HYBRIDOMA Volume 11, Number 2, 1992 Mary Ann Liebert, Inc., Publishers
Monoclonal Antibodies to Human Endothelin-1: Characterization and Utilization in Radioimmunoassay and Immunocytochemistry ABDULMAGED M. TRAISH,1 ELIZABETH MORAN,2 JENNIFER T. DALEY,2 ANTONIO de las MORENAS,2 and IÑIGO SAENZ de TEJADA2 Departments
of'Biochemistry and 2Urology,
Boston University, School
of Medicine, Boston,
MA 02118
ABSTRACT A host of monoclonal antibodies directed against human endothelin-1 (ET-1) has been developed and characterized. The antibodies reacted with ET-1 specifically and with high affinity, as determined by competition analysis and sucrose density gradients. The antibodies did not cross-react with neuropeptide YY, ß-endorphin, calcitonin gene-related peptide, secretin or somatostatin. The antibodies cross-reacted with big endothelin (B-ET), endothelin-2 (ET-2), vasointestinal constrictor peptide (VIC), and endothelin-3 (ET-3) albeit with varying affinity but did not cross-react with sarafotoxin (SRTX-6b). None of the antibodies reacted with the Cterminal hexapeptide (HXPT) of ET-1, indicating that the epitopes are not located within this region of ET-1. The monoclonal antibodies exhibited binding activity in dilutions ranging from 1:1000, to 1:106. The isotypes of the monoclonal antibodies were determined by competition binding assay. Six of the monoclonal antibodies were of the IgG , two were IgM and one of the IgG y2a subclass. The antibodies detected immunoreactive ETs by radioimmunoassay and in immunocytochemical localization, suggesting the potential use of these antibodies as tools to determine the concentration of ETs in biological fluids and in immunocytochemical localization of ETs in specific cell types in various tissues.
INTRODUCTION Endothelins (ETs), a family of 21-amino acid regulatory peptides, possess potent constrictor activity in vascular and nonvascular smooth muscle and mediate distinct biological effects in many cells and tissues (1-6). For instance, ET-1 increases systemic blood pressure (7), stimulates the release of atrial natriuretic peptide (ANP) (8), stimulates steroid secretion (9,10), increases DNA synthesis (11,12), stimulates cytosolic calcium and gonadotropin secretion in anterior pituitary cells (13), and stimulates the release of arginine vasopressin from perfused rat
hypothalamus (14). Although initially demonstrated in porcine aortic endothelial cells, it is now known that several other cell types synthesize and release endothelin (5,6,15,16), suggesting that endothelin may participate in autocrine and paracrine modulation of cell growth and function in many tissues. The role of ETs in regulation of cell function remains the subject of active investigation.
147
Thus far, limited information is available concerning the relationship between the in vivo levels of ET-1 and the various biological responses attributed to ET-1. Elevated endothelin levels have been detected in diabetes (17) and in preeclampsia (18). It is not yet known whether an increase in endothelin levels may contribute to the exacerbation of these diseases. There is considerable interest in the role of endothelin in the pathogenesis of cardiovascular diseases (1,2,5,6) and hypertension (5,19). Thus, the availability of methods for detection and measurement of ET-1 levels in tissue extracts and biological fluids should allow assessment of the possible role of ETs in the pathophysiology of some diseases. Further, the ability to determine the presence of ETs in specific cell types in tissue sections, using immunocytochemistry, is important for our understanding of the synthesis of ETs. The aims of the present study are : a) To develop a host of specific antibodies to ET-1; To b) develop a simple, inexpensive and reliable radioimmunoassay for the measurement of ET in biological fluids; c) To utilize these antibodies in immunocytochemical assay to determine their potential as tools for the localization and distribution of ETs in tissue sections.
MATERIALS AND METHODS
Isotopes and chemicals:
[I23I]endothelins (ET-1, ET-2 and ET-3), [125I]sarafotoxin (SRTX-6b) and [125I]vasoconstrictor intestinal peptide (VIC) were a gift from Dr. Russell Garlick, New England
Nuclear Co.
Billerica, MA. Unlabeled ET-1, ET-2 and ET-3, B-ET, SRTX-6b, and VIC were Inc. (Louisville, KY). Human neuropeptide YY, human ß-endorphin, human secretin, somatostatin (cyclic), and human calcitonin gene related peptide were obtained from Bachern, Inc. (Torrance, CA). All other chemicals were reagent grade and
purchased from Peptide International, were
obtained from commercial
sources.
Buffers and solutions: Phosphate buffered saline (PBS): 0.2 g KH2P04, 8 g NaCl, 0.16 g Na^PO,,.? H20 in one liter of distilled water, pH 7.5. Phosphate buffer/bovine serum albumin (BSA): 50 mM sodium phosphate, pH 7.4 0.1% BSA. ,
Conjugation of endothelin-1 to keyhole limpet hemocyanin (KLH): The conjugation of ET-1
to KLH was carried out as described for other peptides by Traish KLH dissolved in PBS to give a final concentration of 1 mg/ml. was al., (20,21). Briefly, Two mg of pure synthetic ET-1 was then dissolved in 2 ml of the KLH solution. The pH of the mixtures was adjusted to 9 with 0.1 M LiOH. Coupling of ET-1 to the carrier protein was et
initiated by dropwise addition of 6.25% glutaraldehyde to achieve a final concentration of 1%. The mixture was then incubated at 0-4°C for 1 h with gentle agitation. The mixture was transferred into dialysis tubing and dialyzed extensively against four changes of PBS. The dialysate was divided into small aliquots and frozen at -80°C until needed.
Immunization of mice: Five to eight week old female (BALB/C A/J) mice were obtained from Jackson Laboratories, Bar Harbor, ME. Three mice were immunized by subcutaneous injection with 50 pg of the ET-1-KLH conjugate emulsified in Freund's complete adjuvant. Two booster injections were given at 3 week intervals. Serum samples were collected 10 days after immunization and tested for specific antibodies to ET-1 by RIA. Mice with antiserum that recognized ET-1 were injected intraperitoneally (i.p.) one month later with 100 pg of the antigen in PBS. After three days the animals were sacrificed, their spleens were removed and used for fusion. Cells and media: Cultures of mouse myeloma cell line Sp2/0 were maintained in Dulbecco's modified Eagle's medium (DME) (GIBCO, Grand Island, NY) supplemented with fetal bovine serum 148
(FBS, 20%) (Hazelton, Lenexa, KS), penicillin (10 units per ml), streptomycin (100 pg/ml), nonessential amino acids (GIBCO) and glutamine (580 pg/ml). After fusion the cells were plated in DME supplemented with hypoxanthine (0.1 mM), aminopterin (0.4 µ ), thymidine (3 µ ) and concanavalin A (Con A) conditioned medium (10%) (HAT medium). DME medium supplemented with hypoxanthine and thymidine (HT medium) was also prepared for later use. Con A conditioned medium was obtained by incubating BALB/c mouse spleen cells (3xl06 cells/ml) with con A (2 pg/ml), at 37°C in DME under 5.6% C02 for 4 h. The tissue culture supernatants were separated by centrifugation and stored at 4°C. Cell fusion: Cell fusion was carried out as described elsewhere (21,22). Briefly, spleens were excised, fat and mesenteric tissues were removed quickly, and a single cell suspension was made by squeezing the spleen between two glass slides in Hank's balanced salt solution (HBSS) buffered with 0.01 M phosphate, pH 7.2. Red blood cells were Iysed by brief incubation in ammonium chloride lysis buffer. Spleen cells (5xl07) were mixed with Sp2/0 cells (5xl06) in round bottom tubes and pelleted at 700 g for 5 min at 22°C. The cells were resuspended in serum-free DME and centrifuged. After removal of the supernatants, the cell pellets were resuspended for six minutes in 0.5 ml of polyethylene glycol (PEG, 14500 MW, 30% vol/vol) (Baker Chemical Co., Phillipsburg, NJ) followed by addition of 4 ml of serum-free DME to dilute the PEG to a final concentration of 3.3%. The cell suspensions were transferred to petri dishes (100 17 mm), DME containing 20% fetal bovine serum (FBS) was added, and the cultures were incubated at 37°C for 24 h under 5.6% C02. The cells were then plated and resuspended in HAT conditioned medium (106 cells/ml). Aliquots of the cell suspension (0.1 ml) were dispensed into 96-well flatbottom microtiter dishes and incubated at 37°C. Seven days later the cultures were fed with 0.1 ml of conditioned media (DME, HT). After two additional days, clones were screened by RIA for the presence of ET antibodies.
Cloning by limiting dilution: Cells from wells that tested positive by ELISA were cloned by limiting dilution. Cells diluted to 1, 0.3 and 0.1 cell equivalent/100 µ in DME containing 20% FCS and BALB/c peritoneal exúdate cells (5xl04 cells/ml) and then plated in 96-well microtiter plates. After ten days wells with single clones were identified by microscopic examination and tested for the presence of ET antibodies by RIA (see below). Clones that tested positive were expanded in large flasks, spent media were collected and used for immunoprecipitation assay of ET-1. These cells were either used for production of ascites fluid or frozen tor later use. were
Determination of antibody class: The isotypes of the antibodies
were
determined
as
described
previously (21,22).
Ascites production: Female mice (BALB/c A/JF^ were injected i.p. with pristane (0.5 ml) and seven days later the animals were injected with 106 hybridoma cells in 0.2 ml of PBS. Ascites fluid was collected 7-10 days later by insertion of a needle into the peritoneal cavity. The fluid was clarified by centrifugation at 700 80°C until use.
g for 10
min, divided into several vials, and kept frozen
at -
Immunization of rabbits: Two New Zealand White female rabbits (7-9 lb) were obtained from Pine Acre Rabbitry (Norton, MA). Serum was collected from each rabbit by bleeding through the ear artery and designated as pre-immune serum. One day later each animal was injected subcutaneously (sc) at multiple sites along the back with a total of 1 ml of an emulsion made by mixing equal The final emulsion volumes of complete Freund's adjuvant and KLH-conjugated ET-1. contained 200 pg/ml of ET-1. After three weeks the animals were boosted with the antigen in incomplete Freund's adjuvant. Two weeks after the booster shots, the animals were bled, the sera 149
were
collected and tested for the presence of ET-antibodies
below).
by a radioimmunoassay (RIA) (see
Radioimmunoassay of ET by immunoprecipitation of ET-1 by monoclonal antibodies and polyclonal antibody:
Aliquots
of the diluted antisera
or
ascites fluid
were
incubated at
0-2°C for 16 h with
[125I]ET-1 (15,000-20,000 cpm) in the absence (control) or presence of increasing concentrations of ET-1. To precipitate the antibody-[125I]ET-l complexes, aliquots of anti-rabbit or anti-mouse
were added to the incubation and the samples were 4 h with intermittent reincubated 2°C for vortexing. The bound radioactivity was separated from free radioactivity by centrifugation and the pellets were washed three times by resuspension and centrifugation and counted in a -counter with 48% efficiency. Similar assays were performed by using secondary antibodies conjugated to polystyrene beads (Bio-Rad).
IgG-Amerlex conjugate (Amersham) at
Radioimmunoassay of ET with monoclonal antibodies and polyclonal antibody using dextran coated charcoal (DCC) assay:
Aliquots (100 pi in triplicates) of the diluted antibodies (as indicated in each experiment) incubated at 2°C for 16 h with 50 µ of [125I]ET-1 (15,000-20,000 cpm) in the presence or absence of increasing concentrations of ET-1, or other unlabeled competitor, in a final volume of 200 µ . The bound and free radioactivity were separated with addition of 200 µ of DCC and incubated at 2°C for an additional 20 minutes with intermittent vortexing. The charcoal was then separated by centrifugation at 1000 g for 5 minutes and 200 µ aliquots of the supernatants were removed and counted. The bound radioactivity was corrected for dilution by the charcoal suspension and expressed as percent of total added radioactivity. were
Sucrose density gradient (SDG)analysis of 25 -1 binding to the antibodies: Sucrose density gradient analysis was carried out as described (20-22). Briefly, sucrose density gradients (5-20%) were prepared in 50 tnM phosphate buffer, containing 0.4 M KC1 and 10% glycerol, pH 7.4. Samples of each antibody were incubated at 2°C for 16 h with [125I]ET-1 in the absence (total binding) or presence (nonspecific binding) of 1 µ of ET-1. At the end of the incubation, samples were treated with DCC to remove unbound [125I]ET-1 and aliquots were layered on the gradients. The gradients were centrifuged for 18 h at 2°C at 50,000 rpm in a SW 60 rotor, and then fractionated into individual 0.1 ml fractions and counted in a -counter for radioactivity. The specifically bound radioactivity was determined by subtraction of nonspecific binding from total binding and plotted as a function of the fraction number. The profiles represent specifically bound radioactivity.
Immunocytochemical analysis of ET in tissue sections: The detection of ET using monoclonal antibodies TR.48.5 (1:500 dilution) was carried human bowel tissue sections fixed in non-buffered 10% formalin using the avidin-biotin peroxidase complex technique (23). Test sections were incubated with the primary antibody (1:500), washed and then incubated with the biotinylated-secondary antibody. After washing of the unbound antibody, the sections were incubated with avidin-biotin peroxidase complex. To demonstrate that the staining represent specific binding in the same experiment, the antibody was pre-incubated with 4 nmoles of unlabeled ET prior to use in immunocytochemistry. out on
RESULTS
Development and characterization of monoclonal antibodies and polyclonal antibody:
Using ET-1-KLH conjugates as an immunogen, we have obtained eight monoclonal antibodies (TR.ET.8.1; TR.ET.55.5; TR.ET.48.5; TR.ET.49.2; TR.ET.52.4; TR.ET.57.6; TR.ET.33.1; TR.ET.32.1) and one polyclonal antibody (TR.ET.l) which bind ET-1 specifically
150
-10-9
ET-1 Figure
1. Determination of MAb TR.ET.8.1
-
8
-7
-
6
[log M] Binding Activity at Various Dilutions.
of the antibody were diluted and incubated at 0°C for 16 h with [125I]ET-1 in the absence (control) or the presence of increasing concentrations of unlabeled ET-1. At the end of the incubation, free radioactivity was separated from protein-bound radioactivity with DCC and the radioactivity was counted. The protein-bound radioactivity in each dilution was expressed as percent of that bound in the control incubation. The following dilutions were used: 1:100, open squares; 1:1000, solid triangles; 1:5000, open circles; 1:10,000 solid squares; 1:25,000, open triangles; 1:50,000, solid circles; 1:1,000,000, cross sign.
Aliquots
,
high affinity. The antibodies were screened by a RIA in which binding of [125I]ET-1 is displaced by ET-1. The titer of the antibodies was determined using various dilutions of the antibodies and displacement of bound [125I]ET-1 by ET-1. As shown in Figure 1, TR.ET.8.1 MAb was active at dilutions approaching 1:10*. Since the titer of the antibody is very high, no competition was observed at high concentrations of antibody. Analysis of the titer of the other antibodies gave working dilutions ranging from 1:1000 (TR.ET.l, TR.ET.33.1, TR.ET.57.6 and TR.ET.32.1) to 1:100,000 (TR.ET.49.2, TR.ET.52.4 and TR.ET.55.5). Figure 2 shows that the antibodies bound [125I]ET-1 specifically since ET-1 competed effectively for binding of [125I]ET-1. The antibodies bound ET-1 with high affinity since displacement of 50% of the bound [125I]ET-1 was achieved by 0.1-1 nM of ET-1. Similar binding data was obtained with the other antibodies (data not shown). To further demonstrate that the binding of [125I]ET-1 to the antibodies is of high affinity, we have analyzed the antibody-bound [125I]ET-1 on SDG. As shown in Figure 3 (panels A and B), [125I]ET-1 remained bound to the antibodies and with
with minimal dissociation,
even
indicating high affinity binding
after 18 h of centrifugation, under of [125I]ET-1 to the antibodies.
Peptide specificity of the antibodies: As shown in Figure 4, binding of
non-equilibrium conditions,
[125I]ET-1 to polyclonal antibody TR.ET.l was not 0.1 secretin (SEC), ß-endorphin (END), neuropeptide YY (YY), calcitonin gene displaced by µ related peptide (CGRP) or somatostatin (SOM) but was effectively displaced with ET-1. This suggests that this monoclonal antibody is specific to ET. Identical results were obtained with all monoclonal antibodies, confirming their peptide specificity (data not shown).
151
o m
O -11-10-9
-
8
7
-
-
6
[log M]
ET-1
0 -10-9
ET-1
-
8
-
7
-
6
[log M]
Figure 2. Displacement of [1ZSI]ET-1 Binding to the Monoclonal Antibodies and Polyclonal Antibody with Unlabeled ET-1. of diluted antibodies were incubated at 0°C for 16 h with [125I]ET-1 in the absence (control) or presence of increasing concentrations of unlabeled ET-1. At the end of the incubation, the samples were assayed for bound radioactivity with DCC. The protein-bound radioactivity was expressed as percent of bound [125I]ET-1 in the control incubation. The dilutions used in these experiments are 1:100,000 for the antibodies shown in the upper panel and 1:1,000,000 for the antibodies shown in the lower panel except for TR.ET. 1 which was at
Aliquots
1:1000.
152
10
20
30
40 0
10
20
30
Fraction Number Figure 3. Analysis of Antibody [12SI]ET-1
Interactions by Sucrose
Density Gradients.
diluted as described in Figure 2 and incubated at 0°C for 16 h with [I25I]ET-1 in the absence or presence of 1 µ unlabeled ET-1. At the end of the incubation, the samples were treated with DCC and analyzed on sucrose density gradients as described in the "Materials and Methods Section". Specific binding was determined by subtraction of nonspecific binding from total binding in each fraction and plotted as a function of fraction number. The arrow represents sedimentation of [14C]bovine serum albumin (4.6S), used as a sedimentation marker, in an identical gradient in the same experiment. Panel A: TR.ET.l, open triangles; TR.ET.8.1, open circles; TR.ET.55.5, open squares; Panel B: TR.ET.52.4, solid circles; TR.ET.49.2, solid triangles; TR.ET.48.5, solid squares.
Aliquots of each antibody
were
153
o m
O
ET1 ENDCGRPSEC SOM YY
UNLABELED COMPETITOR
(0.1 uM)
O m
0
ET1 ENDCGRPSEC SOM YY
UNLABELED COMPETITOR
Figure 4. Peptide Specificity of the Polyclonal and
(0.1 uM)
Monoclonal Antibodies.
Aliquots of the diluted polyclonal antibody (1:1000) were incubated at 0°C for 16 h with [125I]ET-
1 in the absence (control) or presence of 100 nM of unlabeled ET-1, ß-endorphin (END), calcitonin gene-related peptide (CGRP), secretin (SEC), somatostatin (SOM) or neuropeptide YY (YY). At the end of the incubation, the samples were analyzed for bound [125I]ET-1 with DCC assay. The upper panel represents monoclonal antibody TR.ET. 8.1; the lower panel represents the polyclonal antibody TR.ET.l.
154
o m
-10-9
ET-1
-
8
7
-
-
6
[log M]
Figure S. Cross-reactivity of the Monoclonal Antibodies and Polyclonal Antibody with ET-2 and ET-3.
Aliquots of the diluted MAb (1:100,000) were incubated at 0°C for 16 h with [125I]ET-1 in the absence (control) or the presence of increasing concentrations of ET-1, ET-2 and ET-3. At the end of the incubation, the samples were assayed for bound radioactivity with DCC assay. The bound radioactivity was expressed as percent of that bound in the control incubation.
Cross-reactivity of the antibodies with ET-2 and ET-3: 5 shows the competition analysis when MAb TR.ET 8.1 was incubated with in the absence or presence of increasing concentrations of ET-1, ET-2 or ET-3. ET-1, ET-2, and ET-3 displace the binding of [125I]ET-1 albeit with varying affinity. Similar data was obtained with all other antibodies (data not shown).
Figure
[125I]ET-1
Cross-reactivity of the antibodies with VIC, SRTX-6b and C-terminal hexapeptide: All antibodies bound VIC with varying affinity (Figure 6A). None of the monoclonal antibodies recognized SRTX-6b (< 1% displacement) (data not shown). The polyclonal antibody, however, cross-reacted with SRTX-6b (Figure 6B). Neither the monoclonal antibodies nor the polyclonal antibody recognized the C-terminal hexapeptide of ET-1 (Figure 6B and data not
shown).
Cross-reactivity of the antibodies with big endothelin (B-ET): As shown in Figure 7, the binding of [125I]ET-1 to MAb TR.ET.52.4 was displaced with 1 nM and 100 nM of unlabeled B-ET (figure 7, upper panel). Similar data was obtained with all other monoclonal antibodies (data not shown). In contrast, B-ET did not compete for binding
of [125I]ET-1 to polyclonal antibody TR.ET.l (Figure 7, lower panel) This observation suggests that the monoclonal antibodies and the polyclonal antibody may not share the same epitope (Figure 7, upper and lower panels). Since only representative data were shown, we have summarized the overall characteristics of the antibodies in Table 1. .
155
VIC
Û
[logM]
120 i 100
O m
-7
COMPETITOR
-
6
[logM]
Figure 6. Cross-reactivity of the Monoclonal Antibodies and Polyclonal Antibody with VIC, SRTX-61) and the C-terminal
hexapeptide.
Aliquots of the diluted monoclonal antibodies and polyclonal antibody were incubated at 0°C for 16 h with [I25I]ET-1 in the absence (control) or presence of increasing concentrations of VIC (panel A), SRTX-6b or the C-terminal hexapeptide (panel B). At the end of the incubation, the samples were assayed for bound radioactivity with DCC assay. The bound radioactivity was expressed as percent of that bound in the control incubation. Panel A: open circles, TR.ET.8.1; open triangles, TR.ET.55.5; solid triangles, TR.ET.48.5; open squares, TR.ET.52.4; solid squares, TR.ET.49.2; solid circles, TR.ET.57.6 and cross sign TR.ET.l. Panel B: represents competition for ET-1 with the C-terminal hexapeptide (solid circles) or sarafotoxin-6b (open squares).
156
TR.ET 52.4
0
B-ET
B-ET
ET-1
UNLABELED COMPETITOR 12 10 8 O 6
1nM
lOOnM
TR.ET.1
B-ET
B-ET
ET-1
m
2 0
0
UNLABELED COMPETITOR Figure 7. Cross-reactivity of the Monoclonal Antibodies and Polyclonal Antibody with BET.
Aliquots of the diluted antibodies were incubated at 0°C for
16 h with [125 ] -1 in the absence nM 100 of unlabeled ET-1. At the end of (control) the incubation, the samples were assayed for bound radioactivity with DCC assay. The bound radioactivity was expressed as percent of that bound in the control incubation. Upper panel represents binding of B-ET to MAb TR.ET.52.4 and the lower panel represents binding of polyclonal antibody TR.ET.l. or
presence of 1 nM, 100 nM of B-ET
or
157
CHARACTERISTICS OF ENDOTHELIN MONOCLONAL AND POLYCLONAL ANTIBODIES
Antibody
_cross-reactivity
Titer for ET-1
Affinity
for_
ET-2 ET-3 HXPT SRTX VIC BET
0.1nM
1:106
TR.ET 48.5
0.1nM
1:10s
+
TR.ET 49.2
0.1nM
1:105
+
TR.ET 52.4
0.1nM
1:10s
TR.ET 55.5
0.1nM
TR.ET 57.6
Isotvpe
+
+
IgGyl
+
+
+
IgGyl
+
+
+
IgGyl
+
+
+
+
1:105
+
+
+
+
N.D.
1 :1000
+
+
+
N.D.
TR.ET 33.1
N.D.
1 :1000
+
+
+
+
TR.ET 32.1
N.D.
1:1 ooo
+
+
+
+
lgG71 IgGyl IgM lgG72a IgM
TR.ET.1_
1nM_
1:1000
+
+
TR.ET 8.1
N.D.= not determined ; N.A.= not
+
+
NA
applicable
O m
0
-10-9
ET-1
-8
-7
-6
[log M]
Figure 8. Comparison of ET-1 Measurements by Radioimmunoassay Using Dextran Coated Charcoal and
Immunoprecipitation with Amerlex-conjugated Antibodies.
TR.ET.l were incubated at 0°C for 16 h with [125I]ET-1 in the absence (control) or presence of increasing concentrations of unlabeled ET-1. At the end of the incubation the samples were divided and either assayed by dextran coated charcoal or by reincubation with the secondary antibody conjugates, for additional 16 h as recommended by the instructions from the manufacturer, and the bound radioactivity was separated from free by centrifugation. The bound radioactivity was expressed as percent of that in the control incubation. Open squares represent the DCC assay and the solid circles represent the immunoprecipitation assay.
Aliquots of the polyclonal antibody
158
Measurements of ET-1 by RIA: To determine the concentrations of ET-1 with RIA, we have compared the immunoprecipitation assay method with that in which dextran coated charcoal (DCC) is used to remove unbound ET-1. Samples of the diluted antibody were incubated at 0°C for 16 h with [125I]ET-1 (15,000-20,000 cpm) and increasing concentrations of ET-1. At the end of the incubation, samples were analyzed with one of the following methods: a) immunoprecipitation using Bio-Rad immunobeads or Amerlex-conjugated secondary antibodies; b) treatment with DCC and counting the protein bound radioactivity. The data in Figure 8 compares the binding of ET-1 to polyclonal antibody TR.ET.l, as determined by immunoprecipitation assay and DCC assay. The binding data were comparable in their sensitivity for ET levels. However, the DCC assay is advantageous since it neither requires the secondary antibody conjugates nor the additional incubation time (16-24 h) and the subsequent washing of the precipitated material and centrifugation. The DCC assay is sensitive (1 pmol/ml), reproducible (
Monoclonal Antibodies to Human Endothelin-1: Characterization and Utilization in Radioimmunoassay and Immunocytochemistry ABDULMAGED M. TRAISH,1 ELIZABETH MORAN,2 JENNIFER T. DALEY,2 ANTONIO de las MORENAS,2 and IÑIGO SAENZ de TEJADA2 Departments
of'Biochemistry and 2Urology,
Boston University, School
of Medicine, Boston,
MA 02118
ABSTRACT A host of monoclonal antibodies directed against human endothelin-1 (ET-1) has been developed and characterized. The antibodies reacted with ET-1 specifically and with high affinity, as determined by competition analysis and sucrose density gradients. The antibodies did not cross-react with neuropeptide YY, ß-endorphin, calcitonin gene-related peptide, secretin or somatostatin. The antibodies cross-reacted with big endothelin (B-ET), endothelin-2 (ET-2), vasointestinal constrictor peptide (VIC), and endothelin-3 (ET-3) albeit with varying affinity but did not cross-react with sarafotoxin (SRTX-6b). None of the antibodies reacted with the Cterminal hexapeptide (HXPT) of ET-1, indicating that the epitopes are not located within this region of ET-1. The monoclonal antibodies exhibited binding activity in dilutions ranging from 1:1000, to 1:106. The isotypes of the monoclonal antibodies were determined by competition binding assay. Six of the monoclonal antibodies were of the IgG , two were IgM and one of the IgG y2a subclass. The antibodies detected immunoreactive ETs by radioimmunoassay and in immunocytochemical localization, suggesting the potential use of these antibodies as tools to determine the concentration of ETs in biological fluids and in immunocytochemical localization of ETs in specific cell types in various tissues.
INTRODUCTION Endothelins (ETs), a family of 21-amino acid regulatory peptides, possess potent constrictor activity in vascular and nonvascular smooth muscle and mediate distinct biological effects in many cells and tissues (1-6). For instance, ET-1 increases systemic blood pressure (7), stimulates the release of atrial natriuretic peptide (ANP) (8), stimulates steroid secretion (9,10), increases DNA synthesis (11,12), stimulates cytosolic calcium and gonadotropin secretion in anterior pituitary cells (13), and stimulates the release of arginine vasopressin from perfused rat
hypothalamus (14). Although initially demonstrated in porcine aortic endothelial cells, it is now known that several other cell types synthesize and release endothelin (5,6,15,16), suggesting that endothelin may participate in autocrine and paracrine modulation of cell growth and function in many tissues. The role of ETs in regulation of cell function remains the subject of active investigation.
147
Thus far, limited information is available concerning the relationship between the in vivo levels of ET-1 and the various biological responses attributed to ET-1. Elevated endothelin levels have been detected in diabetes (17) and in preeclampsia (18). It is not yet known whether an increase in endothelin levels may contribute to the exacerbation of these diseases. There is considerable interest in the role of endothelin in the pathogenesis of cardiovascular diseases (1,2,5,6) and hypertension (5,19). Thus, the availability of methods for detection and measurement of ET-1 levels in tissue extracts and biological fluids should allow assessment of the possible role of ETs in the pathophysiology of some diseases. Further, the ability to determine the presence of ETs in specific cell types in tissue sections, using immunocytochemistry, is important for our understanding of the synthesis of ETs. The aims of the present study are : a) To develop a host of specific antibodies to ET-1; To b) develop a simple, inexpensive and reliable radioimmunoassay for the measurement of ET in biological fluids; c) To utilize these antibodies in immunocytochemical assay to determine their potential as tools for the localization and distribution of ETs in tissue sections.
MATERIALS AND METHODS
Isotopes and chemicals:
[I23I]endothelins (ET-1, ET-2 and ET-3), [125I]sarafotoxin (SRTX-6b) and [125I]vasoconstrictor intestinal peptide (VIC) were a gift from Dr. Russell Garlick, New England
Nuclear Co.
Billerica, MA. Unlabeled ET-1, ET-2 and ET-3, B-ET, SRTX-6b, and VIC were Inc. (Louisville, KY). Human neuropeptide YY, human ß-endorphin, human secretin, somatostatin (cyclic), and human calcitonin gene related peptide were obtained from Bachern, Inc. (Torrance, CA). All other chemicals were reagent grade and
purchased from Peptide International, were
obtained from commercial
sources.
Buffers and solutions: Phosphate buffered saline (PBS): 0.2 g KH2P04, 8 g NaCl, 0.16 g Na^PO,,.? H20 in one liter of distilled water, pH 7.5. Phosphate buffer/bovine serum albumin (BSA): 50 mM sodium phosphate, pH 7.4 0.1% BSA. ,
Conjugation of endothelin-1 to keyhole limpet hemocyanin (KLH): The conjugation of ET-1
to KLH was carried out as described for other peptides by Traish KLH dissolved in PBS to give a final concentration of 1 mg/ml. was al., (20,21). Briefly, Two mg of pure synthetic ET-1 was then dissolved in 2 ml of the KLH solution. The pH of the mixtures was adjusted to 9 with 0.1 M LiOH. Coupling of ET-1 to the carrier protein was et
initiated by dropwise addition of 6.25% glutaraldehyde to achieve a final concentration of 1%. The mixture was then incubated at 0-4°C for 1 h with gentle agitation. The mixture was transferred into dialysis tubing and dialyzed extensively against four changes of PBS. The dialysate was divided into small aliquots and frozen at -80°C until needed.
Immunization of mice: Five to eight week old female (BALB/C A/J) mice were obtained from Jackson Laboratories, Bar Harbor, ME. Three mice were immunized by subcutaneous injection with 50 pg of the ET-1-KLH conjugate emulsified in Freund's complete adjuvant. Two booster injections were given at 3 week intervals. Serum samples were collected 10 days after immunization and tested for specific antibodies to ET-1 by RIA. Mice with antiserum that recognized ET-1 were injected intraperitoneally (i.p.) one month later with 100 pg of the antigen in PBS. After three days the animals were sacrificed, their spleens were removed and used for fusion. Cells and media: Cultures of mouse myeloma cell line Sp2/0 were maintained in Dulbecco's modified Eagle's medium (DME) (GIBCO, Grand Island, NY) supplemented with fetal bovine serum 148
(FBS, 20%) (Hazelton, Lenexa, KS), penicillin (10 units per ml), streptomycin (100 pg/ml), nonessential amino acids (GIBCO) and glutamine (580 pg/ml). After fusion the cells were plated in DME supplemented with hypoxanthine (0.1 mM), aminopterin (0.4 µ ), thymidine (3 µ ) and concanavalin A (Con A) conditioned medium (10%) (HAT medium). DME medium supplemented with hypoxanthine and thymidine (HT medium) was also prepared for later use. Con A conditioned medium was obtained by incubating BALB/c mouse spleen cells (3xl06 cells/ml) with con A (2 pg/ml), at 37°C in DME under 5.6% C02 for 4 h. The tissue culture supernatants were separated by centrifugation and stored at 4°C. Cell fusion: Cell fusion was carried out as described elsewhere (21,22). Briefly, spleens were excised, fat and mesenteric tissues were removed quickly, and a single cell suspension was made by squeezing the spleen between two glass slides in Hank's balanced salt solution (HBSS) buffered with 0.01 M phosphate, pH 7.2. Red blood cells were Iysed by brief incubation in ammonium chloride lysis buffer. Spleen cells (5xl07) were mixed with Sp2/0 cells (5xl06) in round bottom tubes and pelleted at 700 g for 5 min at 22°C. The cells were resuspended in serum-free DME and centrifuged. After removal of the supernatants, the cell pellets were resuspended for six minutes in 0.5 ml of polyethylene glycol (PEG, 14500 MW, 30% vol/vol) (Baker Chemical Co., Phillipsburg, NJ) followed by addition of 4 ml of serum-free DME to dilute the PEG to a final concentration of 3.3%. The cell suspensions were transferred to petri dishes (100 17 mm), DME containing 20% fetal bovine serum (FBS) was added, and the cultures were incubated at 37°C for 24 h under 5.6% C02. The cells were then plated and resuspended in HAT conditioned medium (106 cells/ml). Aliquots of the cell suspension (0.1 ml) were dispensed into 96-well flatbottom microtiter dishes and incubated at 37°C. Seven days later the cultures were fed with 0.1 ml of conditioned media (DME, HT). After two additional days, clones were screened by RIA for the presence of ET antibodies.
Cloning by limiting dilution: Cells from wells that tested positive by ELISA were cloned by limiting dilution. Cells diluted to 1, 0.3 and 0.1 cell equivalent/100 µ in DME containing 20% FCS and BALB/c peritoneal exúdate cells (5xl04 cells/ml) and then plated in 96-well microtiter plates. After ten days wells with single clones were identified by microscopic examination and tested for the presence of ET antibodies by RIA (see below). Clones that tested positive were expanded in large flasks, spent media were collected and used for immunoprecipitation assay of ET-1. These cells were either used for production of ascites fluid or frozen tor later use. were
Determination of antibody class: The isotypes of the antibodies
were
determined
as
described
previously (21,22).
Ascites production: Female mice (BALB/c A/JF^ were injected i.p. with pristane (0.5 ml) and seven days later the animals were injected with 106 hybridoma cells in 0.2 ml of PBS. Ascites fluid was collected 7-10 days later by insertion of a needle into the peritoneal cavity. The fluid was clarified by centrifugation at 700 80°C until use.
g for 10
min, divided into several vials, and kept frozen
at -
Immunization of rabbits: Two New Zealand White female rabbits (7-9 lb) were obtained from Pine Acre Rabbitry (Norton, MA). Serum was collected from each rabbit by bleeding through the ear artery and designated as pre-immune serum. One day later each animal was injected subcutaneously (sc) at multiple sites along the back with a total of 1 ml of an emulsion made by mixing equal The final emulsion volumes of complete Freund's adjuvant and KLH-conjugated ET-1. contained 200 pg/ml of ET-1. After three weeks the animals were boosted with the antigen in incomplete Freund's adjuvant. Two weeks after the booster shots, the animals were bled, the sera 149
were
collected and tested for the presence of ET-antibodies
below).
by a radioimmunoassay (RIA) (see
Radioimmunoassay of ET by immunoprecipitation of ET-1 by monoclonal antibodies and polyclonal antibody:
Aliquots
of the diluted antisera
or
ascites fluid
were
incubated at
0-2°C for 16 h with
[125I]ET-1 (15,000-20,000 cpm) in the absence (control) or presence of increasing concentrations of ET-1. To precipitate the antibody-[125I]ET-l complexes, aliquots of anti-rabbit or anti-mouse
were added to the incubation and the samples were 4 h with intermittent reincubated 2°C for vortexing. The bound radioactivity was separated from free radioactivity by centrifugation and the pellets were washed three times by resuspension and centrifugation and counted in a -counter with 48% efficiency. Similar assays were performed by using secondary antibodies conjugated to polystyrene beads (Bio-Rad).
IgG-Amerlex conjugate (Amersham) at
Radioimmunoassay of ET with monoclonal antibodies and polyclonal antibody using dextran coated charcoal (DCC) assay:
Aliquots (100 pi in triplicates) of the diluted antibodies (as indicated in each experiment) incubated at 2°C for 16 h with 50 µ of [125I]ET-1 (15,000-20,000 cpm) in the presence or absence of increasing concentrations of ET-1, or other unlabeled competitor, in a final volume of 200 µ . The bound and free radioactivity were separated with addition of 200 µ of DCC and incubated at 2°C for an additional 20 minutes with intermittent vortexing. The charcoal was then separated by centrifugation at 1000 g for 5 minutes and 200 µ aliquots of the supernatants were removed and counted. The bound radioactivity was corrected for dilution by the charcoal suspension and expressed as percent of total added radioactivity. were
Sucrose density gradient (SDG)analysis of 25 -1 binding to the antibodies: Sucrose density gradient analysis was carried out as described (20-22). Briefly, sucrose density gradients (5-20%) were prepared in 50 tnM phosphate buffer, containing 0.4 M KC1 and 10% glycerol, pH 7.4. Samples of each antibody were incubated at 2°C for 16 h with [125I]ET-1 in the absence (total binding) or presence (nonspecific binding) of 1 µ of ET-1. At the end of the incubation, samples were treated with DCC to remove unbound [125I]ET-1 and aliquots were layered on the gradients. The gradients were centrifuged for 18 h at 2°C at 50,000 rpm in a SW 60 rotor, and then fractionated into individual 0.1 ml fractions and counted in a -counter for radioactivity. The specifically bound radioactivity was determined by subtraction of nonspecific binding from total binding and plotted as a function of the fraction number. The profiles represent specifically bound radioactivity.
Immunocytochemical analysis of ET in tissue sections: The detection of ET using monoclonal antibodies TR.48.5 (1:500 dilution) was carried human bowel tissue sections fixed in non-buffered 10% formalin using the avidin-biotin peroxidase complex technique (23). Test sections were incubated with the primary antibody (1:500), washed and then incubated with the biotinylated-secondary antibody. After washing of the unbound antibody, the sections were incubated with avidin-biotin peroxidase complex. To demonstrate that the staining represent specific binding in the same experiment, the antibody was pre-incubated with 4 nmoles of unlabeled ET prior to use in immunocytochemistry. out on
RESULTS
Development and characterization of monoclonal antibodies and polyclonal antibody:
Using ET-1-KLH conjugates as an immunogen, we have obtained eight monoclonal antibodies (TR.ET.8.1; TR.ET.55.5; TR.ET.48.5; TR.ET.49.2; TR.ET.52.4; TR.ET.57.6; TR.ET.33.1; TR.ET.32.1) and one polyclonal antibody (TR.ET.l) which bind ET-1 specifically
150
-10-9
ET-1 Figure
1. Determination of MAb TR.ET.8.1
-
8
-7
-
6
[log M] Binding Activity at Various Dilutions.
of the antibody were diluted and incubated at 0°C for 16 h with [125I]ET-1 in the absence (control) or the presence of increasing concentrations of unlabeled ET-1. At the end of the incubation, free radioactivity was separated from protein-bound radioactivity with DCC and the radioactivity was counted. The protein-bound radioactivity in each dilution was expressed as percent of that bound in the control incubation. The following dilutions were used: 1:100, open squares; 1:1000, solid triangles; 1:5000, open circles; 1:10,000 solid squares; 1:25,000, open triangles; 1:50,000, solid circles; 1:1,000,000, cross sign.
Aliquots
,
high affinity. The antibodies were screened by a RIA in which binding of [125I]ET-1 is displaced by ET-1. The titer of the antibodies was determined using various dilutions of the antibodies and displacement of bound [125I]ET-1 by ET-1. As shown in Figure 1, TR.ET.8.1 MAb was active at dilutions approaching 1:10*. Since the titer of the antibody is very high, no competition was observed at high concentrations of antibody. Analysis of the titer of the other antibodies gave working dilutions ranging from 1:1000 (TR.ET.l, TR.ET.33.1, TR.ET.57.6 and TR.ET.32.1) to 1:100,000 (TR.ET.49.2, TR.ET.52.4 and TR.ET.55.5). Figure 2 shows that the antibodies bound [125I]ET-1 specifically since ET-1 competed effectively for binding of [125I]ET-1. The antibodies bound ET-1 with high affinity since displacement of 50% of the bound [125I]ET-1 was achieved by 0.1-1 nM of ET-1. Similar binding data was obtained with the other antibodies (data not shown). To further demonstrate that the binding of [125I]ET-1 to the antibodies is of high affinity, we have analyzed the antibody-bound [125I]ET-1 on SDG. As shown in Figure 3 (panels A and B), [125I]ET-1 remained bound to the antibodies and with
with minimal dissociation,
even
indicating high affinity binding
after 18 h of centrifugation, under of [125I]ET-1 to the antibodies.
Peptide specificity of the antibodies: As shown in Figure 4, binding of
non-equilibrium conditions,
[125I]ET-1 to polyclonal antibody TR.ET.l was not 0.1 secretin (SEC), ß-endorphin (END), neuropeptide YY (YY), calcitonin gene displaced by µ related peptide (CGRP) or somatostatin (SOM) but was effectively displaced with ET-1. This suggests that this monoclonal antibody is specific to ET. Identical results were obtained with all monoclonal antibodies, confirming their peptide specificity (data not shown).
151
o m
O -11-10-9
-
8
7
-
-
6
[log M]
ET-1
0 -10-9
ET-1
-
8
-
7
-
6
[log M]
Figure 2. Displacement of [1ZSI]ET-1 Binding to the Monoclonal Antibodies and Polyclonal Antibody with Unlabeled ET-1. of diluted antibodies were incubated at 0°C for 16 h with [125I]ET-1 in the absence (control) or presence of increasing concentrations of unlabeled ET-1. At the end of the incubation, the samples were assayed for bound radioactivity with DCC. The protein-bound radioactivity was expressed as percent of bound [125I]ET-1 in the control incubation. The dilutions used in these experiments are 1:100,000 for the antibodies shown in the upper panel and 1:1,000,000 for the antibodies shown in the lower panel except for TR.ET. 1 which was at
Aliquots
1:1000.
152
10
20
30
40 0
10
20
30
Fraction Number Figure 3. Analysis of Antibody [12SI]ET-1
Interactions by Sucrose
Density Gradients.
diluted as described in Figure 2 and incubated at 0°C for 16 h with [I25I]ET-1 in the absence or presence of 1 µ unlabeled ET-1. At the end of the incubation, the samples were treated with DCC and analyzed on sucrose density gradients as described in the "Materials and Methods Section". Specific binding was determined by subtraction of nonspecific binding from total binding in each fraction and plotted as a function of fraction number. The arrow represents sedimentation of [14C]bovine serum albumin (4.6S), used as a sedimentation marker, in an identical gradient in the same experiment. Panel A: TR.ET.l, open triangles; TR.ET.8.1, open circles; TR.ET.55.5, open squares; Panel B: TR.ET.52.4, solid circles; TR.ET.49.2, solid triangles; TR.ET.48.5, solid squares.
Aliquots of each antibody
were
153
o m
O
ET1 ENDCGRPSEC SOM YY
UNLABELED COMPETITOR
(0.1 uM)
O m
0
ET1 ENDCGRPSEC SOM YY
UNLABELED COMPETITOR
Figure 4. Peptide Specificity of the Polyclonal and
(0.1 uM)
Monoclonal Antibodies.
Aliquots of the diluted polyclonal antibody (1:1000) were incubated at 0°C for 16 h with [125I]ET-
1 in the absence (control) or presence of 100 nM of unlabeled ET-1, ß-endorphin (END), calcitonin gene-related peptide (CGRP), secretin (SEC), somatostatin (SOM) or neuropeptide YY (YY). At the end of the incubation, the samples were analyzed for bound [125I]ET-1 with DCC assay. The upper panel represents monoclonal antibody TR.ET. 8.1; the lower panel represents the polyclonal antibody TR.ET.l.
154
o m
-10-9
ET-1
-
8
7
-
-
6
[log M]
Figure S. Cross-reactivity of the Monoclonal Antibodies and Polyclonal Antibody with ET-2 and ET-3.
Aliquots of the diluted MAb (1:100,000) were incubated at 0°C for 16 h with [125I]ET-1 in the absence (control) or the presence of increasing concentrations of ET-1, ET-2 and ET-3. At the end of the incubation, the samples were assayed for bound radioactivity with DCC assay. The bound radioactivity was expressed as percent of that bound in the control incubation.
Cross-reactivity of the antibodies with ET-2 and ET-3: 5 shows the competition analysis when MAb TR.ET 8.1 was incubated with in the absence or presence of increasing concentrations of ET-1, ET-2 or ET-3. ET-1, ET-2, and ET-3 displace the binding of [125I]ET-1 albeit with varying affinity. Similar data was obtained with all other antibodies (data not shown).
Figure
[125I]ET-1
Cross-reactivity of the antibodies with VIC, SRTX-6b and C-terminal hexapeptide: All antibodies bound VIC with varying affinity (Figure 6A). None of the monoclonal antibodies recognized SRTX-6b (< 1% displacement) (data not shown). The polyclonal antibody, however, cross-reacted with SRTX-6b (Figure 6B). Neither the monoclonal antibodies nor the polyclonal antibody recognized the C-terminal hexapeptide of ET-1 (Figure 6B and data not
shown).
Cross-reactivity of the antibodies with big endothelin (B-ET): As shown in Figure 7, the binding of [125I]ET-1 to MAb TR.ET.52.4 was displaced with 1 nM and 100 nM of unlabeled B-ET (figure 7, upper panel). Similar data was obtained with all other monoclonal antibodies (data not shown). In contrast, B-ET did not compete for binding
of [125I]ET-1 to polyclonal antibody TR.ET.l (Figure 7, lower panel) This observation suggests that the monoclonal antibodies and the polyclonal antibody may not share the same epitope (Figure 7, upper and lower panels). Since only representative data were shown, we have summarized the overall characteristics of the antibodies in Table 1. .
155
VIC
Û
[logM]
120 i 100
O m
-7
COMPETITOR
-
6
[logM]
Figure 6. Cross-reactivity of the Monoclonal Antibodies and Polyclonal Antibody with VIC, SRTX-61) and the C-terminal
hexapeptide.
Aliquots of the diluted monoclonal antibodies and polyclonal antibody were incubated at 0°C for 16 h with [I25I]ET-1 in the absence (control) or presence of increasing concentrations of VIC (panel A), SRTX-6b or the C-terminal hexapeptide (panel B). At the end of the incubation, the samples were assayed for bound radioactivity with DCC assay. The bound radioactivity was expressed as percent of that bound in the control incubation. Panel A: open circles, TR.ET.8.1; open triangles, TR.ET.55.5; solid triangles, TR.ET.48.5; open squares, TR.ET.52.4; solid squares, TR.ET.49.2; solid circles, TR.ET.57.6 and cross sign TR.ET.l. Panel B: represents competition for ET-1 with the C-terminal hexapeptide (solid circles) or sarafotoxin-6b (open squares).
156
TR.ET 52.4
0
B-ET
B-ET
ET-1
UNLABELED COMPETITOR 12 10 8 O 6
1nM
lOOnM
TR.ET.1
B-ET
B-ET
ET-1
m
2 0
0
UNLABELED COMPETITOR Figure 7. Cross-reactivity of the Monoclonal Antibodies and Polyclonal Antibody with BET.
Aliquots of the diluted antibodies were incubated at 0°C for
16 h with [125 ] -1 in the absence nM 100 of unlabeled ET-1. At the end of (control) the incubation, the samples were assayed for bound radioactivity with DCC assay. The bound radioactivity was expressed as percent of that bound in the control incubation. Upper panel represents binding of B-ET to MAb TR.ET.52.4 and the lower panel represents binding of polyclonal antibody TR.ET.l. or
presence of 1 nM, 100 nM of B-ET
or
157
CHARACTERISTICS OF ENDOTHELIN MONOCLONAL AND POLYCLONAL ANTIBODIES
Antibody
_cross-reactivity
Titer for ET-1
Affinity
for_
ET-2 ET-3 HXPT SRTX VIC BET
0.1nM
1:106
TR.ET 48.5
0.1nM
1:10s
+
TR.ET 49.2
0.1nM
1:105
+
TR.ET 52.4
0.1nM
1:10s
TR.ET 55.5
0.1nM
TR.ET 57.6
Isotvpe
+
+
IgGyl
+
+
+
IgGyl
+
+
+
IgGyl
+
+
+
+
1:105
+
+
+
+
N.D.
1 :1000
+
+
+
N.D.
TR.ET 33.1
N.D.
1 :1000
+
+
+
+
TR.ET 32.1
N.D.
1:1 ooo
+
+
+
+
lgG71 IgGyl IgM lgG72a IgM
TR.ET.1_
1nM_
1:1000
+
+
TR.ET 8.1
N.D.= not determined ; N.A.= not
+
+
NA
applicable
O m
0
-10-9
ET-1
-8
-7
-6
[log M]
Figure 8. Comparison of ET-1 Measurements by Radioimmunoassay Using Dextran Coated Charcoal and
Immunoprecipitation with Amerlex-conjugated Antibodies.
TR.ET.l were incubated at 0°C for 16 h with [125I]ET-1 in the absence (control) or presence of increasing concentrations of unlabeled ET-1. At the end of the incubation the samples were divided and either assayed by dextran coated charcoal or by reincubation with the secondary antibody conjugates, for additional 16 h as recommended by the instructions from the manufacturer, and the bound radioactivity was separated from free by centrifugation. The bound radioactivity was expressed as percent of that in the control incubation. Open squares represent the DCC assay and the solid circles represent the immunoprecipitation assay.
Aliquots of the polyclonal antibody
158
Measurements of ET-1 by RIA: To determine the concentrations of ET-1 with RIA, we have compared the immunoprecipitation assay method with that in which dextran coated charcoal (DCC) is used to remove unbound ET-1. Samples of the diluted antibody were incubated at 0°C for 16 h with [125I]ET-1 (15,000-20,000 cpm) and increasing concentrations of ET-1. At the end of the incubation, samples were analyzed with one of the following methods: a) immunoprecipitation using Bio-Rad immunobeads or Amerlex-conjugated secondary antibodies; b) treatment with DCC and counting the protein bound radioactivity. The data in Figure 8 compares the binding of ET-1 to polyclonal antibody TR.ET.l, as determined by immunoprecipitation assay and DCC assay. The binding data were comparable in their sensitivity for ET levels. However, the DCC assay is advantageous since it neither requires the secondary antibody conjugates nor the additional incubation time (16-24 h) and the subsequent washing of the precipitated material and centrifugation. The DCC assay is sensitive (1 pmol/ml), reproducible (
