7.4, containing 50% FCS, was added and the tubes were incubated with shaking for 2 h at room temperature. The immunotubes were washed once with 0.1 M imidazole . HCl, pH 7.4, containing 0.1% Tween 20. The peroxidase-conjugated MAb 13Gll (400 ~1) in 0.1 M imidazole . HCl, pH 7.4, containing 4% FCS was added and the tubes were incubated with shaking for 18 h at 4” C. Washing and detection were performed as described in the one-step IEMA. 2.10. Detection of plasma prorenin Blood from healthy volunteers was collected at room temperature in tubes containing EDTA, centrifuged and immediately stored at -20” C. Trypsin activation was performed according to Sealey et al. (1979), with addition of trypsin (Sigma, cat. no. T-8253) to plasma to give a final concentration of 1 mg/ml and with incubation for 1 h at -4” C before performing the immunoassay. Preparation of renin-free plasma

The anti-total renin MAb lA12 previously described by Galen et al. (1984) was coupled to cyanogen bromide-activated Sepharose 4B according to the method described by March et al. (1974). A pool of plasma was applied to this gel and total plasma renin was then separated from the plasma as described by Galen et al. (1984). The total renin-free plasma was collected and stored at -20°C. Plasma prorenin was assayed by the one-step IRMA procedure. The standard curve was diluted in the renin-free plasma. The tracer was diluted in 0.1 M imidazole . HCl, pH 7.4, containing 0.2% casein. Immunotubes were coated with one of the anti-prorenin MAbs, rinsed and saturated as previously described. Prorenin (300 ~1, 0.2-20 ng/ml), trypsin activated plasma (300 ~11 or non activated plasma (300 ~1) were added with the iodinated MAb 3E8 (100 ~1, 200 000

95

counts/min). The tubes were then incubated with shaking for 18 h at 4” C. Finally, the tubes were washed three times with 0.1 M imidazole . HCI, pH 7.4, containing 0.1% Tween 20, and the bound radioactivity was counted with a y counter. 2.11. Renin inhibitor The renin inhibitor the incubation in the ent procedures at a PM as recommended

SR 43845, included during assay, was tested in differfinal concentration of 100 by Derkx et al. (1992).

One-step IRM4 and one-step IEM.

These assays were performed according to the method used for the detection of plasma prorenin with acidified MAb 13Gll (Conradie et al., 1983) as immobilized antibody and MAb 3E8 as second antibody (iodinated or peroxidase-conjugated). The acidification step was used to increase the density of MAb on the surface of the tube. Two-step IRMA and two-step IEMA

The pair tested consisted of immobilized MAb 3E8 with MAb 13Gll used as second antibody (iodinated or peroxidase-conjugated).

3. Results 3.1. Selection of the immunized mouse for subsequen t cell fusion

To obtain anti-prorenin MAbs, two different immunization protocols were performed. Mouse sera were assayed for antibodies directed against SP91681 and specifically against prorenin (not against active renin). The mice that were only immunized by injections of prorenin were not selected because their sera crossreacted with active renin. The sera of mice that received injections of immunogen (SP91681 coupled to OVA) and one injection of prorenin, specifically recognized SP91681 and prorenin, but did not recognize active renin. The serum titer of the mouse selected for cell fusion (giving a signal equivalent to twice the nonspecific binding) was 1:SOOOOfor binding both to SP91681 and to prorenin.

96

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of Immunological Methods 184 (1995) 91-100

3.2. M4b specificity After fusion, culture supernatants were tested in the different enzyme immunoassays formats for antibodies against prorenin, active renin and peptide SP91681. 17 hybridomas were selected (Table 1). Six of them (group A) produced IgG antibodies exclusively directed against peptide SP91681. Five other hybridomas produced IgM antibodies (group B) directed against both SP91681 and total renin. Finally, six hybridomas producing IgG monoclonal antibodies (group C) directed against both SP91681 and prorenin were obtained.

Table 1 Specificity of MAbs obtained by immunizing a mouse with several injections of ovalbumin-peptide SP91681 conjugate and one injection of prorenin MAb

Peptide SP91681 Prorenin

group

Anti-y

Anti-k

A(6) B(5) C(6)

+ +

+ -

Renin

Anti-y _

Anti-p -

Anti-y -

Anti-F _

+

+ _

-

+ _

Culture supernatants were tested by enzyme immunoassays of peptide SP91681. prorenin or active renin according to the procedures described in the materials and methods section. To detect IgG and IgM antibodies, a peroxidase-conjugated anti-mouse y heavy chain (anti-y) and a peroxidase-conjugated anti-mouse p heavy chain (anti-p.) were used. The number of MAbs obtained in each group is indicated in parentheses.

3.3. MAb characterization The anti-prorenin MAb-secreting hybridomas (group C) were subcloned, produced and purified from ascitic fluids. The equilibrium affinity constants of these MAbs, determined by the biosensor BIAcore system, ranged from 3.2 X lo8 to 5.7 x lo9 l/mol (data not shown). The characterization of the MAbs was further investigated using the enzyme immunoassays for prorenin and for active renin, the indirect solid phase enzyme immunoassay for peptide SP91681, and the RIA for peptide SP91681 in solution. Enzyme immunoassays for prorenin and for active renin

The specificity of the MAbs was confirmed by the total lack of binding to active renin and their binding to prorenin. The most active prorenin antibodies were MAbs 13Gll and 2C6 (data not shown). Peptide SP91681

The purified anti-prorenin MAbs recognized both immobilized SP91681 and iodinated SP91681 in solution but not to the same extent. The greatest activity for the immobilized peptide was observed for MAbs lOC3 and lOC2, while the peptide in solution was best recognized by MAbs 2C6 and 13Gll. Similarly, the MAbs 2C6 and 13Gll showed the greatest reactivity with prorenin in the IEMA format (data not shown).

3.4. IRMA for prorenin This two-site immunoassay could not be performed using two of the anti-prorenin MAbs obtained because they all recognized the same epitopic region. So, the MAb pairs tested were composed of the anti-total renin MAb 3E8 and one of the anti-prorenin MAbs. For each MAb pair, the lack of binding to active renin was verified. Furthermore, the signals fell to background levels when trypsin-activated plasma was tested. These two sets of results confirmed the specificity of the MAbs for prorenin. The direct IRMA was performed in two ways: either the immobilized antibody was MAb 3E8 and the tracer MAb was an anti-prorenin MAb or the immobilized MAb was one of the anti-prorenin MAbs and the tracer MAb was the MAb 3E8. The IRMA for prorenin was carried out in a one-step procedure or in a two-step procedure. When the assay was performed with the pairs composed of the immobilized MAb 3E8 and one of the anti-prorenin MAbs as tracer antibody, the two-step procedure gave the highest signals (Fig. 1). In contrast, when an immobilized anti-prorenin MAb and MAb 3E8 as tracer were tested, the one-step procedure was preferable to the two-step procedure (Fig. 2). The optimal signals for the pairs of immobilized MAb 3E8 and iodinated anti-prorenin MAb in the

I? Cohen et al. /Journal

1R

97

of Immunological Methods 184 (19951 91- 100

7E12

Fig. 1. Comparison of the one-step (0 ) and the two-step ( n 1 IRMA for prorenin using an anti-prorenin MAb as tracer. The MAb pairs comprised the anti-total renin MAb 3E8 as immobilized antibody and one of the anti-prorenin MAbs as iodinated tracer. The data presented were obtained with 20 rig/ml of prorenin.

two-step procedure and for the pairs of immobilized anti-prorenin MAb and iodinated MAE, 3E8 in the one-step procedure were different depending on the MAb tested. The maximal values for 20 ng/ml prorenin were observed when MAbs 13Gll and 2C6 were used as tracer antibodies, and when MAbs lOC2 and lOC3 were immobi-

To determine the influence of temperature and incubation time on the signals, the IRMA for prorenin was performed under various conditions. The assays tested were the one-step procedure using immobilized MAb lOC2 and MAb 3E8 as tracer antibody, and the two-step procedure using immobilized MAb 3ES and MAb 13Gll as tracer antibody. In all cases, the best results were obtained with a long incubation period (18 h). The optimal temperature was 4” C for the onestep procedure and indifferently 4” C or room temperature for the two-step procedure. Assays of immobilized acidified MAb were performed by the one-step IRMA, and the best signals for prorenin detection were obtained using acidified MAb 13Gll (data not shown). 3.5. Effect of SR 43845 on the prorenin detection The influence of the renin inhibitor SR 43845 (100 PM) on the detection of prorenin in buffer was tested in the one-step IEMA and IRMA procedures using acidified MAb 13Gll as immobilized antibody and MAb 3E8 as second antibody. The addition of the inhibitor increased the signals by a factor of about two (Fig. 31, whereas the nonspecific signals remained unchanged. A similar increase in the signal was also observed for the detection of prorenin in plasma samples

ONE-STEP

rmlA

ONE-STEP IRMA

50 000

40 000

30 000

low

J+_I 11,, 13Gll

hlmobalrod

2c4

lfl

7E12

20 000

10000

,3

4c1

anilwdy

Fig. 2. Comparison of the one-step (0) and the two-step ( W) IRMA for prorenin using an anti-prorenin MAb as immobilized antibody. The MAb pairs consisted of one of the antiprorenin MAbs as immobilized antibody and the anti-total renin MAb 3E8 as iodinated tracer. The data presented were obtained with 20 ng/ml of prorenin.

PRORENIN

CONCENTRATION

Fig. 3. Effect of the renin inhibitor SR 43845 on the detection of prorenin in the one-step IEMA and IRMA. These assays were performed with acidified MAb 13Gll as immobilized antibody and MAb 3E8 as tracer antibody (peroxidase-conjugated or iodinated) in the absence (01 or presence (~1 of 100 PM SR 43845.

98

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of Immunological Methods 184 (1995) 91-l 00

in the one-step IRMA. This signal was weak, however, and can be explained by the fact that the prorenin concentration in plasma (< 200 pg/ml) is very low (Schumacher et al., 1992; Daneman et al., 1994). All these observations were confirmed by BIAcore experiments (data not shown). Curiously, when MAb 3E8 was used as the immobilized antibody in the two-step IEMA or in the two-step IRMA, the addition of SR 43845 had no effect.

4. Discussion In this study, we have demonstrated that the injection of prorenin into BALB/c mice led to the production of antibodies that strongly crossreacted with active renin whereas mice immunized with peptide corresponding to the sequence (- 17 to +9> of the prorenin prosegment produced specific anti-prorenin MAbs. Hirose et al. (1985) and Kim et al. (1985) obtained different polyclonal antibodies to prosegment peptides and found that an antibody (anti-pro31 directed against the C-terminal part of the prosegment (peptide - 15 to - 1) recognized prorenin. Gaillard et al. (1986) produced monoclonal antibodies to sequences ( - 19 to - 7) and (- 26 to - 17) of the prorenin prosegment, and only an anti-( - 19 to - 7) MAb reacted with inactive renin. Schumacher et al. (1992) used two synthetic peptides corresponding to the N-terminal portion and the C-terminal portion of the prorenin prosegment to obtain polyclonal antibodies, and only the peptide reflecting the Cterminal sequence (- 15 to - 1) of the prorenin prosegment generated antibodies specific for prorenin. Hirose et al. (1985) suggested that plasma prorenin might lack a large portion of the N-terminal part of the profragment and as such might exist in the plasma in the form of a proteolyzed activation intermediate. Gaillard et al. (1986) proposed another hypothesis according to which antibodies directed against the N-terminus or the central part of the prosegment cannot recognize prorenin because this part of the molecule is buried inside prorenin.

In the present study, the choice of the peptide used for immunization was based on these previous observations and corresponded to the (-17 to +9) sequence of prorenin. Mice immunized using several injections of immunogen (peptide SP91681 coupled to OVA) and one prorenin injection allowed us to obtain three groups of MAbs. One group was composed of anti-peptide and anti-prorenin IgG MAbs, confirming the peptide (- 17 to +9) immunization strategy. A second group composed of only anti-peptide IgG MAbs was obtained, suggesting that a part of the immunogen presented a folding state different from the prosegment on the total molecule, or that the epitope recognized by these MAbs was inaccessible in the native molecule. Thirdly, anti-peptide and anti-total renin IgM MAbs were obtained, suggesting they were directed against the (+ 1 to + 9) region of the renin. The isotype of these antibodies can probably be attributed to the injection of a single dose of prorenin. The direct prorenin IRMA values showed that the highest signals were obtained using the twostep procedure with the anti-total renin MAb as immobilized antibody and using the one-step procedure with this MAb as tracer. These results demonstrate a cooperative effect between the anti-total renin MAb and the anti-prorenin MAbs, suggesting that the prorenin molecule must first be captured by MAb 3E8 in order to adopt a conformational state allowing a greater affinity for the anti-prorenin MAbs. Derkx et al. (1992) showed that high affinity active site-directed renin inhibitors permitted prorenin recognition by the MAb 4G1, an antibody that binds to active renin but not to inactive prorenin. Apparently, complex formation with the renin inhibitors was associated with a conformational change in prorenin and the unmasking of the epitope on the renin part of the molecule recognized by MAb 4Gl. Derkx and colleagues suggested that by interfering with the interaction of the propart segment with the renin part of the prorenin molecule, renin inhibitors may trigger destabilization of the prosegment, so that the prorenin molecule assumes its active configuration. This conformational change also occurred

P. Cohen et al. /Journal

of Immunological Methods 184 (1995) 91-100

after acid activation of a prorenin molecule that also became recognizable by MAb 4Gl. Price-Jones et al. (1993) obtained similar results with polyclonal anti-prorenin antibodies directed against synthetic peptides of the prosegment sequence and proposed that detection of the respective epitopes in the prosegment of the prorenin required prior acid activation to partially unfold the prorenin molecule by dislodging the prosegment from the main body of the protein. Our observations are in accordance with the results of the above-mentioned studies and demonstrate that the addition of the renin inhibitor SR 43845 increased prorenin detection in the one-step IEMA and IRMA, confirming an inhibitor-induced conformational change of the prorenin molecule allowing a better recognition of the prosegment by the anti-prorenin MAbs. In summary, we have obtained and characterized six anti-prorenin MAbs. A conformational change of the prorenin molecule appears to be induced or stabilized by its interaction with the anti-total renin MAb 3E8. Furthermore, this effect is increased in the presence of a renin inhibitor in the one-step immunocapture assay using MAb 3E8 as second antibody.

Acknowledgements

The authors wish to thank Dr. J.A. Fehrentz for helpful discussions concerning the choice of the peptide sequence and Dr. S.L. Salhi, Faculte de Pharmacie, Montpellier, for critical reading of the manuscript.

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