HYBRIDOMA Volume 11, Number 4, 1992 Mary Ann Liebert, Inc., Publishers
Production and Characterization of Four Anti-Neuropeptide Y Monoclonal Antibodies ERIC GROUZMANN,' ETIENNE COMOY,2 PHILIPPE WALKER,1 MICHEL BURNIER,1 CLAUDE BOHUON,2 BERNARD WAEBER,1 and HANS BRUNNER1
'Hypertension Division and Cardiovascular Research Group, University Hospital, Lausanne, 2Departement de Biologie Clinique, Institut Gustave Roussy, Villejuif, France
Switzerland
ABSTRACT
neuropeptide Y (hNPY) is a potent vasoconstrictor peptide of 36 aminoacid residues. We isolated hybridomas secreting four monoclonal antibodies directed against various epitopes of neuropeptide Y and studied their cross-reactivity with peptide YY (PYY) and the pancreatic polypeptide (PP), two peptides sharing sequence homologies with hNPY (respectively 70% and 50%). The antibody NPY02 is an IgGl with a Ka of 5.5 1010 liters/mol. It binds to the 11-24 region of NPY (IC50 2 10-7M), does not recognize PP but cross-reacts weakly with PYY. Antibodies NPY03 and NPY05 are IgG2 with respective Ka's of 6.7 109 and 2.5 1010 liters/mol. They interact with a C-terminal epitope on NPY (NPY 27-34, IC50 2 10-9 M for NPY03 and NPY 3236, IC50 1 10-9 M for NPY05). These two antibodies cross-react with PYY whereas only NPY05 binds PP. NPY05 is unable to bind the free acid form of neuropeptide Y. The 32-36 COOH free subpeptide is recognized 50,000 less efficiently by NPY05 than its amidated form. Antibody NPY04 is an IgG3 with a Ka of 3.8 108 liters/mol. It recognizes a N-terminal epitope between aminoacids 1 and 12 (IC50 2·5 x 10-6 M). NPY04 interacts weakly with PYY but not detectably with PP. These Human
=
=
=
=
results obtained with 4 different monoclonal antibodies demonstrate the presence of at least four epitopes on hNPY, two of them being continuous. These antibodies will be used to study the interaction of NPY with its receptor and to develop sensitive and specific assays for determination of NPY concentrations in
biological fluids
.
INTRODUCTION
Neuropeptide Y (NPY) is a 36 amino-acid peptide first discovered by Tatemoto et al. in 1982 (1). This peptide is widely distributed in the central and peripheral nervous system (2). It possesses direct vasoconstrictor properties and also potentiates the contractile effect of various stimuli including norepinephrine and angiotensin II (3,4). Like many peptides processed from precursors, NPY has an 409
amidated C-terminal amino-acid (1). It is a member of a peptide family sharing the "PP fold" structure such as pancreatic peptide (PP) and peptide YY (PYY) (5). Several authors have prepared polyclonal antibodies directed against NPY. These antibodies have been used mainly for immuno-histochemical studies and for the determination of NPY concentration in biological fluids (6-12). Regarding circulating levels of NPY, different values have been measured depending on the polyclonal antibodies used. In some assays, a problem of cross-reactivity with structurally related peptides may exist. Recently, we have isolated 4 hybridomas secreting anti-NPY monoclonal antibodies. Two of them have been partially described in an earlier report and employed to set up an immuno-radiometric assay (13). In this paper, we describe the production and characterization of these four monoclonal antibodies as well as the mapping of their epitopes. MATERIAL AND METHODS
Peptides Peptide synthesis was performed according to Merrifield (14) using an automated synthesizer (Applied Biosystem, model 430A, Foster City, CA). The solid phase support was a benzyl-hydrylamine resin. Total human NPY (hNPY) and the following fragments were synthesized:l-12, 11-24, 18-24, 23-36, 27-36, 32-36 and the 32-36 acid-free peptide. The peptides were then deprotected and cleaved from the resin with anhydrous hydrofluoric acid. They were purified by gel filtration on biogel P/4 (BioRad). The purity of the peptides was tested by reverse phase high pressure liquid chromatography and the amino-acid composition was confirmed by amino acid analysis on an LKB Alpha Analyzer. Additional peptides were purchased. Porcine NPY (pNPY), human PP (hPP), bovine PP (bPP), avian PP (aPP) and rat PP (rPP) were obtained from Sigma (Sigma Ine, St Louis, USA). Salmon PP (sPP), porcine NPY16-36 (pNPY16-36), NPY20-36 (pNPY20-36), NPY22-36 (pNPY22-36), NPY26-36 (pNPY26-36) were from Peninsula (Peninsula, Belmont, CA). Porcine NPY 10- 18 (pNPY10-18), NPY14-23 (pNPY14-23), NPY19-28 (pNPY19-28), NPY24-36 (pNPY24-36) were from Novabiochem (Laufelfingen, Switzerland).PYY (pPYY), human PP31-36 (hPP31-36) and its free acid C- terminal analog were ordered from Bachern (Bachern Bubendorf, Switzerland). Human NPY13-36 (hNPY13-36) was kindly supplied by Dr. Kamper (Ciba-Geigy AG, Basel). Ten pNPY 1-36 analogs, in which residues 1 through 10 were substituted by an alanine were a gift from Dr A. Fournier (IRSC, Quebec). The Pro34 NPY was a kind gift from T. Schwarz (Copenhagen, Denmark). pNPY 5-20 was kindly provided by Dr. A. Beck.- Sickinger(Tiibingen, RFA). Immunization NPY 1-36
conjugated to keyhole limpet hemocyanin (KLH, Sigma, St Louis) using glutaraldehyde were injected subcutaneously to six week-old mice of different strains (Biozzi, C3H). Fifteen µg to 30 µg of the various conjugated antigens were administered together with the same amount of Freund complete adjuvant.The injections were repeated at monthly intervals with the same amount of immunogen in Freund's incomplete adjuvant.Response to immunization was evaluated 10 days after each boost by measuring the antibody levels in the mouse sera using the radioimmunoassay or
NPY 27-36
410
(RIA) described below.When the presence of antibody was detected (60 % binding or more with the diluted 1/2), two additional doses of immunogen were given, one intraperitoneally and the other one intravenously nine and three days before performing the fusion, respectively. mouse serum
Production of monoclonal antibodies Cell fusion,
hybridoma selection, cloning and ascites production were performed according to the procedure described by Bidart et al (15). Three weeks after the last injection, myeloma cells (P3-NS1-1Ag4-1 (NS1) were fused with splenocytes in 40% (w/v) polyethylene glycol (MW=1000). The ratio of NS1 cells to splenocytes was about 1 to 4. Anti-NPY antibody producing hybridoma were also detected with the RIA described below. After cloning, 2x106 cells were injected intraperitoneally to nude mice and ascites fluid collected after two weeks. Detection of antibodies The presence of anti-NPY antibodies in mouse sera and hybridoma supernatants was tested by RIA. Ten microliter serum or hybridoma supernatant were incubated at 20 ° C for two hours with 125l-NFY (3000 cpm /100 µ , 2000 Ci/mmol, Amersham GB) and 500 µ of assay buffer (50 mM phosphate buffer, 20 mM EDTA, 0.3% bovine serum albumin, 0.005% aprotinine, pH 7.5). The incubation was stopped by adding 250 µ of a charcoal-dextran suspension. After centrifugation (2000 g, 15 min) the
radioactivity in the pellet (P, free NPY) and in the supernatant (S, bound NPY) was counted and the ratio (S) / (S)+(P) was calculated. Non-specific binding was evaluated using a non-relevant monoclonal antibody. Hybridoma supernatants showing more than 20% *25 _ binding were further analysed for the presence of an anti-NPY antibody using an enzyme linked immunosorbent assay (ELISA). Microtiter plates (96-wells, NUNC) were coated for 24 hours at 4° C with 100 µ of a 10 mg/1 solution of hNPY dissolved in 0.1M phosphate buffered saline (PBS), pH 7.4. The wells were saturated by incubation for 18 hours at 4° C in the same buffer containing 1% BSA. After four washes with 10 mM PBS, containing 150 mM NaCl (pH 7.4), 100 µ of the culture supernatants were added to the wells and the plates were incubated for another hour at 37° C. After incubation, the wells were washed again four times and a 1000 fold dilution of peroxidase-conjugated rabbit anti-mouse IgG antiserum (200 µ , Nordic, Tilburg, The Netherlands) was added. After 45 min incubation at 37° C and four additional washes with the 10 mM PBS, pH 7.4, containing 150 mM NaCl, peroxidase activity was revealed by addition of 200 µ of a 50 mM citrate/phosphate buffer solution containing 500 mg/1 o-phenylene diamine and 0.007% H2O2. The reaction was stopped 20 min later by adding 50 µ of a 12.5% H2SO4 solution. The final concentration of NPY bound antibody was determined by measuring the absorbance at 492
nm on a
Titertek Multiskan ELISA reader (Flow Laboratories, Rockville,
MD).
Purification of antibodies Monoclonal antibodies were purified from
mouse
ascites
411
by amonium sulfate precipitation and protein A
chromatography as described by Ey et al (16) For IgGl purification, anion exchange chromatography (DEAE-Sephacel, Pharmacia) was performed prior to the affinity separation. The presence of anti-NPY immunoglobulins was monitored by the above described RIA. Protein was measured with the BioRad protein assay. Isotvpe identification The ELISA described above for the antibody detection was modified as follows: after coating with NPY, the wells were saturated, washed and 100 µ (20 ng) purified monoclonal antibody was added per well .The plates were then incubated for 1 h at 37° C. Then the wells were washed with PBS and rabbit antibodies (150 µ , 1/5000 diluted ) directed against mouse IgM, IgGl, IgG2 or IgG3 (Nordic,
Tilburgh, The Netherlands) were added in separate wells. After a 45 min incubation at 37° C and subsequent washing, 150 µ of peroxidase conjugated swine anti-rabbit antibody 1/2000 diluted ( Nordic), was added. Peroxidase activity was revealed over a 45 min incubation period. Determination of affinity constant The modified RIA procedure described by Van Heyningen et al. (17) was used for the affinity study. The NPY (or related peptides) concentration resulting in 50% tracer binding inhibition for each antibody
(IC50) was calculated. Identification of epitopes
by RIA. A constant amount of tracer (3000 cpm of ¡-^Sj NPY) was incubated with various quantities of antibody ( Ab) and with increasing amounts of the different peptides (10-11 to IO-4 M). Non-specific binding was evaluated using an irrelevant monoclonal antibody (IAb). Percentage inhibition was calculated according to the following formula: 100-(A Al/B Bl). In this formula, A and Al represent respectively the percentage binding obtained with Ab and IAb in the presence of peptide and and 1 the percentage binding obtained with the same antibodies incubated without peptide. The IC50 for each peptide was derived from the percentage inhibition and the peptide concentration.
The
capacity of the peptides to bind monoclonal antibodies
-
was
tested
-
Immunoradiometric assay (IRMA)
Polystyrene beads were coated with a first anti-NPY antibody ( capture antibody ). The beads ( 4.6 mm diameter ) were incubated overnight at room temperature with 1/750 to 1/1000 dilutions of ascitic fluid in PBS
(400 µ / bead ) under gentle shaking. The beads were then washed with deionized water and incubated for 18 hours with 250 µ of sample and 50 µ of an iodinated second antibody ( indicator antibody; 100 00 cpm; 16 mCi/mg ). The labelling was performed using iodogen (18). After incubation, the beads
were
washed twice with
pairs of capture and indicator antibodies
were
water
and
analyzed. 412
A
were
counted in
signal was
a gamma counter. Various obtained in the presence of several
NPY related peptides while noise
determined by incubating medium without peptide. The signal-toability of the peptides to bind to various sandwich systems. In our designation of pairs of antibodies, the first number indicates the unlabelled antibody (capture antibody) and the second the radiolabelled antibody (indicator antibody) as for example 02/05. was
noise ratio characterized the
RESULTS A total of 23 mice were immunized with NPY 1-36 and NPY 27-36. The splenocytes of six of them were used for fusions. Table 1 describes the three fusions that led to the production of four monoclonal
antibodies. The number of cultures positive for anti-hNPY activity were respectively 71 and 36 for the two mice injected with total NPY, and 4 for the one injected with the 27-36 NPY fragment (see table 1). Three cultures were considered positive as their 1/10 diluted supernatant was able to bind more than 20% of 125| NPY in the RIA. The antibodies secreted by these hybridomas were named NPY02, NPY03 and NPY05. In addition, each culture supernatant was tested for its ability to bind selected NPY
fragments (hNPYl-36, pNPYl-12, hNPYll-24, pNPY23-36, pNPY27-36). An additional hybridoma was retained because it recognizes preferentially the pNPYl-12 fragment (see below). The antibody secreted by this hybridoma was named NPY04. Immunization of several mice with KLH-conjugated 112, 11-24, 18-24, or 19-28 NPY fragments were unsuccessful after three sub-cutaneous injections at 2 month intervals.
Characteristics of the anti-NPY monoclonal antibodies The characteristics of the four anti-NPY monoclonal antibodies are presented in Table 2. The isotypes of each antibody were determined using an ELISA. NPY02 belongs to the IgGl sub-class whereas
NPY03 and NPY05 belong to the IgG2 sub-class. NPY04 is an IgG3. The affinity constant calculated from the RIA was very similar for NPY02 (Ka 5.5 1010 liters/mole) and NPY05 (2.5 1010 liters/mole). The poorest affinity was observed with NPY04 (Ka 3.8 108 liters/mole). =
=
Characterization of the anti genie determinants recognized by the monoclonal antibodies The antigenic determinants recognized by the different monoclonal antibodies were mapped by studying the binding characteristics of various NPY related peptides and NPY fragments. Figure 1 displays the inhibition of 125j NPY binding by selected competitors for each antibody. The IC50 values obtained
listed in Table 3. IC50's greater than -4 M were considered non-significant. The four antibodies bind human and porcine NPY with an IC50 in the nanomolar range. NPY02 binds the hNPY13-36 fragment merely 10 fold less efficiently ( IC50 8 nM ) than the whole NPY whereas the hNPY 16-36 is not significantly recognized by this antibody ( IC50 > 100 00 nM ). Among all other NPY fragment tested, only the hNPY 11-24 sub-peptide exhibits significant affinity for NPY02 ( IC50 200 nM ). are
=
=
Unlike NPY02, NPY03 cross-reacts with pPYY, sPP and aPP.The NPY03 antibody binds efficiently NPY C-terminal fragments. No significant loss of affinity is seen using C-terminal NPY fragments 413
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using diluted ascitic fluids and 125j_
Characteristics of the Anti-hNPY MonoclonalAntibodies
Hybridoma
Isotype
Epitope Binding Region
IgGl IgG2b IgG3 IgG2a
11-24
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NPY03 NPY04 *NPY05
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Ka
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). On the other hand, NPY03 does
with pNPY 32-36. NPY03 does not bind (Pro-34) NPY. of NPY 1-36 for NPY04 ( 9.0 nM) is 10 fold higher than for the other monoclonal IC50 antibodies.Only one member of the "PP fold" family ,the sPP cross-reacts efficiently with NPY04 antibody ( IC50 45 nM ). Among all NPY sub-peptides tested, only pNPYl-12 and pNPY5-20 were not cross-react
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significantly recognized by NPY04 (IC50 =2500 nM and 20 nM respectively ). To define more precisely the NPY04 epitope, we used ten different NPY analogs as competitors in which any of the 1st through the "1 residues were individually replaced by an alanine. These analogs are recognized by NPY04 with IC50 comparable with the one observed for native NPY (data not shown). All the members of the "PP-fold family of peptides tested in our assay cross-react with NPY05 (IC50 0.8 to 100 nM). Because NPY05 was obtained following immunization with NPY27-36, it is not surprising that C-terminal NPY fragments hNPY 13-36 to hNPY 27-36 are recognized by this antibody. Amidation of the C-terminal fragments is essential for NPY05 antibody recognition. The "
=
IC50 of the amidated form of NPY32-36 is identical with the one observed for native NPY, whereas its free acid form is very poorly reactive (IC50 50000 nM).The Morphin Modulating Neuropeptide, another amidated peptide showing some structural homology with the C-terminus of NPY, is weakly recognized by NPY05 ( IC50 8000 nM ). =
=
Immunoradiometric assay The availability of monoclonal antibodies with well defined epitopes makes it possible to measure NPY biological fluids using an immunoradiometric assay (IRMA). As described in material and methods, this type of assay requires two antibodies with non-overlapping epitopes ( capture and concentration in
indicator antibodies). With four monoclonal antibodies, twelve pairs of antibodies were theoretically possible. Monoclonal antibodies NPY02, NPY03, and NPY04 were chosen as possible capture antibodies. Monoclonal antibodies NPY02, NPY03 and NPY05 were used as possible indicator antibodies. Thus the following pairs of capture/indicator antibodies were tested: 04/02, 03/05, 04/03, 415
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Production and Characterization of Four Anti-Neuropeptide Y Monoclonal Antibodies ERIC GROUZMANN,' ETIENNE COMOY,2 PHILIPPE WALKER,1 MICHEL BURNIER,1 CLAUDE BOHUON,2 BERNARD WAEBER,1 and HANS BRUNNER1
'Hypertension Division and Cardiovascular Research Group, University Hospital, Lausanne, 2Departement de Biologie Clinique, Institut Gustave Roussy, Villejuif, France
Switzerland
ABSTRACT
neuropeptide Y (hNPY) is a potent vasoconstrictor peptide of 36 aminoacid residues. We isolated hybridomas secreting four monoclonal antibodies directed against various epitopes of neuropeptide Y and studied their cross-reactivity with peptide YY (PYY) and the pancreatic polypeptide (PP), two peptides sharing sequence homologies with hNPY (respectively 70% and 50%). The antibody NPY02 is an IgGl with a Ka of 5.5 1010 liters/mol. It binds to the 11-24 region of NPY (IC50 2 10-7M), does not recognize PP but cross-reacts weakly with PYY. Antibodies NPY03 and NPY05 are IgG2 with respective Ka's of 6.7 109 and 2.5 1010 liters/mol. They interact with a C-terminal epitope on NPY (NPY 27-34, IC50 2 10-9 M for NPY03 and NPY 3236, IC50 1 10-9 M for NPY05). These two antibodies cross-react with PYY whereas only NPY05 binds PP. NPY05 is unable to bind the free acid form of neuropeptide Y. The 32-36 COOH free subpeptide is recognized 50,000 less efficiently by NPY05 than its amidated form. Antibody NPY04 is an IgG3 with a Ka of 3.8 108 liters/mol. It recognizes a N-terminal epitope between aminoacids 1 and 12 (IC50 2·5 x 10-6 M). NPY04 interacts weakly with PYY but not detectably with PP. These Human
=
=
=
=
results obtained with 4 different monoclonal antibodies demonstrate the presence of at least four epitopes on hNPY, two of them being continuous. These antibodies will be used to study the interaction of NPY with its receptor and to develop sensitive and specific assays for determination of NPY concentrations in
biological fluids
.
INTRODUCTION
Neuropeptide Y (NPY) is a 36 amino-acid peptide first discovered by Tatemoto et al. in 1982 (1). This peptide is widely distributed in the central and peripheral nervous system (2). It possesses direct vasoconstrictor properties and also potentiates the contractile effect of various stimuli including norepinephrine and angiotensin II (3,4). Like many peptides processed from precursors, NPY has an 409
amidated C-terminal amino-acid (1). It is a member of a peptide family sharing the "PP fold" structure such as pancreatic peptide (PP) and peptide YY (PYY) (5). Several authors have prepared polyclonal antibodies directed against NPY. These antibodies have been used mainly for immuno-histochemical studies and for the determination of NPY concentration in biological fluids (6-12). Regarding circulating levels of NPY, different values have been measured depending on the polyclonal antibodies used. In some assays, a problem of cross-reactivity with structurally related peptides may exist. Recently, we have isolated 4 hybridomas secreting anti-NPY monoclonal antibodies. Two of them have been partially described in an earlier report and employed to set up an immuno-radiometric assay (13). In this paper, we describe the production and characterization of these four monoclonal antibodies as well as the mapping of their epitopes. MATERIAL AND METHODS
Peptides Peptide synthesis was performed according to Merrifield (14) using an automated synthesizer (Applied Biosystem, model 430A, Foster City, CA). The solid phase support was a benzyl-hydrylamine resin. Total human NPY (hNPY) and the following fragments were synthesized:l-12, 11-24, 18-24, 23-36, 27-36, 32-36 and the 32-36 acid-free peptide. The peptides were then deprotected and cleaved from the resin with anhydrous hydrofluoric acid. They were purified by gel filtration on biogel P/4 (BioRad). The purity of the peptides was tested by reverse phase high pressure liquid chromatography and the amino-acid composition was confirmed by amino acid analysis on an LKB Alpha Analyzer. Additional peptides were purchased. Porcine NPY (pNPY), human PP (hPP), bovine PP (bPP), avian PP (aPP) and rat PP (rPP) were obtained from Sigma (Sigma Ine, St Louis, USA). Salmon PP (sPP), porcine NPY16-36 (pNPY16-36), NPY20-36 (pNPY20-36), NPY22-36 (pNPY22-36), NPY26-36 (pNPY26-36) were from Peninsula (Peninsula, Belmont, CA). Porcine NPY 10- 18 (pNPY10-18), NPY14-23 (pNPY14-23), NPY19-28 (pNPY19-28), NPY24-36 (pNPY24-36) were from Novabiochem (Laufelfingen, Switzerland).PYY (pPYY), human PP31-36 (hPP31-36) and its free acid C- terminal analog were ordered from Bachern (Bachern Bubendorf, Switzerland). Human NPY13-36 (hNPY13-36) was kindly supplied by Dr. Kamper (Ciba-Geigy AG, Basel). Ten pNPY 1-36 analogs, in which residues 1 through 10 were substituted by an alanine were a gift from Dr A. Fournier (IRSC, Quebec). The Pro34 NPY was a kind gift from T. Schwarz (Copenhagen, Denmark). pNPY 5-20 was kindly provided by Dr. A. Beck.- Sickinger(Tiibingen, RFA). Immunization NPY 1-36
conjugated to keyhole limpet hemocyanin (KLH, Sigma, St Louis) using glutaraldehyde were injected subcutaneously to six week-old mice of different strains (Biozzi, C3H). Fifteen µg to 30 µg of the various conjugated antigens were administered together with the same amount of Freund complete adjuvant.The injections were repeated at monthly intervals with the same amount of immunogen in Freund's incomplete adjuvant.Response to immunization was evaluated 10 days after each boost by measuring the antibody levels in the mouse sera using the radioimmunoassay or
NPY 27-36
410
(RIA) described below.When the presence of antibody was detected (60 % binding or more with the diluted 1/2), two additional doses of immunogen were given, one intraperitoneally and the other one intravenously nine and three days before performing the fusion, respectively. mouse serum
Production of monoclonal antibodies Cell fusion,
hybridoma selection, cloning and ascites production were performed according to the procedure described by Bidart et al (15). Three weeks after the last injection, myeloma cells (P3-NS1-1Ag4-1 (NS1) were fused with splenocytes in 40% (w/v) polyethylene glycol (MW=1000). The ratio of NS1 cells to splenocytes was about 1 to 4. Anti-NPY antibody producing hybridoma were also detected with the RIA described below. After cloning, 2x106 cells were injected intraperitoneally to nude mice and ascites fluid collected after two weeks. Detection of antibodies The presence of anti-NPY antibodies in mouse sera and hybridoma supernatants was tested by RIA. Ten microliter serum or hybridoma supernatant were incubated at 20 ° C for two hours with 125l-NFY (3000 cpm /100 µ , 2000 Ci/mmol, Amersham GB) and 500 µ of assay buffer (50 mM phosphate buffer, 20 mM EDTA, 0.3% bovine serum albumin, 0.005% aprotinine, pH 7.5). The incubation was stopped by adding 250 µ of a charcoal-dextran suspension. After centrifugation (2000 g, 15 min) the
radioactivity in the pellet (P, free NPY) and in the supernatant (S, bound NPY) was counted and the ratio (S) / (S)+(P) was calculated. Non-specific binding was evaluated using a non-relevant monoclonal antibody. Hybridoma supernatants showing more than 20% *25 _ binding were further analysed for the presence of an anti-NPY antibody using an enzyme linked immunosorbent assay (ELISA). Microtiter plates (96-wells, NUNC) were coated for 24 hours at 4° C with 100 µ of a 10 mg/1 solution of hNPY dissolved in 0.1M phosphate buffered saline (PBS), pH 7.4. The wells were saturated by incubation for 18 hours at 4° C in the same buffer containing 1% BSA. After four washes with 10 mM PBS, containing 150 mM NaCl (pH 7.4), 100 µ of the culture supernatants were added to the wells and the plates were incubated for another hour at 37° C. After incubation, the wells were washed again four times and a 1000 fold dilution of peroxidase-conjugated rabbit anti-mouse IgG antiserum (200 µ , Nordic, Tilburg, The Netherlands) was added. After 45 min incubation at 37° C and four additional washes with the 10 mM PBS, pH 7.4, containing 150 mM NaCl, peroxidase activity was revealed by addition of 200 µ of a 50 mM citrate/phosphate buffer solution containing 500 mg/1 o-phenylene diamine and 0.007% H2O2. The reaction was stopped 20 min later by adding 50 µ of a 12.5% H2SO4 solution. The final concentration of NPY bound antibody was determined by measuring the absorbance at 492
nm on a
Titertek Multiskan ELISA reader (Flow Laboratories, Rockville,
MD).
Purification of antibodies Monoclonal antibodies were purified from
mouse
ascites
411
by amonium sulfate precipitation and protein A
chromatography as described by Ey et al (16) For IgGl purification, anion exchange chromatography (DEAE-Sephacel, Pharmacia) was performed prior to the affinity separation. The presence of anti-NPY immunoglobulins was monitored by the above described RIA. Protein was measured with the BioRad protein assay. Isotvpe identification The ELISA described above for the antibody detection was modified as follows: after coating with NPY, the wells were saturated, washed and 100 µ (20 ng) purified monoclonal antibody was added per well .The plates were then incubated for 1 h at 37° C. Then the wells were washed with PBS and rabbit antibodies (150 µ , 1/5000 diluted ) directed against mouse IgM, IgGl, IgG2 or IgG3 (Nordic,
Tilburgh, The Netherlands) were added in separate wells. After a 45 min incubation at 37° C and subsequent washing, 150 µ of peroxidase conjugated swine anti-rabbit antibody 1/2000 diluted ( Nordic), was added. Peroxidase activity was revealed over a 45 min incubation period. Determination of affinity constant The modified RIA procedure described by Van Heyningen et al. (17) was used for the affinity study. The NPY (or related peptides) concentration resulting in 50% tracer binding inhibition for each antibody
(IC50) was calculated. Identification of epitopes
by RIA. A constant amount of tracer (3000 cpm of ¡-^Sj NPY) was incubated with various quantities of antibody ( Ab) and with increasing amounts of the different peptides (10-11 to IO-4 M). Non-specific binding was evaluated using an irrelevant monoclonal antibody (IAb). Percentage inhibition was calculated according to the following formula: 100-(A Al/B Bl). In this formula, A and Al represent respectively the percentage binding obtained with Ab and IAb in the presence of peptide and and 1 the percentage binding obtained with the same antibodies incubated without peptide. The IC50 for each peptide was derived from the percentage inhibition and the peptide concentration.
The
capacity of the peptides to bind monoclonal antibodies
-
was
tested
-
Immunoradiometric assay (IRMA)
Polystyrene beads were coated with a first anti-NPY antibody ( capture antibody ). The beads ( 4.6 mm diameter ) were incubated overnight at room temperature with 1/750 to 1/1000 dilutions of ascitic fluid in PBS
(400 µ / bead ) under gentle shaking. The beads were then washed with deionized water and incubated for 18 hours with 250 µ of sample and 50 µ of an iodinated second antibody ( indicator antibody; 100 00 cpm; 16 mCi/mg ). The labelling was performed using iodogen (18). After incubation, the beads
were
washed twice with
pairs of capture and indicator antibodies
were
water
and
analyzed. 412
A
were
counted in
signal was
a gamma counter. Various obtained in the presence of several
NPY related peptides while noise
determined by incubating medium without peptide. The signal-toability of the peptides to bind to various sandwich systems. In our designation of pairs of antibodies, the first number indicates the unlabelled antibody (capture antibody) and the second the radiolabelled antibody (indicator antibody) as for example 02/05. was
noise ratio characterized the
RESULTS A total of 23 mice were immunized with NPY 1-36 and NPY 27-36. The splenocytes of six of them were used for fusions. Table 1 describes the three fusions that led to the production of four monoclonal
antibodies. The number of cultures positive for anti-hNPY activity were respectively 71 and 36 for the two mice injected with total NPY, and 4 for the one injected with the 27-36 NPY fragment (see table 1). Three cultures were considered positive as their 1/10 diluted supernatant was able to bind more than 20% of 125| NPY in the RIA. The antibodies secreted by these hybridomas were named NPY02, NPY03 and NPY05. In addition, each culture supernatant was tested for its ability to bind selected NPY
fragments (hNPYl-36, pNPYl-12, hNPYll-24, pNPY23-36, pNPY27-36). An additional hybridoma was retained because it recognizes preferentially the pNPYl-12 fragment (see below). The antibody secreted by this hybridoma was named NPY04. Immunization of several mice with KLH-conjugated 112, 11-24, 18-24, or 19-28 NPY fragments were unsuccessful after three sub-cutaneous injections at 2 month intervals.
Characteristics of the anti-NPY monoclonal antibodies The characteristics of the four anti-NPY monoclonal antibodies are presented in Table 2. The isotypes of each antibody were determined using an ELISA. NPY02 belongs to the IgGl sub-class whereas
NPY03 and NPY05 belong to the IgG2 sub-class. NPY04 is an IgG3. The affinity constant calculated from the RIA was very similar for NPY02 (Ka 5.5 1010 liters/mole) and NPY05 (2.5 1010 liters/mole). The poorest affinity was observed with NPY04 (Ka 3.8 108 liters/mole). =
=
Characterization of the anti genie determinants recognized by the monoclonal antibodies The antigenic determinants recognized by the different monoclonal antibodies were mapped by studying the binding characteristics of various NPY related peptides and NPY fragments. Figure 1 displays the inhibition of 125j NPY binding by selected competitors for each antibody. The IC50 values obtained
listed in Table 3. IC50's greater than -4 M were considered non-significant. The four antibodies bind human and porcine NPY with an IC50 in the nanomolar range. NPY02 binds the hNPY13-36 fragment merely 10 fold less efficiently ( IC50 8 nM ) than the whole NPY whereas the hNPY 16-36 is not significantly recognized by this antibody ( IC50 > 100 00 nM ). Among all other NPY fragment tested, only the hNPY 11-24 sub-peptide exhibits significant affinity for NPY02 ( IC50 200 nM ). are
=
=
Unlike NPY02, NPY03 cross-reacts with pPYY, sPP and aPP.The NPY03 antibody binds efficiently NPY C-terminal fragments. No significant loss of affinity is seen using C-terminal NPY fragments 413
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using diluted ascitic fluids and 125j_
Characteristics of the Anti-hNPY MonoclonalAntibodies
Hybridoma
Isotype
Epitope Binding Region
IgGl IgG2b IgG3 IgG2a
11-24
Clone *NPY02
NPY03 NPY04 *NPY05
hNPY 13-36
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hNPY 27-36
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27-34 1-12 32-36
competitors ( IC50
=
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Ka
(liters/mol)
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1010 109 IO» 1010
). On the other hand, NPY03 does
with pNPY 32-36. NPY03 does not bind (Pro-34) NPY. of NPY 1-36 for NPY04 ( 9.0 nM) is 10 fold higher than for the other monoclonal IC50 antibodies.Only one member of the "PP fold" family ,the sPP cross-reacts efficiently with NPY04 antibody ( IC50 45 nM ). Among all NPY sub-peptides tested, only pNPYl-12 and pNPY5-20 were not cross-react
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significantly recognized by NPY04 (IC50 =2500 nM and 20 nM respectively ). To define more precisely the NPY04 epitope, we used ten different NPY analogs as competitors in which any of the 1st through the "1 residues were individually replaced by an alanine. These analogs are recognized by NPY04 with IC50 comparable with the one observed for native NPY (data not shown). All the members of the "PP-fold family of peptides tested in our assay cross-react with NPY05 (IC50 0.8 to 100 nM). Because NPY05 was obtained following immunization with NPY27-36, it is not surprising that C-terminal NPY fragments hNPY 13-36 to hNPY 27-36 are recognized by this antibody. Amidation of the C-terminal fragments is essential for NPY05 antibody recognition. The "
=
IC50 of the amidated form of NPY32-36 is identical with the one observed for native NPY, whereas its free acid form is very poorly reactive (IC50 50000 nM).The Morphin Modulating Neuropeptide, another amidated peptide showing some structural homology with the C-terminus of NPY, is weakly recognized by NPY05 ( IC50 8000 nM ). =
=
Immunoradiometric assay The availability of monoclonal antibodies with well defined epitopes makes it possible to measure NPY biological fluids using an immunoradiometric assay (IRMA). As described in material and methods, this type of assay requires two antibodies with non-overlapping epitopes ( capture and concentration in
indicator antibodies). With four monoclonal antibodies, twelve pairs of antibodies were theoretically possible. Monoclonal antibodies NPY02, NPY03, and NPY04 were chosen as possible capture antibodies. Monoclonal antibodies NPY02, NPY03 and NPY05 were used as possible indicator antibodies. Thus the following pairs of capture/indicator antibodies were tested: 04/02, 03/05, 04/03, 415
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