BLOOD PRESSURE
1992; 1 181-1 86
Radioimmunoassay of Endothelin in Human Plasma PETER HAVE RASMUSSEN,' INGER GRAVES PLUM,' NIELS ESKE BRUUN,' HARRIET DIGE-PETERSEN,' THOMAS HEDNER,2 JAN HEDNER2 and JORN GIESE3 From the 'Department of Clinical Physiology and Nuclear Medicine, Glostrup Hospital, University of Copenhagen, DK-2600 Glostrup, Denmark, 2Department of Clinical Pharmacology, Sahlgrenska Hospital, University of Goteborg. 9 4 1 3 45 Goteborg. Sweden, and University Institute of Experimental Medicine, Division of Human Pathophysiology. The Panum Institute, DK-2200 Copenhagen N,Denmark
'
Blood Press Downloaded from informahealthcare.com by University of Toronto on 01/16/15 For personal use only.
Rasmussen PH, Plum IG, Bruun NE, Dige-Petersen H, Hedner T, Hedner J, Giese J. Radioimmunoassay ofendothe/in in human plasma. Blood Pressure 1992; 1 : 18 I - 186.
A specific and sensitive radioimmunoassay (RIA) for determination of endothelin-l (ET-1) in human plasma has been developed. Antibodies were raised in rabbits using synthetic ET-I conjugated to thyroglobulin as immunogen. The antibodies obtained were used at a final dilution of 1 :300,000 yielding maximum binding of 61.7 & 3.0%(mean f 1 SD, n = 20) of IZ5I-ET1. The IDSO (inhibitory dose 50%) was 4.5 k0.6 fmo1/100 pl (mean k 1 SD, n = 20). The sensitivity of the RIA was 0.33 fmol/IOO pl standard solution. No cross reactivity was observed with endothelin-3, big-endothelin-I, atrial natriuretic factor, angiotensin 1 or angiotensin 11. The cross-reactivity with endothelin-2 was 100%.Endothelin was extracted from acidified plasma with Sep-pak c18 cartridgesand recovery of ET-I added to normal plasma was 70.9 k 10.3%(mean 1 SD, n = 12). The concentration of ET-1 in plasma from normal subjects was I .5 i0.4pmol/l (mean F 1 SD, n = I I) ranging from 1.O to 2.2 pmol/ 1. Extracts of normal human plasma subjected to high performance liquid chromatography on a reverse phase c18 column showed one peak of immunoreactivity co-eluting with the standard for ET-I. From these data it is concluded that the immunoreactive material measured in normal plasma with the present RIA is identical to ET-I. Key words: endothelin, radioimmunoassay, human plasma. reverse-phase HPLC.
INTRODUCTION Endothelin-1 (ET-1) is a 21 amino acid peptide (molecular weight: 2490) with powerful vasoconstrictive properties, synthesized by endothelial cells [ 11. The existence of two other endothelins, ET-2 and ET-3 has also been predicted from analysis of human genomic DNA [2]. However, only ET-1 has been shown to be present in human blood. Recently, various radioimmunoassays for measurement of ET-1 in human plasma have been developed [381, and it is generally accepted that the peptide is present at low concentrations in plasma. However, the reported concentration of ET-1 in plasma from healthy subjects varies considerably between laboratories, ranging from 0.1 pmol/l [3] to 5.2 pmol/l [4]. An explanation for this variability is not yet available but could relate to different methods of endothelin extraction and differences in the specificity of antibodies employed. In this paper we report the development of a sensitive solid-phase double-antibody radioimmunoassay for the determination of ET-1 in human plasma. MATERIALS AND METHODS Reagents ET-1 (human, porcine) (Peptide Institute, Osaka, Japan); '251-labelled endothelin (Amersham, U.K.); ET-2 (human), ET-3 (human, rat) and big-endothelin- 1 (human) (Peninsula, Belmont, USA); atrial natriuretic factor (Bachem, Switzerland); angiotensin I and angio-
tensin I1 (Schwarz-Mann, St Louis, USA); human serum albumin ('reinst', Behring Institute, FRG); aprotinin (Novo, Denmark); Sep-pak Cl8 cartridges (Millipore-Waters, USA); Sac-cel (Solid-phase second antibody-coated cellulose suspension) (Wellcome Diagnostics, UK); thyroglobulin (Koch-Light Laboratories, UK); thyroglobulin (Koch-Light Laboratories, UK); l-ethyl-3(3-dimethylaminopropyl)-carbodiimide (CDI) (Fluka, FRG); Na-'251(Amersham, UK); P-2 for gel filtration (Bio-Rad, UK). All other reagents were of analytical grade. Preparation of immunogen Synthetic ET-1 was conjugated to thyroglobulin by the method of Sofroniew et al. [9]. In brief, 9 mg of thyroglobulin and 2 mg of ET-I were dissolved in 3300 p1 of water. Five hundred microliters of CDI (0.6 g/l) were then added dropwise to the solution. To control the yield of the coupling reaction, 100 pl of radioiodinated ET-1 (9. lo6cpm) were also added. The reaction mixture was kept for 18 h at room temperature. Removal of excess coupling agent was accomplished by 48 h dialysis against 1 1 of demineralized water at 4°C with two changes of the water. Percentage conjugation was calculated by measuring the radioactivity in the conjugate. In two experiments, the coupling of IZ5I-ET1 to thyroglobulin was 73% and 86%, respectively. The conjugate was divided into 10 aliquots of 450 p1 and subsequently stored a t - 20°C.
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182
P . Have Rasmussen et al.
Immunization Antibodies against ET-I were raised in 4 rabbits. Each animal was immunized four times at 2-week intervals with 0.1 ml emulsion consisting of 0.05 ml conjugate and 0.05 ml of Freund's incomplete adjuvant. After the 4th immunization, the animals received booster injections of the same emulsion at 4-week intervals for one year, with bleeds 12 days after each immunization. All antisera were tested to assess the titer, defined as the dilution of antiserum at which 50% of a constant amount of labelled ET-1 (5000 cpm) was bound to the antiserum. For the determination of antisera titers, our in-house tracer was used. Iodination of ET-1 was performed by the lactoperoxidase method according to Morrison & Bayse [lo]. The labelled ET-1 was purified by gel filtration on a P-2 column. Preparation of standards The ET-1 standard was kept at -80°C as a stock solution of 0.4 mmol/l (1 mg/ml) in 0. I mol/l acetic acid. The stock solution was diluted with RIA buffer (vide infra) and stored at a concentration of 84 pmol/l. Standards covering a range from 0.33 fmol to 20.9 fmol per 100 p1 were freshly prepared in the buffer for radioimmunoassay . Radioimmunoassay RIA buffer consisted of a 0.1 mol/l sodium phosphate buffer pH 7.4 containing 0.3% NaCl, 0.1% human serum albumin, 0.1 % Triton X- 100,O.1 % trifluoroacetic acid (TFA) and 0.02% sodium azide. Incubation was carried out in polyethylene tubes (70 x 11 mm, Nunc, Denmark). Assay was performed on three aliquots of 50 pl, 75 p1 and 100 p1 of plasma extract, respectively. One hundred microliters of standards (0.33 fmol-20.9 fmol per 100 pl) or plasma extract with RIA buffer (total volume 100 pl) was mixed with 100 pl antiserum diluted 1 : 100,000 in RIA buffer and incubated for 24 h at 4°C. '251-ET-l(5000 cpm of a freshly prepared tracer) in 100 pl RIA buffer was then added and incubation continued for 24 h at 4°C. Separation of free from antibody-bound radioactivity was achieved by addition of 100 p1 Sac-cel suspension to each tube. After vertical shaking for 3 h at room temperature, the tubes were incubated for 5 days at 4°C and centrifuged at 4°C for 2 min at 1600 g. The supernatant was then discharged by decanting and the precipitate counted in a gamma counter. All standard determinations were performed in duplicate. Collection of blood samples Blood (10 ml) was drawn into precooled 30 ml plastic
containers (Sterilin, UK) containing 250 pl sodium EDTA (5%) and 30p1 aprotinin (2.5%) and kept on ice. Plasma was obtained by centrifugation for 20 min at 1000 g and stored at - 18°C until extraction of ET-I from plasma. Extraction Plasma (2.5 ml) was acidified with 5 ml of 6% acetic acid. Six ml of this solution was applied to a Sep-pak CIS cartridge, prewashed with 5 ml 4% acetic acid in 86% ethanol, 5 ml methanol, 5 ml distilled water and 5 ml4% acetic acid. The cartridge was then washed with 2 x 5 ml 4% acetic acid in 10% ethanol. ET-I was eluted into plastic tubes (14 x 75 mm, Hansac plastic, Denmark) with 2 ml4% acetic acid in 86% ethanol. The eluate was dried with a flow of dry nitrogen at 45°C or in a vacuum centrifuge (Savant, USA) at 37°C dissolved in 250 pl RIA buffer and stored at - 18°C until assayed. High-performance liquid chromatography High-performance liquid chromatography (HPLC) was performed with a 5 pm CIScolumn (0.46*25 cm) (Vydac, USA). Two eluents were used: A and B. Eluent A consisted of acetonitrile: water:methanol (20: 70: 10 v/v/v) with 0.1 % (w/v) of TFA. Eluent B consisted of acetonitrile: water:methanol (50:40: 10 v/v/v) with 0.1 YO(w/v) of TFA. Plasma was extracted by Sep-pak Cl*cartridges as described above. The dried extract was dissolved into 100 p1 of eluent A and passed through a Millex-HV filter (Millipore, USA). The extract was then loaded onto the column, which was equilibrated for 20 minutes with eluent A. In the same way standards of ET- 1 and ET-2 were dissolved in eluent A, and 20 fmol of ET-1 and 42 fmol of ET-2, respectively, were injected into the column. For elution, a linear gradient from 100% of eluent A to 100% of eluent B was applied over 60 min. The flow rate was 1 ml/min and 1 ml fractions were collected. Samples of each fraction were dried in a vacuum centrifuge (Savant, USA), reconstituted in RIA buffer and assayed. Stability of ET-I in blood samples The routine procedure for sample collection was modified to test the stability of both endogenous ET-1 and exogenous ET-1 added to whole blood in a concentration of 4 pmol/l. Blood samples were kept on ice or at room temperature for 13, 30, 60 and 180 min prior to processing. Stability of ET-1 in plasma samples during storage The stability of endogenous ET-1 and exogenous ET-1
Radioimmunoassay of endothelin
added to plasma in a concentration of 8 pmol/l was studied during storage. The plasma samples were stored at - 18°C for 3 days, 1 week, 1 month, 3 months and 6 months, respectively.
183
BIT (%) 70
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60
RESULTS Ant isera All 4 rabbits produced antibodies to ET-1. The most suitable antiserum with respect to titer and sensitivity was obtained 32 weeks after the initial immunization and could be used in a final dilution of 1 : 30,000 (inhouse standard). IDsowas 16 fmol/lOO pl. In addition, this antiserum has been tested with the use of the tracer from Amersham, allowing a final dilution of 1 : 300,000. The IDSowas 3.6 fmo1/100 pl. Thus, a much higher titer and sensitivity, as expressed by a lower IDsO, was obtained with the Amersham tracer as compared with our in-house ET-1 tracer. In this study no further attempts were made to increase the quality of our inhouse tracer and therefore the tracer from Amersham has been used for the development of the radioimmunoassay for ET- 1. Radioimmunoassay A typical standard curve for ET- 1 is shown in Fig. 1. The minimum amount of ET-1 distinguishable from zero was 0.33 fmol per 100 pl (p
1992; 1 181-1 86
Radioimmunoassay of Endothelin in Human Plasma PETER HAVE RASMUSSEN,' INGER GRAVES PLUM,' NIELS ESKE BRUUN,' HARRIET DIGE-PETERSEN,' THOMAS HEDNER,2 JAN HEDNER2 and JORN GIESE3 From the 'Department of Clinical Physiology and Nuclear Medicine, Glostrup Hospital, University of Copenhagen, DK-2600 Glostrup, Denmark, 2Department of Clinical Pharmacology, Sahlgrenska Hospital, University of Goteborg. 9 4 1 3 45 Goteborg. Sweden, and University Institute of Experimental Medicine, Division of Human Pathophysiology. The Panum Institute, DK-2200 Copenhagen N,Denmark
'
Blood Press Downloaded from informahealthcare.com by University of Toronto on 01/16/15 For personal use only.
Rasmussen PH, Plum IG, Bruun NE, Dige-Petersen H, Hedner T, Hedner J, Giese J. Radioimmunoassay ofendothe/in in human plasma. Blood Pressure 1992; 1 : 18 I - 186.
A specific and sensitive radioimmunoassay (RIA) for determination of endothelin-l (ET-1) in human plasma has been developed. Antibodies were raised in rabbits using synthetic ET-I conjugated to thyroglobulin as immunogen. The antibodies obtained were used at a final dilution of 1 :300,000 yielding maximum binding of 61.7 & 3.0%(mean f 1 SD, n = 20) of IZ5I-ET1. The IDSO (inhibitory dose 50%) was 4.5 k0.6 fmo1/100 pl (mean k 1 SD, n = 20). The sensitivity of the RIA was 0.33 fmol/IOO pl standard solution. No cross reactivity was observed with endothelin-3, big-endothelin-I, atrial natriuretic factor, angiotensin 1 or angiotensin 11. The cross-reactivity with endothelin-2 was 100%.Endothelin was extracted from acidified plasma with Sep-pak c18 cartridgesand recovery of ET-I added to normal plasma was 70.9 k 10.3%(mean 1 SD, n = 12). The concentration of ET-1 in plasma from normal subjects was I .5 i0.4pmol/l (mean F 1 SD, n = I I) ranging from 1.O to 2.2 pmol/ 1. Extracts of normal human plasma subjected to high performance liquid chromatography on a reverse phase c18 column showed one peak of immunoreactivity co-eluting with the standard for ET-I. From these data it is concluded that the immunoreactive material measured in normal plasma with the present RIA is identical to ET-I. Key words: endothelin, radioimmunoassay, human plasma. reverse-phase HPLC.
INTRODUCTION Endothelin-1 (ET-1) is a 21 amino acid peptide (molecular weight: 2490) with powerful vasoconstrictive properties, synthesized by endothelial cells [ 11. The existence of two other endothelins, ET-2 and ET-3 has also been predicted from analysis of human genomic DNA [2]. However, only ET-1 has been shown to be present in human blood. Recently, various radioimmunoassays for measurement of ET-1 in human plasma have been developed [381, and it is generally accepted that the peptide is present at low concentrations in plasma. However, the reported concentration of ET-1 in plasma from healthy subjects varies considerably between laboratories, ranging from 0.1 pmol/l [3] to 5.2 pmol/l [4]. An explanation for this variability is not yet available but could relate to different methods of endothelin extraction and differences in the specificity of antibodies employed. In this paper we report the development of a sensitive solid-phase double-antibody radioimmunoassay for the determination of ET-1 in human plasma. MATERIALS AND METHODS Reagents ET-1 (human, porcine) (Peptide Institute, Osaka, Japan); '251-labelled endothelin (Amersham, U.K.); ET-2 (human), ET-3 (human, rat) and big-endothelin- 1 (human) (Peninsula, Belmont, USA); atrial natriuretic factor (Bachem, Switzerland); angiotensin I and angio-
tensin I1 (Schwarz-Mann, St Louis, USA); human serum albumin ('reinst', Behring Institute, FRG); aprotinin (Novo, Denmark); Sep-pak Cl8 cartridges (Millipore-Waters, USA); Sac-cel (Solid-phase second antibody-coated cellulose suspension) (Wellcome Diagnostics, UK); thyroglobulin (Koch-Light Laboratories, UK); thyroglobulin (Koch-Light Laboratories, UK); l-ethyl-3(3-dimethylaminopropyl)-carbodiimide (CDI) (Fluka, FRG); Na-'251(Amersham, UK); P-2 for gel filtration (Bio-Rad, UK). All other reagents were of analytical grade. Preparation of immunogen Synthetic ET-1 was conjugated to thyroglobulin by the method of Sofroniew et al. [9]. In brief, 9 mg of thyroglobulin and 2 mg of ET-I were dissolved in 3300 p1 of water. Five hundred microliters of CDI (0.6 g/l) were then added dropwise to the solution. To control the yield of the coupling reaction, 100 pl of radioiodinated ET-1 (9. lo6cpm) were also added. The reaction mixture was kept for 18 h at room temperature. Removal of excess coupling agent was accomplished by 48 h dialysis against 1 1 of demineralized water at 4°C with two changes of the water. Percentage conjugation was calculated by measuring the radioactivity in the conjugate. In two experiments, the coupling of IZ5I-ET1 to thyroglobulin was 73% and 86%, respectively. The conjugate was divided into 10 aliquots of 450 p1 and subsequently stored a t - 20°C.
Blood Press Downloaded from informahealthcare.com by University of Toronto on 01/16/15 For personal use only.
182
P . Have Rasmussen et al.
Immunization Antibodies against ET-I were raised in 4 rabbits. Each animal was immunized four times at 2-week intervals with 0.1 ml emulsion consisting of 0.05 ml conjugate and 0.05 ml of Freund's incomplete adjuvant. After the 4th immunization, the animals received booster injections of the same emulsion at 4-week intervals for one year, with bleeds 12 days after each immunization. All antisera were tested to assess the titer, defined as the dilution of antiserum at which 50% of a constant amount of labelled ET-1 (5000 cpm) was bound to the antiserum. For the determination of antisera titers, our in-house tracer was used. Iodination of ET-1 was performed by the lactoperoxidase method according to Morrison & Bayse [lo]. The labelled ET-1 was purified by gel filtration on a P-2 column. Preparation of standards The ET-1 standard was kept at -80°C as a stock solution of 0.4 mmol/l (1 mg/ml) in 0. I mol/l acetic acid. The stock solution was diluted with RIA buffer (vide infra) and stored at a concentration of 84 pmol/l. Standards covering a range from 0.33 fmol to 20.9 fmol per 100 p1 were freshly prepared in the buffer for radioimmunoassay . Radioimmunoassay RIA buffer consisted of a 0.1 mol/l sodium phosphate buffer pH 7.4 containing 0.3% NaCl, 0.1% human serum albumin, 0.1 % Triton X- 100,O.1 % trifluoroacetic acid (TFA) and 0.02% sodium azide. Incubation was carried out in polyethylene tubes (70 x 11 mm, Nunc, Denmark). Assay was performed on three aliquots of 50 pl, 75 p1 and 100 p1 of plasma extract, respectively. One hundred microliters of standards (0.33 fmol-20.9 fmol per 100 pl) or plasma extract with RIA buffer (total volume 100 pl) was mixed with 100 pl antiserum diluted 1 : 100,000 in RIA buffer and incubated for 24 h at 4°C. '251-ET-l(5000 cpm of a freshly prepared tracer) in 100 pl RIA buffer was then added and incubation continued for 24 h at 4°C. Separation of free from antibody-bound radioactivity was achieved by addition of 100 p1 Sac-cel suspension to each tube. After vertical shaking for 3 h at room temperature, the tubes were incubated for 5 days at 4°C and centrifuged at 4°C for 2 min at 1600 g. The supernatant was then discharged by decanting and the precipitate counted in a gamma counter. All standard determinations were performed in duplicate. Collection of blood samples Blood (10 ml) was drawn into precooled 30 ml plastic
containers (Sterilin, UK) containing 250 pl sodium EDTA (5%) and 30p1 aprotinin (2.5%) and kept on ice. Plasma was obtained by centrifugation for 20 min at 1000 g and stored at - 18°C until extraction of ET-I from plasma. Extraction Plasma (2.5 ml) was acidified with 5 ml of 6% acetic acid. Six ml of this solution was applied to a Sep-pak CIS cartridge, prewashed with 5 ml 4% acetic acid in 86% ethanol, 5 ml methanol, 5 ml distilled water and 5 ml4% acetic acid. The cartridge was then washed with 2 x 5 ml 4% acetic acid in 10% ethanol. ET-I was eluted into plastic tubes (14 x 75 mm, Hansac plastic, Denmark) with 2 ml4% acetic acid in 86% ethanol. The eluate was dried with a flow of dry nitrogen at 45°C or in a vacuum centrifuge (Savant, USA) at 37°C dissolved in 250 pl RIA buffer and stored at - 18°C until assayed. High-performance liquid chromatography High-performance liquid chromatography (HPLC) was performed with a 5 pm CIScolumn (0.46*25 cm) (Vydac, USA). Two eluents were used: A and B. Eluent A consisted of acetonitrile: water:methanol (20: 70: 10 v/v/v) with 0.1 % (w/v) of TFA. Eluent B consisted of acetonitrile: water:methanol (50:40: 10 v/v/v) with 0.1 YO(w/v) of TFA. Plasma was extracted by Sep-pak Cl*cartridges as described above. The dried extract was dissolved into 100 p1 of eluent A and passed through a Millex-HV filter (Millipore, USA). The extract was then loaded onto the column, which was equilibrated for 20 minutes with eluent A. In the same way standards of ET- 1 and ET-2 were dissolved in eluent A, and 20 fmol of ET-1 and 42 fmol of ET-2, respectively, were injected into the column. For elution, a linear gradient from 100% of eluent A to 100% of eluent B was applied over 60 min. The flow rate was 1 ml/min and 1 ml fractions were collected. Samples of each fraction were dried in a vacuum centrifuge (Savant, USA), reconstituted in RIA buffer and assayed. Stability of ET-I in blood samples The routine procedure for sample collection was modified to test the stability of both endogenous ET-1 and exogenous ET-1 added to whole blood in a concentration of 4 pmol/l. Blood samples were kept on ice or at room temperature for 13, 30, 60 and 180 min prior to processing. Stability of ET-1 in plasma samples during storage The stability of endogenous ET-1 and exogenous ET-1
Radioimmunoassay of endothelin
added to plasma in a concentration of 8 pmol/l was studied during storage. The plasma samples were stored at - 18°C for 3 days, 1 week, 1 month, 3 months and 6 months, respectively.
183
BIT (%) 70
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60
RESULTS Ant isera All 4 rabbits produced antibodies to ET-1. The most suitable antiserum with respect to titer and sensitivity was obtained 32 weeks after the initial immunization and could be used in a final dilution of 1 : 30,000 (inhouse standard). IDsowas 16 fmol/lOO pl. In addition, this antiserum has been tested with the use of the tracer from Amersham, allowing a final dilution of 1 : 300,000. The IDSowas 3.6 fmo1/100 pl. Thus, a much higher titer and sensitivity, as expressed by a lower IDsO, was obtained with the Amersham tracer as compared with our in-house ET-1 tracer. In this study no further attempts were made to increase the quality of our inhouse tracer and therefore the tracer from Amersham has been used for the development of the radioimmunoassay for ET- 1. Radioimmunoassay A typical standard curve for ET- 1 is shown in Fig. 1. The minimum amount of ET-1 distinguishable from zero was 0.33 fmol per 100 pl (p
