EVALUATION OF A RADIOIMMUNOASSAY FOR PLASMA ADRENOCORTICOTROPHIN E. STARK, KATALIN MER\l=E'\TEY D. SCHULSTER *Institute ofExperimental Medicine, Hungarian Academy of Sciences and ^National Institute for Rheumatism, Budapest, Hungary; \Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton, BN1 9QG J. MARTON,
AND
(Received 16 August 1977) SUMMARY
radioimmunoassay for plasma ACTH has been described and evaluated. Rabbit antiproduced by immunization with Asp25, Ala26, Gly27,]-\g=a\h-corticotrophin-(1\p=n-\28)\x=req-\ octacosa-peptide (a sequence analogue of \g=a\h1-28-ACTH) bovine gamma globulin conjugate was used. The antiserum is specific for the NH2-terminal portion of the ACTH molecule and cross-reactivity of human, porcine and rat ACTH in the system has been demonstrated. Reasonable agreement was found between estimates obtained by bioassay and radioimmunoassay of the ACTH content of rat pituitary gland incubation media, indicating a close relationship between the sequence of ACTH recognized by the antibodies and the sequence possessing the steroidogenic activity. Measurement of the amount of ACTH in the plasma required the preliminary extraction and concentration of the hormone. Over a range of concentrations between 3\m=.\5and 3600 pg/ml, extraction recovery was independent of the initial concentration of ACTH in the plasma. Extraction gave rise to no changes in the immunological properties of standard ACTH. The concentration of immunoreactive ACTH in rat plasma was 48 \m=+-\3\m=.\6(s.e.m.) pg/ml in the morning and 106 \m=+-\9\m=.\9pg/ml in the afternoon. Exposure to ether for 5 min and subsequent laparotomy gave rise to a significant increase in the concentration of immunoreactive ACTH in the plasma. The resting level of ACTH and the ACTH response to stress were both significantly higher 1 and 7 days after adrenalectomy. Intravenous injection of a hypothalamic extract elicited a considerable rise in the concentration of immunoreactive ACTH in the plasma, but no response was seen after oral administration of this partially purified extract. The sensitivity, precision and specificity of this ACTH radioimmunoassay make it a useful tool for studying pituitary\p=n-\ adrenal physiology. A
serum
introduction
Apart from a few exceptions (Berson & Yalow, 1968; Matsukura, West, Ichikawa, Jubiz, Harada & Tyler, 1971; Galskov, 1972), most sensitive radioimmunoassay techniques for the measurement of adrenocorticotrophin (ACTH) in plasma require a preliminary extrac¬ tion of the hormone. Extraction procedures are based either on the acid-acetone precipita¬ tion of ACTH (Yalow, Glick, Roth & Berson, 1964; Demura, West, Nugent, Nakagawa & Tyler, 1966), or on applying solid-phase adsorbents such as oxycellulose (Sydnor & Sayers, 1952), ion-exchange resins (Williams, Island, Oldfield & Liddle, 1961 ; Orth, Island, Nicholson, Abe & Woodham, 1968), Fuller's earth (Landon & Greenwood, 1968; Horgan § Present address and address for reprint requests : Department of Anatomy, Semmelweis University Medical School, 1094 Budapest, Tüzoltó u 58, Hungary.
& Riley, 1974), silicic-acid (Donald, 1967; Donald, Espiner & Beaven, 1972; Liotta & Krieger, 1975), QUSO-precipitated silica granules (Voigt, Fehm, Reck & Pfeiffer, 1974) or porous silica glass beads (Ratcliffe & Edwards, 1971 ; Rees, Cook, Kendall, Allen, Kramer, Ratcliffe & Knight, 1971; Genazzani, Fraioli, Conti & Fioretti, 1974). Extraction results in the concentration of the hormone and the elimination of proteolytic enzymes or other possible plasma constituents which may interfere with the assay. In this study a radioimmunoassay for ACTH is described, which uses an antiserum raised in rabbits (Marton, Merétey, Stark & Schulster, 1977) and the extraction procedures of Ratcliffe & Edwards (1971) and Rees et al. (1971). The concentration of ACTH in rat plasma was determined under resting conditions, under stress, after adrenalectomy and after administration of a hypothalamic extract. The radioimmunoassay is suitable for the measurement of the level of ACTH in the plasma at rest. materials and methods
Materials the International Working Standard for corticotrophin (3rd ocp-Adrenocorticotrophin, IWS), was obtained from the WHO International Laboratory for Biological Standards, London and was used as standard in these studies. Polystyrene tubes (16 110 mm Henley's Medical Supplies Ltd, London) and polystyrene assay tubes (8 60 mm Luckham's Ltd, Burgess Hill), Norit A, 2-mercaptoethanol and silicic acid (SIL-R; Sigma Chemical Co. Ltd, London), Vycor leached silica glass (code 7930, Corning Glass Works, Corning, New York, U.S.A.), human serum albumin (Lister Institute, Elstree, Herts.), Dextran 70 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and collagenase (P-L Biochemicals Inc., Milwaukee, Wisconsin, U.S.A.) were also used. ah-Adrenocorticotrophin was donated by Dr P. J. Lowry (St Bartholomew's Hospital, London). Extraction of plasma and pituitary ACTH The methods of Ratcliffe & Edwards (1971) and Rees et al. (1971) were used with minor modifications. Plasma containing no ACTH was obtained from hypophysectomized rats 24-48 h after the operation. Plasma blanks containing no ACTH, a series of standards prepared by the addition of increasing amounts of 3rd IWS to plasma from hypophysecto¬ mized rats and quality control samples (5 ml samples of pooled rat plasma and unknown plasma samples, or a recorded smaller volume of each) were extracted in 16 110 mm polystyrene tubes. Fifty milligrams Vycor leached silica glass were added to all tubes and adsorption was accomplished by mixing the tubes on a rotary mixer for 30 min at room temperature. After centrifugation the supernatant fraction was discarded, the precipitate was washed twice with 5 ml portions of distilled water and once with 5 ml 1 m-HCI. The ACTH was eluted with 1 ml acetone: water (1:1, v/v) by mixing the tubes for 30 min on the rotary mixer. The eluates were carefully aspirated and put into polystyrene tubes.The adsorbent was resuspended in 0-5 ml acetone : water by thorough vortexing and the com¬ bined eluates were dried at 60 °C in a water bath, under a fine stream of nitrogen. The dry extracts were taken up in the radioimmunoassay buffer. In the studies in which silicic acid adsorbent was used, the procedure of Liotta & Krieger (1975) was followed. Pituitary extracts were made from glands removed rapidly after the rats had been decapitated. The anterior lobe was dissected and extracted with 1 ml 0-1 m-HCI. The extracts were neutralized before assay.
Radioimmunoassay procedure Iodination of ACTH and the production, selection and assessment of the antisera used in this assay have been described by Marton et al. (1977). Incubation was carried out in 8 60 mm polystyrene tubes at 4°C. Standard ACTH 3rd IWS, unknown plasma extracts,
125I-labelled ACTH and antiserum
or diluted in a phosphate buffer (50 albumin and 0-5% 2-mercaptoethanol. The total volume of incubation was 300 µ . The tubes were incubated for 24 or 72 h with the antiserum at a final dilution of 1 : 40 000 or 1 : 80 000 in the presence of 10 or 5 pg 125I-labelled ACTH respectively. Bound and free radioactivities were separated by the addition of 1 ml dextran-charcoal suspension containing 0-3% Norit A, 0-3% dextran 70 and 1% human serum albumin in phosphate buffer (50 mmol/1, pH 7-6). After vortexmixing and centrifugation (20 min, 2500 g, A °C), the supernatant fraction was discarded and the sediment counted. The method of Rodbard & Lewald (1970) was used for the analysis of assay data on a Wang 2200 computer. Statistical quality control was performed according to Rodbard (1974). were
dissolved
mmol/1, pH 7-6) containing 0-25% human
serum
Animal studies and collection of blood samples Male Sprague-Dawley rats weighing 250-300 g were housed in a room maintained at 24 °C, with 55-75% relative humidity and free access to a standard rat diet and tap water. In the stress experiments the animals were subjected to daily handling for 1 week before application of the Stressor. Adrenalectomy was performed from a bilateral dorsal incision, hypophysectomy by the transauricular method (Gay, 1967) under ether anaesthesia. The adrenalectomized rats were given 0-9% saline and the hypophysectomized rats were given 5% glucose solution instead of drinking water. Blood samples were collected in heparinized polystyrene tubes kept on ice. Resting levels of ACTH were determined in samples of trunk blood collected after decapitation. Exposure to ether lasted for 5 min and blood was withdrawn from the abdominal aorta over a period of 40-60 s. A porcine hypothalamic extract, chromatographed on Sephadex G-50 (Cooper, Synetos, Christie & Schulster, 1976; 50 mg/100 g), was either injected into the jugular vein of dexamethasone-blocked (50 µg/ 100 g, i.p., 24, 12 and 4 h earlier; total 150 µg) and hypophysectomized rats, or given orally to another group of dexamethasone-blocked animals, each under pentobarbitone anaes¬ thesia (3-5 mg/100 g, i.p.). Blood was withdrawn from the abdominal aorta 15 min after intravenous and 30 min after oral administration of the extract. In all experiments blood was collected between 09.00 and 11.00 h except for the afternoon samples which were taken at 18.00 h. The samples were immediately centrifuged at 4 °C and the plasma was frozen and kept at —20 °C until assayed. Mann-Whitney and Kruskal-Wallis tests were used for
statistical analysis.
Bioassay of ACTH The method is based on the measurement of corticosterone production by collagenase disaggregated isolated rat adrenal cells and has been described previously (Richardson & Schulster, 1972). Medium in which isolated pituitary cells had been incubated with a hypothalamic extract (Cooper et al. 1976) was assayed by bioassay and radioimmunoassay without extraction. results
Extraction procedure with labelled ACTH it was shown that maximum adsorption of 20 pg 125I-labelled ACTH from plasma from hypophysectomized rats took place in 30 min with 50 mg or more Vycor glass adsorbent. Recovery of various amounts of unlabelled standard ACTH from plasma samples was studied by radioimmunoassay. Graded amounts of 3rd IWS corticotrophin were added to and extracted from hypophysectomized rat plasma with Vycor glass adsorbent. The ACTH content of the extracts was assayed. In Fig. la the standard curve for unextracted ACTH is compared with the standard curve obtained for ACTH extracted with Vycor glass in the In
a
pilot study
assay. The two curves are parallel indicating that recovery of native porcine ACTH in the extraction procedure was the same over the whole range of physiological hormone concentrations. Silicic acid extraction of ACTH from plasma did not show a similar concentration-independent recovery (Fig. lb), there was a decrease in extraction efficiency as the concentration of ACTH in the plasma increased.
same
05
100 ACTH
100 ACTH
1000
10 000
(pg/tube)
1000
10000
(pg/tube) Fig. 1. (a) Vycor glass extraction of plasma ACTH : comparison of extracted (O) and non-extracted (·) standard curves in the same radioimmunoassay (1 : 80 000 final dilution of the antiserum, 72 h of incubation, 5 pg 125l-labelled ACTH). Graded amounts of 3rd IWS ACTH were added to 5 ml aliquots of plasma from hypophysectomized rats in triplicate, at all dose levels to yield a range of 3-5-3600 pg/ml of initial plasma ACTH concentrations. The samples were extracted with 50 mg Vycor glass and the extracts were assayed together with non-extracted aliquots of the same standard. B¡B0: the bound to initial bound ratio, where initial means zero dose, i.e. in the absence of unlabelled antigen. Inset: logit-log plot of the same data, (b) Silicic acid extraction of plasma ACTH: comparison of extracted (O) and non-extracted (·) standard curves in the same radio¬ immunoassay. Further details as in (a) with the exception that 60 mg silicic acid were used instead of the Vycor glass adsorbent.
Vycor glass was used for the extraction of ACTH from plasma in all further studies. The average recovery of 250 pg 3rd IWS from 5 ml hypophysectomized rat plasma was about 62% (0-6196 ±0-0997, s.e.m.) in ten consecutive assays. In Fig. 2 the serial dilutions of different extracts were compared with a standard curve constructed with the unextracted 3rd IWS ACTH in buffer medium. The slope of the curve obtained with serial dilutions of
from a rat under ether stress, or with the 3rd IWS ACTH extracted from from a hypophysectomized rat was parallel to that of the standard, while the extract of ACTH free plasma from a hypophysectomized rat gave a flat response when serially diluted, which did not differ from the blank in buffer medium.
plasma plasma
extract
1000 100 ACTH (pg/tube)
10000
Fig. 2. Serial dilutions of ACTH-free hypophysectomized rat plasma extract (a), stressed rat plasma extract (x), 3rd IWS ACTH extracted from hypophysectomized rat plasma (v) were assayed against non-extracted 3rd IWS in buffer medium (·) (24 h, 10 pg 125I-labelled ACTH, 1 : 40 000 final dilution of the antiserum, B/B0 is as detailed in the legend to Fig. 1). The
radioimmunoassay system
Sensitivity The limit of detection of the assay was related to the dilution of the antiserum, the amount of tracer added, the duration of the incubation and the variation in the response parameter due to experimental error. The limit of detection was 20 pg ACTH/assay tube in a 24 h assay with 10 pg 125I-labelled ACTH and a 1 :40 000 final dilution of the antiserum and 5 pg/assay tube in a 3-day assay with 5 pg 125I-labelled ACTH added 24 h after starting the incubation, with a 1 : 80 000 final dilution of the antiserum.
Specificity The antiserum used is highly specific for the NH2-terminal portion of the ACTH molecule (Marton et al. 1977). The antiserum cannot differentiate between porcine or human ACTH and rat pituitary extract. The standard curves are superimposable over a wide dose range, indicating that the antiserum is suitable for assaying ACTH in all three species.
Precision
Duplicates of 72 unknown rat plasma samples were run in the same assay (Fig. 3) and duplicates of 28 samples were run in different assays (Fig. 4). A significant correlation was found between-the estimates of the concentration of ACTH in rat plasma both within and between assays (P< 0-001). The coefficients of variation for means of duplicates of four quality control sera were 9-9, 9-2, 6-4 and 10-1 within and 22-2, 8-5, 14-8 and 7-9 between assays over a range of concentrations between 15 and 910 pg ACTH/ml plasma in ten subsequent assays.
10000
CU
" . 1000
ca
"-^
a.
H
u.
-S
.V
100 r=
û·
;i
=
0-9879 72
10
100 10 000 1000 Second replicate Immunoreactive ACTH (pg/ml plasma) 10
Fig. 3. Immunoreactive ACTH concentration of unknown rat plasma samples. Within assay reproducibility: hormone concentration of the same plasma samples were determined in the same assays (r, coefficient of correlation; n, number of samples).
10000
100 1000 Second assay Immunoreactive ACTH (pg/ml plasma)
10000
Fig. 4. Immunoreactive ACTH concentration of unknown rat plasma samples. Between assay reproducibility: hormone concentrations, in duplicate, of the same plasma samples were deter¬ mined in different assays (r, coefficient of correlation; n, number of samples).
Comparison of bioassay and radioimmunoassay potency estimates The ACTH content of 40 samples of isolated rat pituitary cell incubation media was esti¬ mated by a bioassay and the above radioimmunoassay system. The same standard 3rd
IWS ACTH was used as reference in both assays. Estimates of the concentrations of bioactive and immunoreactive ACTH in the samples are compared in Fig. 5. The obvious trend indicated a broad agreement of potency estimates obtained from the two assays.
1000
10 000
Immunoreactive ACTH (pg/ml plasma) Fig. 5. Comparison of bioactive and immunoreactive ACTH concentrations of the same samples of isolated rat pituitary cell incubation media. Unextracted aliquots of the pituitary media were assayed in a bioassay using collagenase disaggregated rat adrenocortical cells, or in the radio¬ immunoassay. In both assays the 3rd IWS ACTH was used as standard (coefficient of correla¬ tion (r) 0-7978; number of samples (n) 40). =
=
Plasma ACTH in rats: experimental studies The level of ACTH in the plasma of resting rats at 09.00 h was 48 ± 3-6 pg/ml (n 10). The mean resting concentration from samples collected at 18.00 h, 106 ± 9-9 pg/ml (n 11), was significantly higher (P
AND
(Received 16 August 1977) SUMMARY
radioimmunoassay for plasma ACTH has been described and evaluated. Rabbit antiproduced by immunization with Asp25, Ala26, Gly27,]-\g=a\h-corticotrophin-(1\p=n-\28)\x=req-\ octacosa-peptide (a sequence analogue of \g=a\h1-28-ACTH) bovine gamma globulin conjugate was used. The antiserum is specific for the NH2-terminal portion of the ACTH molecule and cross-reactivity of human, porcine and rat ACTH in the system has been demonstrated. Reasonable agreement was found between estimates obtained by bioassay and radioimmunoassay of the ACTH content of rat pituitary gland incubation media, indicating a close relationship between the sequence of ACTH recognized by the antibodies and the sequence possessing the steroidogenic activity. Measurement of the amount of ACTH in the plasma required the preliminary extraction and concentration of the hormone. Over a range of concentrations between 3\m=.\5and 3600 pg/ml, extraction recovery was independent of the initial concentration of ACTH in the plasma. Extraction gave rise to no changes in the immunological properties of standard ACTH. The concentration of immunoreactive ACTH in rat plasma was 48 \m=+-\3\m=.\6(s.e.m.) pg/ml in the morning and 106 \m=+-\9\m=.\9pg/ml in the afternoon. Exposure to ether for 5 min and subsequent laparotomy gave rise to a significant increase in the concentration of immunoreactive ACTH in the plasma. The resting level of ACTH and the ACTH response to stress were both significantly higher 1 and 7 days after adrenalectomy. Intravenous injection of a hypothalamic extract elicited a considerable rise in the concentration of immunoreactive ACTH in the plasma, but no response was seen after oral administration of this partially purified extract. The sensitivity, precision and specificity of this ACTH radioimmunoassay make it a useful tool for studying pituitary\p=n-\ adrenal physiology. A
serum
introduction
Apart from a few exceptions (Berson & Yalow, 1968; Matsukura, West, Ichikawa, Jubiz, Harada & Tyler, 1971; Galskov, 1972), most sensitive radioimmunoassay techniques for the measurement of adrenocorticotrophin (ACTH) in plasma require a preliminary extrac¬ tion of the hormone. Extraction procedures are based either on the acid-acetone precipita¬ tion of ACTH (Yalow, Glick, Roth & Berson, 1964; Demura, West, Nugent, Nakagawa & Tyler, 1966), or on applying solid-phase adsorbents such as oxycellulose (Sydnor & Sayers, 1952), ion-exchange resins (Williams, Island, Oldfield & Liddle, 1961 ; Orth, Island, Nicholson, Abe & Woodham, 1968), Fuller's earth (Landon & Greenwood, 1968; Horgan § Present address and address for reprint requests : Department of Anatomy, Semmelweis University Medical School, 1094 Budapest, Tüzoltó u 58, Hungary.
& Riley, 1974), silicic-acid (Donald, 1967; Donald, Espiner & Beaven, 1972; Liotta & Krieger, 1975), QUSO-precipitated silica granules (Voigt, Fehm, Reck & Pfeiffer, 1974) or porous silica glass beads (Ratcliffe & Edwards, 1971 ; Rees, Cook, Kendall, Allen, Kramer, Ratcliffe & Knight, 1971; Genazzani, Fraioli, Conti & Fioretti, 1974). Extraction results in the concentration of the hormone and the elimination of proteolytic enzymes or other possible plasma constituents which may interfere with the assay. In this study a radioimmunoassay for ACTH is described, which uses an antiserum raised in rabbits (Marton, Merétey, Stark & Schulster, 1977) and the extraction procedures of Ratcliffe & Edwards (1971) and Rees et al. (1971). The concentration of ACTH in rat plasma was determined under resting conditions, under stress, after adrenalectomy and after administration of a hypothalamic extract. The radioimmunoassay is suitable for the measurement of the level of ACTH in the plasma at rest. materials and methods
Materials the International Working Standard for corticotrophin (3rd ocp-Adrenocorticotrophin, IWS), was obtained from the WHO International Laboratory for Biological Standards, London and was used as standard in these studies. Polystyrene tubes (16 110 mm Henley's Medical Supplies Ltd, London) and polystyrene assay tubes (8 60 mm Luckham's Ltd, Burgess Hill), Norit A, 2-mercaptoethanol and silicic acid (SIL-R; Sigma Chemical Co. Ltd, London), Vycor leached silica glass (code 7930, Corning Glass Works, Corning, New York, U.S.A.), human serum albumin (Lister Institute, Elstree, Herts.), Dextran 70 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and collagenase (P-L Biochemicals Inc., Milwaukee, Wisconsin, U.S.A.) were also used. ah-Adrenocorticotrophin was donated by Dr P. J. Lowry (St Bartholomew's Hospital, London). Extraction of plasma and pituitary ACTH The methods of Ratcliffe & Edwards (1971) and Rees et al. (1971) were used with minor modifications. Plasma containing no ACTH was obtained from hypophysectomized rats 24-48 h after the operation. Plasma blanks containing no ACTH, a series of standards prepared by the addition of increasing amounts of 3rd IWS to plasma from hypophysecto¬ mized rats and quality control samples (5 ml samples of pooled rat plasma and unknown plasma samples, or a recorded smaller volume of each) were extracted in 16 110 mm polystyrene tubes. Fifty milligrams Vycor leached silica glass were added to all tubes and adsorption was accomplished by mixing the tubes on a rotary mixer for 30 min at room temperature. After centrifugation the supernatant fraction was discarded, the precipitate was washed twice with 5 ml portions of distilled water and once with 5 ml 1 m-HCI. The ACTH was eluted with 1 ml acetone: water (1:1, v/v) by mixing the tubes for 30 min on the rotary mixer. The eluates were carefully aspirated and put into polystyrene tubes.The adsorbent was resuspended in 0-5 ml acetone : water by thorough vortexing and the com¬ bined eluates were dried at 60 °C in a water bath, under a fine stream of nitrogen. The dry extracts were taken up in the radioimmunoassay buffer. In the studies in which silicic acid adsorbent was used, the procedure of Liotta & Krieger (1975) was followed. Pituitary extracts were made from glands removed rapidly after the rats had been decapitated. The anterior lobe was dissected and extracted with 1 ml 0-1 m-HCI. The extracts were neutralized before assay.
Radioimmunoassay procedure Iodination of ACTH and the production, selection and assessment of the antisera used in this assay have been described by Marton et al. (1977). Incubation was carried out in 8 60 mm polystyrene tubes at 4°C. Standard ACTH 3rd IWS, unknown plasma extracts,
125I-labelled ACTH and antiserum
or diluted in a phosphate buffer (50 albumin and 0-5% 2-mercaptoethanol. The total volume of incubation was 300 µ . The tubes were incubated for 24 or 72 h with the antiserum at a final dilution of 1 : 40 000 or 1 : 80 000 in the presence of 10 or 5 pg 125I-labelled ACTH respectively. Bound and free radioactivities were separated by the addition of 1 ml dextran-charcoal suspension containing 0-3% Norit A, 0-3% dextran 70 and 1% human serum albumin in phosphate buffer (50 mmol/1, pH 7-6). After vortexmixing and centrifugation (20 min, 2500 g, A °C), the supernatant fraction was discarded and the sediment counted. The method of Rodbard & Lewald (1970) was used for the analysis of assay data on a Wang 2200 computer. Statistical quality control was performed according to Rodbard (1974). were
dissolved
mmol/1, pH 7-6) containing 0-25% human
serum
Animal studies and collection of blood samples Male Sprague-Dawley rats weighing 250-300 g were housed in a room maintained at 24 °C, with 55-75% relative humidity and free access to a standard rat diet and tap water. In the stress experiments the animals were subjected to daily handling for 1 week before application of the Stressor. Adrenalectomy was performed from a bilateral dorsal incision, hypophysectomy by the transauricular method (Gay, 1967) under ether anaesthesia. The adrenalectomized rats were given 0-9% saline and the hypophysectomized rats were given 5% glucose solution instead of drinking water. Blood samples were collected in heparinized polystyrene tubes kept on ice. Resting levels of ACTH were determined in samples of trunk blood collected after decapitation. Exposure to ether lasted for 5 min and blood was withdrawn from the abdominal aorta over a period of 40-60 s. A porcine hypothalamic extract, chromatographed on Sephadex G-50 (Cooper, Synetos, Christie & Schulster, 1976; 50 mg/100 g), was either injected into the jugular vein of dexamethasone-blocked (50 µg/ 100 g, i.p., 24, 12 and 4 h earlier; total 150 µg) and hypophysectomized rats, or given orally to another group of dexamethasone-blocked animals, each under pentobarbitone anaes¬ thesia (3-5 mg/100 g, i.p.). Blood was withdrawn from the abdominal aorta 15 min after intravenous and 30 min after oral administration of the extract. In all experiments blood was collected between 09.00 and 11.00 h except for the afternoon samples which were taken at 18.00 h. The samples were immediately centrifuged at 4 °C and the plasma was frozen and kept at —20 °C until assayed. Mann-Whitney and Kruskal-Wallis tests were used for
statistical analysis.
Bioassay of ACTH The method is based on the measurement of corticosterone production by collagenase disaggregated isolated rat adrenal cells and has been described previously (Richardson & Schulster, 1972). Medium in which isolated pituitary cells had been incubated with a hypothalamic extract (Cooper et al. 1976) was assayed by bioassay and radioimmunoassay without extraction. results
Extraction procedure with labelled ACTH it was shown that maximum adsorption of 20 pg 125I-labelled ACTH from plasma from hypophysectomized rats took place in 30 min with 50 mg or more Vycor glass adsorbent. Recovery of various amounts of unlabelled standard ACTH from plasma samples was studied by radioimmunoassay. Graded amounts of 3rd IWS corticotrophin were added to and extracted from hypophysectomized rat plasma with Vycor glass adsorbent. The ACTH content of the extracts was assayed. In Fig. la the standard curve for unextracted ACTH is compared with the standard curve obtained for ACTH extracted with Vycor glass in the In
a
pilot study
assay. The two curves are parallel indicating that recovery of native porcine ACTH in the extraction procedure was the same over the whole range of physiological hormone concentrations. Silicic acid extraction of ACTH from plasma did not show a similar concentration-independent recovery (Fig. lb), there was a decrease in extraction efficiency as the concentration of ACTH in the plasma increased.
same
05
100 ACTH
100 ACTH
1000
10 000
(pg/tube)
1000
10000
(pg/tube) Fig. 1. (a) Vycor glass extraction of plasma ACTH : comparison of extracted (O) and non-extracted (·) standard curves in the same radioimmunoassay (1 : 80 000 final dilution of the antiserum, 72 h of incubation, 5 pg 125l-labelled ACTH). Graded amounts of 3rd IWS ACTH were added to 5 ml aliquots of plasma from hypophysectomized rats in triplicate, at all dose levels to yield a range of 3-5-3600 pg/ml of initial plasma ACTH concentrations. The samples were extracted with 50 mg Vycor glass and the extracts were assayed together with non-extracted aliquots of the same standard. B¡B0: the bound to initial bound ratio, where initial means zero dose, i.e. in the absence of unlabelled antigen. Inset: logit-log plot of the same data, (b) Silicic acid extraction of plasma ACTH: comparison of extracted (O) and non-extracted (·) standard curves in the same radio¬ immunoassay. Further details as in (a) with the exception that 60 mg silicic acid were used instead of the Vycor glass adsorbent.
Vycor glass was used for the extraction of ACTH from plasma in all further studies. The average recovery of 250 pg 3rd IWS from 5 ml hypophysectomized rat plasma was about 62% (0-6196 ±0-0997, s.e.m.) in ten consecutive assays. In Fig. 2 the serial dilutions of different extracts were compared with a standard curve constructed with the unextracted 3rd IWS ACTH in buffer medium. The slope of the curve obtained with serial dilutions of
from a rat under ether stress, or with the 3rd IWS ACTH extracted from from a hypophysectomized rat was parallel to that of the standard, while the extract of ACTH free plasma from a hypophysectomized rat gave a flat response when serially diluted, which did not differ from the blank in buffer medium.
plasma plasma
extract
1000 100 ACTH (pg/tube)
10000
Fig. 2. Serial dilutions of ACTH-free hypophysectomized rat plasma extract (a), stressed rat plasma extract (x), 3rd IWS ACTH extracted from hypophysectomized rat plasma (v) were assayed against non-extracted 3rd IWS in buffer medium (·) (24 h, 10 pg 125I-labelled ACTH, 1 : 40 000 final dilution of the antiserum, B/B0 is as detailed in the legend to Fig. 1). The
radioimmunoassay system
Sensitivity The limit of detection of the assay was related to the dilution of the antiserum, the amount of tracer added, the duration of the incubation and the variation in the response parameter due to experimental error. The limit of detection was 20 pg ACTH/assay tube in a 24 h assay with 10 pg 125I-labelled ACTH and a 1 :40 000 final dilution of the antiserum and 5 pg/assay tube in a 3-day assay with 5 pg 125I-labelled ACTH added 24 h after starting the incubation, with a 1 : 80 000 final dilution of the antiserum.
Specificity The antiserum used is highly specific for the NH2-terminal portion of the ACTH molecule (Marton et al. 1977). The antiserum cannot differentiate between porcine or human ACTH and rat pituitary extract. The standard curves are superimposable over a wide dose range, indicating that the antiserum is suitable for assaying ACTH in all three species.
Precision
Duplicates of 72 unknown rat plasma samples were run in the same assay (Fig. 3) and duplicates of 28 samples were run in different assays (Fig. 4). A significant correlation was found between-the estimates of the concentration of ACTH in rat plasma both within and between assays (P< 0-001). The coefficients of variation for means of duplicates of four quality control sera were 9-9, 9-2, 6-4 and 10-1 within and 22-2, 8-5, 14-8 and 7-9 between assays over a range of concentrations between 15 and 910 pg ACTH/ml plasma in ten subsequent assays.
10000
CU
" . 1000
ca
"-^
a.
H
u.
-S
.V
100 r=
û·
;i
=
0-9879 72
10
100 10 000 1000 Second replicate Immunoreactive ACTH (pg/ml plasma) 10
Fig. 3. Immunoreactive ACTH concentration of unknown rat plasma samples. Within assay reproducibility: hormone concentration of the same plasma samples were determined in the same assays (r, coefficient of correlation; n, number of samples).
10000
100 1000 Second assay Immunoreactive ACTH (pg/ml plasma)
10000
Fig. 4. Immunoreactive ACTH concentration of unknown rat plasma samples. Between assay reproducibility: hormone concentrations, in duplicate, of the same plasma samples were deter¬ mined in different assays (r, coefficient of correlation; n, number of samples).
Comparison of bioassay and radioimmunoassay potency estimates The ACTH content of 40 samples of isolated rat pituitary cell incubation media was esti¬ mated by a bioassay and the above radioimmunoassay system. The same standard 3rd
IWS ACTH was used as reference in both assays. Estimates of the concentrations of bioactive and immunoreactive ACTH in the samples are compared in Fig. 5. The obvious trend indicated a broad agreement of potency estimates obtained from the two assays.
1000
10 000
Immunoreactive ACTH (pg/ml plasma) Fig. 5. Comparison of bioactive and immunoreactive ACTH concentrations of the same samples of isolated rat pituitary cell incubation media. Unextracted aliquots of the pituitary media were assayed in a bioassay using collagenase disaggregated rat adrenocortical cells, or in the radio¬ immunoassay. In both assays the 3rd IWS ACTH was used as standard (coefficient of correla¬ tion (r) 0-7978; number of samples (n) 40). =
=
Plasma ACTH in rats: experimental studies The level of ACTH in the plasma of resting rats at 09.00 h was 48 ± 3-6 pg/ml (n 10). The mean resting concentration from samples collected at 18.00 h, 106 ± 9-9 pg/ml (n 11), was significantly higher (P
