GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
25, 313-322 (1975)
A Radioimmunoassay for Plasma Corticotropin in Frogs (Rana esculenta L.) H. VAUDRY, P. VAGUE, W. DUPONT, F. LEBOULENGERAND R. VAILLANT Laboratoire
d’Endocrinoiogie, FacultC des Sciences ef des Techniques. Mont-Saint-Aignan, France and Clinique M&dicale A, HGpital de la Timone, 13 385 Marseille, France
76130
Accepted October 8, 1974 A radioimmunoassay technique has been developed for measuring frog plasma corticotropin (ACTH) without prior extraction. Using synthetic porcine ACTH as a reference standard, ‘3’I-labeled synthetic human ACTH (sp act > 500 mCi/mg) as tracer and rabbit anti-porcine ACTH serum, the lower measurable value was estimated at about 4 pg ACTH. Only human and porcine ACTH, ‘-*‘ACTH, and frog pituitary ACTH reacted with the rabbit anti-porcine ACTH serum. No cross-reactivity has been found with synthetic 1--16**7-39ACTH, aMSH, and bovine BMSH. Appearance of damaged Y-h ACTH components after storage in plasma solutions was followed for 7 days. The conditions making it possible to reduce ACTH damage have been ascertained. The average plasma corticotropin level (*CI) was found to be 38.8 ‘- 7.8 pg/ml without any significant difference between males and females. These results suggest that frog ACTH secretion has much in common with mammalian secretions.
Previous radioimmunoassays have made it possible to assess a number of hormone levels in the plasma of lower vertebrates: insulin (Patent et al., 197 I), gonadotropins (Breton et al., 1971, 1972; Crim et al., 1970), and calcitonin (Deftos et al., 1974) in fishes; prolactin and growth hormone (McKeown, 1972) testosterone, estradiol, and estrone (d’istriat et al., 1973) in amphibians. On the contrary, the ACTH plasma radioimmunoassay has been carried out only in mammals: the rat (Matsuyama et al., 1971; Ress et al., 1971; Cook et al., 1972); the sheep (Boddy et al., 1974); the pig (Donald, 1968); and man (Berson et al., 1968; Rayyis et al., 1969; Weiss et al., 1969; Vague et al., 1972; Ichikawa et al., 1972; Galskov, 1972; Guenazzani et al., 1973; Proeschel et al., 1974). On the other hand, immunocytological studies have shown that the corticotropins both of mammals and amphibians have
related immunological properties (DoerrSchott et al., 1970, 1972b, 1972~; Vaudry et al., 1972). Taking advantage of this lack of species specificity, we have recently demonstrated that the corticotropin extracted from the frog’s pituitary has compared with anti-ACTH porcine antibodies, reactions similar to the human ACTH (Vaudry et al., 1974). Under these conditions, a radioimmunoassay in a heterologous system was a practical propositon. In this paper, we submit the findings of this experiment in the green frog (Rana esculenta L.). MATERIALS
Collection
Samples
The frogs were stunned and blood was removed by heart puncture into 5-ml polystyrene tubes containing 100 ~1 2% EDTA Naz for every 1.5 ml blood. The blood samples were immediately centrifuged (4”C, 2OOg, 15 min). The plasma was transferred to a polystyrene tube, processed by heat (6O”C, 40 min) in 313
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Plasma
AND METHODS
314
VAUDRY
order to prevent damage from proteolytic enzymes (Proeschel ez al., 1974), and stored at -25°C.
Antigens Synthetic human (rma9hACTH), kindly supplied by G. Richter (Budapest, Hungary), was used for labeling procedure. Synthetic porcine (1-39p ACTH) was used as standard hormone. Synthetic 3-16~1-*4J--39pACTH fragments as well as (uMSH and bovine pMSH, generously provided by CIBA (Basel, Switzerland) were used to study the specificity of the antibodies. Frog ACTH was extracted from pituitaries as previously described (Vaudry et al., 1974).
ET AL.
sample. They made possible the determination of the amount of damaged ACTH.
RESULTS
(1) Appearance of Damaged Components from [ 1311]ACTH After Storage in a Solution of Frog Plasma
The appearance of damaged components from labeled human ACTH was recorded for 7 days in different media (Fig. 1). The analysis, after talc separation of both impaired and unimpaired fractions of labeled ACTH showed that ACTH damage was a complex phenomenon. Antibodies Damage velocity is uneven. It varies Anti-porcine ACTH serum 22 B, obtained by imwith the type of medium, temperature, and munizing rabbits with pituitary porcine ACTH (Cros et al., 1970), was a gift from Dr. R. Depieds. The the time of incubation. final dilution was usually l/500,000. Under standard conditions of temperature and buffer, the damage is much faster ACTH Radioimmunoassay in a solution of untreated plasma (A) than Labeling ACTH. ACTH labeling procedure was in a solution of heated plasma. carried out according to Berson er al. (1968) modified Proteolysis inhibitors (zymofren and as follows. As a polystyrene tube (5 ml) containing 2 benzamidine) considerably reduce damage pg h ACTH and 25 ~1 0.5 M, pH 7.4, phosphate velocity, especially with incubations carbuffer was shaken gently, the following were added: ried out at 4 or 37°C. 1.4 mCi r311 from CEA, 15 ~1 of a 2-mg/ml chloraDamage velocity does not vary in promine T solution, 25 ~1 of a 2-mglml sodium metabisulfic solution, and finally 300 ~1 0.5 M, pH 7.6, portion to temperature. While low temperphosphate buffer. ature (4°C) always reduces damage, the Purification of labeled ACTH. One milliliter latter reaches a maximum at 18°C and conhuman plasma and 7 mg Quso GtZ (Philadelphia siderably decreases during a 37°C incubaQuartz Co., Philadelphia) were added to the labeled tion. This phenomenon finds its explanamixture. A rotating agitation was kept for 2 min. Then the tube was centrifuged. The precipitate was tion when it is borne in mind that the washed in buffered water (3 ml) and eluted with HCI optimal temperature of the green frog’s 0.1 N (3 ml). The tracer was stored at WC, diluted in metabolic activity is 18°C. Above 30°C a 0.3% albumin, 0.02 M, pH 8.6, Verona1 buffer. the enzymatic reactions, in particular, Preparation of incubation mixtures. In 5-ml polystyrene tubes were incubated the 22 B serum with proteolysis, fall down rapidly. l/500,000 final dilution (100 PI), 4 pg Ia11 h ACTH Therefore, three precautions are neces(100 PI), and given amounts of standard p ACTH, sary to reduce damage in a plasma soluranging from 4 to 200 pg ACTH. The final incubation tion: (1) Operate at low temperature and volume was usually 2.5 ml (0.02 M veronal buffer). store the tubes at 4°C throughout the inAt the end of the incubation period (3 days, 4”C), the free hormone (F) was separated from the antibodycubation period. (2) Process all plasmas by bound hormone (B) by adsorption to talc (50 mg) ac- heat (60°C 40 min). If this operation is cording to Rosselin et a/., 1968. carried out immediately after the centrifuFor each plasma sample, three test tubes, congation of blood samples, the plasmas may taining 500 /*I of frog plasma without prior extraction, be stored, without damage, at -25°C. (3) was incubated with 100 ~1 of anti-ACTH serum (l/500,000) and 4 pg 1311 h ACTH. Two control Add two antienzymes to the incubation tubes, without antibodies, were prepared for each buffer: mercaptoethanol (2 pi/ml) and
RADIOIMMUNOASSAY
L 1
i
I 3
I 4
OF
I 6
ACTH
IN
FROG
315
.
FIG. 1. Appearance of damaged ACTH in several incubation media at 4°C (O-.-.-O), 18°C (A---A), and 37°C (o-0). Four picograms 1311h ACTH were incubated with 500 ~1 fresh frog plasma (A) or 500 ~1 heated frog plasma(B). The final incubation volume (2.5 ml) was reached by adding 0.3% human albumin 0.02 M veronal buffer (VAB). In order to determine the effects of proteolytic inhibitors, some incubations were performed in Verona1 buffer containing 0.2% mercaptoethanol and Trasylol-1000 NJ/ml (VMTAB), mercaptoethanol and 0.1 M benzamidine (VMBAB), or mercaptoethanol and 0.01 M benzamidine (VMbAB).
zymofren (1000 U/ml) or benzamidine (0.1 Iv). Under these conditions, plasma ACTH level may be assessed without prior extraction. (2) Assessment of 22 B Serum Titer and Sensitivity The titer of 22 B serum was assessed by placing constant amounts of labeled antigen (4 pg) with decreasing dilutions of
immune serum. The media were incubated for 3 days at 4°C. The analysis of the 22 B serum titer (Fig. 2) demonstrates again its very high titer already pointed out by several authors (Cros et a/., 1970; Oliver, 1972). With a 2 X lo+ dilution, this serum binds over 30% of the labeled hormone and still shows a marked incorporation of tracer with 5 x 10e7 dilution. To assess the sensitivity of the an-
316
VAUDRY
ET AL.
mune serum dilutions ranging from 12.5 x IO-fi to 1 x 10-6. The percentage of bound radioactivity displacement as a proportion to the amounts of standard hormone added to the medium is ascertained (Fig. 4). Antiserum optimum sensitivity corresponds to a 2 X. 10m6 tinal dilution.
‘\ -\
(3) Assessment of 22B Serum Specificity The specificity of 22 B serum (Fig. 5) “‘VLV was sought, using six different hormones (1-3gp ACTH, p 1-24 ACTH, l-l6 ACTH, 17-3sp ACTH, (rMSH, PMSH) with equimolar concentrations. Each had the same FIG. 2. 22 B serum titration curve showing the tracer 1311 h ACTH and a constant anexceptionally high titer of this antiserum. tiserum dilution (2 x lo+). The whole p ACTH molecule and lPz4ACTH involve a marked displacement tiserum, 10, 20, and 50 pg of standard hor(> 30%) of bound radioactivity. With the mone were added to the incubation same concentrations, l-l6 and 17-3sACTH medium containing the antibodies and the fragments involve only a very slight detracer. crease in the B/T ratio (T is the total radioThe study of the resulting curves (Fig. activity in each incubation tube). There3) shows a maximum sensitivity for imfore, the 24 -N terminal -amino acids of the ACTH molecule include the antigenic site(s) which the 22 B serum can bind. As, on the other hand, each of the l-l6 and 17-39polypeptide has only a very slight antigenic capacity, the simultaneous presence of the 16 and 17 amino acids (Ly9Arg17) is a precondition to the antigenicity of the molecule. The behavior of the aMSH molecule in front of the 22 B serum is like that of l-l6 and 17-3sACTH. A study of the curves shows that 40 x 10-15M inaMSH and 1 X 10-15M 1-3sp ACTH volve a similar displacement of bound radioactivity. The cross reactions between (YMSH and the antiserum are, therefore, comparatively negligible and do not prevent an ACTH radioimmunoassay. Conversely, the 22 B serum has no avidity for the pMSH molecule, which again FIG. 3. Assessment of 22 B serum sensitivity. Antiserum dilution curves using doubling dilutions of the bears witness to the presence of an anlo-24 sequence on the ACTH serum incubated with constant amounts (4 pg) of IS11 tigenic Moreover, a study of Fig. 6 h ACTH (tracer) and SO (O-----O), 20 (a-.-.-@), 10 molecule. (V---V), or 0 (S-S) pg of p ACTH (standard). shows that the frog ACTH has, with the
i
RADIOIMMUNOASSAY
[AS]
ACTH
IN
317
FROG
=12.5~10-~
10
[AS]
OF
20
10
%PL
=2x10-6
[AS]=
20
50 PI7
10-b
15.2
10
20
50 PO
10
20
50 PI
Sensitivitv of 22 B serum at four different antiserum dill 3tions. The higher sensitivity is obtained between 2 X lo-” and lo-” dilutions. FIG.
4.
22 B serum, similar immunological tions to those of mammal ACTH.
reac-
(4) ACTH Assay in a Plasma Medium and Plasma Processing by Heat
The influence of plasma on frog ACTH assays was checked by comparing the standard curves (Fig. 7) under the following conditions: (1) . buffer only; (2) buffer + 500 ~1 human plasma ACTH-free patient); (dexamethasone-treated (3) buffer + 500 ~1 frog plasma ACTH-free (6 days’ hypophysectomized animals). The addition of plasma causes the B/T ratio to vary about 2%. These variations involve errors in the assessment of the ACTH rate which make it necessary to set up the standard curve in a solution of hypophysectomized frog plasma. To study the influence of heat treatment
on ACTH antigenicity, two standard curves had been established in human plasma medium: one with unheated given ACTH amounts, the other with the same ACTH concentrations treated by heat (60°C 40 min). Both curves are very alike (Fig. 7). Therefore, plasma heat processing does not considerably modify the antigenic properties of ACTH. (5) Standard Curve
Drawn from nine reference points, the standard curve is regarded as satisfactory under the following conditions: (1) The ACTH damage in the control tubes remains below 12%; (2) the B/T ratio, after deducting the damage rate, is over 30% ; the initial B/T ratio falls by over 70%, after adding 50 pg standard hormone.
318
VAUDRY
ET AL.
AS 228 1/500.000
lo-‘= M Pepttde
5. Assessment of 22 B serum specificity. Inhibition curves obtained with p ACTH antiserum and different ACTH-related peptides. Only the whole p ACTH molecule and the 24 (N terminal) amino acid sequence involve an important displacement of the bound radioactivity while a and /3 MSH do not crossreact. W-m, B p MSH; C-O, (Y MSH. V-V’, ‘+‘p ACTH; A-A, B ‘-**ACTH; + -+, ‘r=p ACTH; 0-0, ‘-16ACTH. FIG.
FIG. 7. Effects of heat treatment and plasma addition on the standard curve. Comparison between standard curves established with fresh ACTH in buffer medium (*), fresh ACTH in frog, ACTH-free, plasma (O), fresh ACTH in human, ACTH-free, plasma (O), heat-treated ACTH in human, ACTH-free plasma (0).
AS 22 0 1/500.000 ‘?I
L
,
4
20
40
60
hACTH 419
I
100
*
200 PB ACCTR
FIG. 6. Comparison of immunological reactivity of human ACTH (A-A), porcine ACTH (*-*), and frog pituitary extract (0-O). There is a close similarity between human and frog ACTH and an important cross-reaction between porcine and frog ACTH.
Under these conditions, the assay sensitivity threshold reaches 4 pg ACTH and the ACTH rate assessment is correct between 0 and 60 pg (Fig. 8). The ACTH rate was determined with the heterologous system indicated above (tracer, 1311 h ACTH; antibody, 22 B serum anti-p ACTH; standard, p ACTH). The results are recorded in Table 1. There is no statistically significant difference (P = 0.05) between males and females. The distribution histogram (Fig. 9) points out that two plasma pools in three have an ACTH rate ranging from 30 to 50 pglml. Only one pool shows a figure above 100 pg/mI (130 pg/ml). Two samples have nondetectable ACTH.
RADIOIMMUNOASSAY
OF
ACTH
319
IN FROG
TABLE PLASMA
Sex d 0
3 and 0
ACTH
LEVELS
1 IN NORMAL
FROGS
Number of plasma pools”
ACTH* Cpg/ml)
6 6 32
44.4 + 9.3 41.0 + 11.7 38.8’ k 7.8
‘Each pool of plasma corresponds to four or five animals. b Mean and confidence interval (P = 0.05). ’ This value is the mean of 32 pools of plasma issued from 130 animals.
DISCUSSION
100
.
200 PO l ACTW
FIG. 8. Standard curve. Displacement by unlabeled synthetic porcine ACTH of lzlI human ACTH. Antiserum dilution, 1500,000; amount of labeled hormone, 2 pg.
NUMBER OF POOLS IN EACH CLASS t
ACTH RATE pg/ml FIG. 9. Distribution histogram of plasma ACTH rate in 32 pools of frog plasma. Two samples have nondetectable ACTH (ND); two samples in three have 30-50 pg/ml ACTH rate; one sample has more than 100 pglml ACTH.
Even though it is theoretically possible to measure very small amounts of ACTH with the recent biological techniques (Kitabchi et al., 1970; Sayers er al., 1971), a radioimmunoassay alone makes it currently possible to carry out a series of simultaneous measures. Moreover, its very low sensitivity threshold admits of tests on very small plasma volumes (100-500 ~1). It is consequently especially appropriate for the measure of the ACTH rate in small-size low vertebrates. However, no plasma ACTH assay has yet been performed in the Poi’kilotherms. As a consequence, the frog ACTH rates can only be compared to those recorded for the mammals (Donald et a/., 1968; Matsuyama ef al., 1971; Oliver, 1971; Proeschel et al., 1974; Boddy et al., 1974). Whatever the species under study, these figures, in resting subjects, generally range from 0 to 100 pg/ml and are often below 50 pglml. Our findings on the plasma ACTH rate in the normal frogs are thus comparable to those already recorded for the rat, the sheep, the pig, and man. These figures also tally with those determined by biological means in man (Vance et al., 1962; Espiner et al., 1963; Girard et al., 1966; Kitabschi et al., 1971), in the hedgehog (Hoo-Paris, 1971) and in the rat (Mialhe et al., 1973). Yet, these results can meet with criticism.
320
VAUDRY
ET AL.
Allowing for the small quantities of rate, to multiply the recorded figures by a blood which can be drawn from a subject, factor K so that all the assays have been carried out on KJ$ plasma pools coming from different anK > 1, Y imals. Therefore, the assays do not take into consideration the individual variations where K, is the association constant which have been clearly shown in man between the serum and the standard (por(Berson ef al., 1968; Oliver, 197 1; Gelzer, cine ACTH) and K, is the association con1972). On the other hand, we have proved stant between the serum and frog plasma ACTH. that the ACTH rate is not significantly Moreover, the synthetic ACTH strucinfluenced by the sex of the animals. These tures (porcine standard and human tracer) findings agree with those of Binoux et al. (1965) and Oliver (1971) in man. determined by Shepherd et al. (1956, p ACTH) and Lee et al. (1961, h ACTH) A current systematic study of the daily and used in our studies are erroneous. Rinand seasonal ACTH plasma secretion iker er al. (1972) have found four anomrhythms in the frog during the hibernation or sexually active periods may point out alies in the 25-31 sequence of these polyimportant variations. For, in man, the peptides. Though the new molecule (h plasma ACTH rate varies throughout the ACTH) synthetized by Sieber et ul. ( 1972) has the same biological properties as that day (Binoux er al., 1965; Berson et al., used as a tracer in our study (Tesser et ul., 1968; Oliver, 1971), during the menstrual 1973; Schenkel-Hulliger et al., 1974), its cycle and pregnancy (Genazzani et al., properties may notably be 1973). Nycthemeral variations of the immunological ACTH rate have also been shown in the different. Nevertheless, the development of the rat (Matsuyama et al., 1971; Rees et al., radioimmunoassay has already 1971). In the same way, there is a cir- ACTH cadian rhythm of ACTH rate among the made possible the assessment, in the frog, of the hormone concentration in the whole hibernating wild animals (Hoo-Paris, adenohypophysis, in the pars distalis and 1971). in the pars intermedia (Vaudry et al., Moreover, an amphibian ACTH ra1974). dioimmunoassay in a heterologous system The assessment of the plasma ACTH may bring two types of errors. rate will shortly be useful when following We have noticed that the ACTH plasma the evolution of the corticotropic function concentrations do not vary linearly with under various experimental conditions the plasma dilutions in the incubation (stress, interrenalectomy, metyrapone intubes. According to Yalow et al. (1971), jections, total or partial hypophysectomy). this phenomenon is inherent to hetThus, the direct assessment of the erologous immunoassays. Yet, the presACTH rate, will give valuable indications ence of antigenic fragments in the samples which will supplement the numerous cytounder study or a structural heterogeneity logical and ultrastructural studies available in the plasma ACTH may also bring about on the amphibian pituitaries. the same variations. The plasma ACTH rate assessed during our tests cannot be taken as accurate figACKNOWLEDGMENTS ures, because the avidity of 22 B serum is We are indebted to Dr. R. Depieds, Laboratoires lower for frog ACTH than for porcine d’Endocrinologie, Facultk des Sciences Nord, MarACTH. It would, therefore, be desirable, seille, France, for his generous gift of serum 22 B, to to get an exact assessment of the ACTH Dr. P. A. Desaulles and Dr. W. Rittel, CIBA Lab-
RADIOIMMUNOASSAY oratoires, Base], Switzerland, and Dr. G. Fekete, Richter Laboratoires, Budapest, Hungary for their supplies of polypeptide hormones, to Dr. A. Capet and M. C. Simon for constructive technical assistance, and to J. Lecourt for secretarial assistance. The present studies were supported by grants from the Direction GCnerale de la Recherche Scientifique et Technique (73.7-1853).
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S. S., AND BETHUNE, J. E. (1969). Radioimmunoassayable ACTH in dexamethasone nonsuppressible Cushing’s syndrome. J. C/in. Endocrinol. Metab. 29, 1231-1237. REES, L. H., RATCLIFFE, J. G., KENDALL, J. W., KNIGHT, R. A., ALLEN, C., AND COOK, D. M. (1971). A radioimmunoassay for rat plasma corticotrophin. J. Endocrinol. 51, 3-4. ROSSELIN, G., ASSAN, R., YALLOW, R. S., AND BERSON, S. A. (1966). Separation of antibody bound and unbound peptide hormones labelled with iodine- 13 1 by talcum powder and precipitated silica. Nature (London) 212, 355-358. RINIKER, B., SIEBER, P., RITTEL, W., AND ZUBBER, H. (1972). Revised amino-acid sequences for porcine and human adrenocorticotrophic hormone. Nature New Biol. 235, 114-115. SAYERS, G., SWALLOW, R. L., AND GIORDANO, N. D. (1971). An improved technique for the preparation of isolated rat adrenal cells: a sensitive accurate and specific method for the assay of ACTH. Endocrinology 88, 1063-1068. SCHENKEL-HULLIGER, L., MAIER, R., BARTHE, P. L., DESAULLES, P. A., JARRET, A., RINIKER, B., RITTEL, W., AND SIEBER, P. (1974). Biological activity of synthetic human corticotrophin with RAYYIS,
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SHEPHERD, R. G., HOWARD, K. S., BELL, P. H., CACCIOLA, A. R., CHILD, R. G., DAVIES, M. D., ENGLISH, J. P., FINN, B. M., MEISENHELDER, J. H., MOYER, A. W., AND VAN DEK SCHERR, J. ( 1956). Studies with corticotropin. I. Isolation, purification and properties of p-corticotropin. J. Amer. Chem. Sot. 78, 5051-5059. SIEBER, P., RITTEL, W., AND RINIKER, B. (1972). Die Synthese von memschlichem adrenocorticotropem Hormon (h-ACTH) mit revidierter AminosPuresequenz. Helv. Chim. Acta 55, 1243-1248. TESSER, G. I., MAIER, R., SCHENKEL-HULLIGER, L., BARTHE, P. L., KAMBER, B., AND RITTEL, W. (1973). Biological activity of corticotrophin peptides with homo-arginine, lysine, or omithine substituted for arginine in position 8. Acta Endocrinol.
74,
56-66.
VAGUE, P., AND OLIVER, C. (1972). Le dosage radioimmunologique de l’ACTH plasmatique. In “Techniques Radioimmunologiques,” pp. 3 5 l366. SCminaire INSERM, Paris. VAUDRY, H., DUPONT, W., AND LEBOULENGER, F. (1972). Mise en Cvidence des cellules corticotropes de l’hypophyse de la Grenouille verte, Rana esculenta L., par la technique d’immunofluorescence. C. R. Sot. Biol. 166, 1362-1365. VAUDRY, H., LEBOULENGER, F., DUPONT, W., AND VAILLANT, R. (1974). Assessment of pituitary ACTH rate in the green frog Rana esculenta L. by radioimmunological means. C. R. Sot. Biol., in press. WEISS, E. R., RAYYIS, S. S., NELSON, D. H., AND BETHUNE, J. E. (1969). Evaluation of stimulation and suppression tests in the etiological diagnosis of Cushing’s syndrome. Ann. Intern. Med. 71, 941-949.
YALOW, R. S., AND BERSON, S. A. (1971). Problems of validation of radioimmunoassays. In “Principles of Competitive Protein Binding Assays,” pp. 374-400. Lippincott, Philadelphia.
AND
COMPARATIVE
ENDOCRINOLOGY
25, 313-322 (1975)
A Radioimmunoassay for Plasma Corticotropin in Frogs (Rana esculenta L.) H. VAUDRY, P. VAGUE, W. DUPONT, F. LEBOULENGERAND R. VAILLANT Laboratoire
d’Endocrinoiogie, FacultC des Sciences ef des Techniques. Mont-Saint-Aignan, France and Clinique M&dicale A, HGpital de la Timone, 13 385 Marseille, France
76130
Accepted October 8, 1974 A radioimmunoassay technique has been developed for measuring frog plasma corticotropin (ACTH) without prior extraction. Using synthetic porcine ACTH as a reference standard, ‘3’I-labeled synthetic human ACTH (sp act > 500 mCi/mg) as tracer and rabbit anti-porcine ACTH serum, the lower measurable value was estimated at about 4 pg ACTH. Only human and porcine ACTH, ‘-*‘ACTH, and frog pituitary ACTH reacted with the rabbit anti-porcine ACTH serum. No cross-reactivity has been found with synthetic 1--16**7-39ACTH, aMSH, and bovine BMSH. Appearance of damaged Y-h ACTH components after storage in plasma solutions was followed for 7 days. The conditions making it possible to reduce ACTH damage have been ascertained. The average plasma corticotropin level (*CI) was found to be 38.8 ‘- 7.8 pg/ml without any significant difference between males and females. These results suggest that frog ACTH secretion has much in common with mammalian secretions.
Previous radioimmunoassays have made it possible to assess a number of hormone levels in the plasma of lower vertebrates: insulin (Patent et al., 197 I), gonadotropins (Breton et al., 1971, 1972; Crim et al., 1970), and calcitonin (Deftos et al., 1974) in fishes; prolactin and growth hormone (McKeown, 1972) testosterone, estradiol, and estrone (d’istriat et al., 1973) in amphibians. On the contrary, the ACTH plasma radioimmunoassay has been carried out only in mammals: the rat (Matsuyama et al., 1971; Ress et al., 1971; Cook et al., 1972); the sheep (Boddy et al., 1974); the pig (Donald, 1968); and man (Berson et al., 1968; Rayyis et al., 1969; Weiss et al., 1969; Vague et al., 1972; Ichikawa et al., 1972; Galskov, 1972; Guenazzani et al., 1973; Proeschel et al., 1974). On the other hand, immunocytological studies have shown that the corticotropins both of mammals and amphibians have
related immunological properties (DoerrSchott et al., 1970, 1972b, 1972~; Vaudry et al., 1972). Taking advantage of this lack of species specificity, we have recently demonstrated that the corticotropin extracted from the frog’s pituitary has compared with anti-ACTH porcine antibodies, reactions similar to the human ACTH (Vaudry et al., 1974). Under these conditions, a radioimmunoassay in a heterologous system was a practical propositon. In this paper, we submit the findings of this experiment in the green frog (Rana esculenta L.). MATERIALS
Collection
Samples
The frogs were stunned and blood was removed by heart puncture into 5-ml polystyrene tubes containing 100 ~1 2% EDTA Naz for every 1.5 ml blood. The blood samples were immediately centrifuged (4”C, 2OOg, 15 min). The plasma was transferred to a polystyrene tube, processed by heat (6O”C, 40 min) in 313
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Plasma
AND METHODS
314
VAUDRY
order to prevent damage from proteolytic enzymes (Proeschel ez al., 1974), and stored at -25°C.
Antigens Synthetic human (rma9hACTH), kindly supplied by G. Richter (Budapest, Hungary), was used for labeling procedure. Synthetic porcine (1-39p ACTH) was used as standard hormone. Synthetic 3-16~1-*4J--39pACTH fragments as well as (uMSH and bovine pMSH, generously provided by CIBA (Basel, Switzerland) were used to study the specificity of the antibodies. Frog ACTH was extracted from pituitaries as previously described (Vaudry et al., 1974).
ET AL.
sample. They made possible the determination of the amount of damaged ACTH.
RESULTS
(1) Appearance of Damaged Components from [ 1311]ACTH After Storage in a Solution of Frog Plasma
The appearance of damaged components from labeled human ACTH was recorded for 7 days in different media (Fig. 1). The analysis, after talc separation of both impaired and unimpaired fractions of labeled ACTH showed that ACTH damage was a complex phenomenon. Antibodies Damage velocity is uneven. It varies Anti-porcine ACTH serum 22 B, obtained by imwith the type of medium, temperature, and munizing rabbits with pituitary porcine ACTH (Cros et al., 1970), was a gift from Dr. R. Depieds. The the time of incubation. final dilution was usually l/500,000. Under standard conditions of temperature and buffer, the damage is much faster ACTH Radioimmunoassay in a solution of untreated plasma (A) than Labeling ACTH. ACTH labeling procedure was in a solution of heated plasma. carried out according to Berson er al. (1968) modified Proteolysis inhibitors (zymofren and as follows. As a polystyrene tube (5 ml) containing 2 benzamidine) considerably reduce damage pg h ACTH and 25 ~1 0.5 M, pH 7.4, phosphate velocity, especially with incubations carbuffer was shaken gently, the following were added: ried out at 4 or 37°C. 1.4 mCi r311 from CEA, 15 ~1 of a 2-mg/ml chloraDamage velocity does not vary in promine T solution, 25 ~1 of a 2-mglml sodium metabisulfic solution, and finally 300 ~1 0.5 M, pH 7.6, portion to temperature. While low temperphosphate buffer. ature (4°C) always reduces damage, the Purification of labeled ACTH. One milliliter latter reaches a maximum at 18°C and conhuman plasma and 7 mg Quso GtZ (Philadelphia siderably decreases during a 37°C incubaQuartz Co., Philadelphia) were added to the labeled tion. This phenomenon finds its explanamixture. A rotating agitation was kept for 2 min. Then the tube was centrifuged. The precipitate was tion when it is borne in mind that the washed in buffered water (3 ml) and eluted with HCI optimal temperature of the green frog’s 0.1 N (3 ml). The tracer was stored at WC, diluted in metabolic activity is 18°C. Above 30°C a 0.3% albumin, 0.02 M, pH 8.6, Verona1 buffer. the enzymatic reactions, in particular, Preparation of incubation mixtures. In 5-ml polystyrene tubes were incubated the 22 B serum with proteolysis, fall down rapidly. l/500,000 final dilution (100 PI), 4 pg Ia11 h ACTH Therefore, three precautions are neces(100 PI), and given amounts of standard p ACTH, sary to reduce damage in a plasma soluranging from 4 to 200 pg ACTH. The final incubation tion: (1) Operate at low temperature and volume was usually 2.5 ml (0.02 M veronal buffer). store the tubes at 4°C throughout the inAt the end of the incubation period (3 days, 4”C), the free hormone (F) was separated from the antibodycubation period. (2) Process all plasmas by bound hormone (B) by adsorption to talc (50 mg) ac- heat (60°C 40 min). If this operation is cording to Rosselin et a/., 1968. carried out immediately after the centrifuFor each plasma sample, three test tubes, congation of blood samples, the plasmas may taining 500 /*I of frog plasma without prior extraction, be stored, without damage, at -25°C. (3) was incubated with 100 ~1 of anti-ACTH serum (l/500,000) and 4 pg 1311 h ACTH. Two control Add two antienzymes to the incubation tubes, without antibodies, were prepared for each buffer: mercaptoethanol (2 pi/ml) and
RADIOIMMUNOASSAY
L 1
i
I 3
I 4
OF
I 6
ACTH
IN
FROG
315
.
FIG. 1. Appearance of damaged ACTH in several incubation media at 4°C (O-.-.-O), 18°C (A---A), and 37°C (o-0). Four picograms 1311h ACTH were incubated with 500 ~1 fresh frog plasma (A) or 500 ~1 heated frog plasma(B). The final incubation volume (2.5 ml) was reached by adding 0.3% human albumin 0.02 M veronal buffer (VAB). In order to determine the effects of proteolytic inhibitors, some incubations were performed in Verona1 buffer containing 0.2% mercaptoethanol and Trasylol-1000 NJ/ml (VMTAB), mercaptoethanol and 0.1 M benzamidine (VMBAB), or mercaptoethanol and 0.01 M benzamidine (VMbAB).
zymofren (1000 U/ml) or benzamidine (0.1 Iv). Under these conditions, plasma ACTH level may be assessed without prior extraction. (2) Assessment of 22 B Serum Titer and Sensitivity The titer of 22 B serum was assessed by placing constant amounts of labeled antigen (4 pg) with decreasing dilutions of
immune serum. The media were incubated for 3 days at 4°C. The analysis of the 22 B serum titer (Fig. 2) demonstrates again its very high titer already pointed out by several authors (Cros et a/., 1970; Oliver, 1972). With a 2 X lo+ dilution, this serum binds over 30% of the labeled hormone and still shows a marked incorporation of tracer with 5 x 10e7 dilution. To assess the sensitivity of the an-
316
VAUDRY
ET AL.
mune serum dilutions ranging from 12.5 x IO-fi to 1 x 10-6. The percentage of bound radioactivity displacement as a proportion to the amounts of standard hormone added to the medium is ascertained (Fig. 4). Antiserum optimum sensitivity corresponds to a 2 X. 10m6 tinal dilution.
‘\ -\
(3) Assessment of 22B Serum Specificity The specificity of 22 B serum (Fig. 5) “‘VLV was sought, using six different hormones (1-3gp ACTH, p 1-24 ACTH, l-l6 ACTH, 17-3sp ACTH, (rMSH, PMSH) with equimolar concentrations. Each had the same FIG. 2. 22 B serum titration curve showing the tracer 1311 h ACTH and a constant anexceptionally high titer of this antiserum. tiserum dilution (2 x lo+). The whole p ACTH molecule and lPz4ACTH involve a marked displacement tiserum, 10, 20, and 50 pg of standard hor(> 30%) of bound radioactivity. With the mone were added to the incubation same concentrations, l-l6 and 17-3sACTH medium containing the antibodies and the fragments involve only a very slight detracer. crease in the B/T ratio (T is the total radioThe study of the resulting curves (Fig. activity in each incubation tube). There3) shows a maximum sensitivity for imfore, the 24 -N terminal -amino acids of the ACTH molecule include the antigenic site(s) which the 22 B serum can bind. As, on the other hand, each of the l-l6 and 17-39polypeptide has only a very slight antigenic capacity, the simultaneous presence of the 16 and 17 amino acids (Ly9Arg17) is a precondition to the antigenicity of the molecule. The behavior of the aMSH molecule in front of the 22 B serum is like that of l-l6 and 17-3sACTH. A study of the curves shows that 40 x 10-15M inaMSH and 1 X 10-15M 1-3sp ACTH volve a similar displacement of bound radioactivity. The cross reactions between (YMSH and the antiserum are, therefore, comparatively negligible and do not prevent an ACTH radioimmunoassay. Conversely, the 22 B serum has no avidity for the pMSH molecule, which again FIG. 3. Assessment of 22 B serum sensitivity. Antiserum dilution curves using doubling dilutions of the bears witness to the presence of an anlo-24 sequence on the ACTH serum incubated with constant amounts (4 pg) of IS11 tigenic Moreover, a study of Fig. 6 h ACTH (tracer) and SO (O-----O), 20 (a-.-.-@), 10 molecule. (V---V), or 0 (S-S) pg of p ACTH (standard). shows that the frog ACTH has, with the
i
RADIOIMMUNOASSAY
[AS]
ACTH
IN
317
FROG
=12.5~10-~
10
[AS]
OF
20
10
%PL
=2x10-6
[AS]=
20
50 PI7
10-b
15.2
10
20
50 PO
10
20
50 PI
Sensitivitv of 22 B serum at four different antiserum dill 3tions. The higher sensitivity is obtained between 2 X lo-” and lo-” dilutions. FIG.
4.
22 B serum, similar immunological tions to those of mammal ACTH.
reac-
(4) ACTH Assay in a Plasma Medium and Plasma Processing by Heat
The influence of plasma on frog ACTH assays was checked by comparing the standard curves (Fig. 7) under the following conditions: (1) . buffer only; (2) buffer + 500 ~1 human plasma ACTH-free patient); (dexamethasone-treated (3) buffer + 500 ~1 frog plasma ACTH-free (6 days’ hypophysectomized animals). The addition of plasma causes the B/T ratio to vary about 2%. These variations involve errors in the assessment of the ACTH rate which make it necessary to set up the standard curve in a solution of hypophysectomized frog plasma. To study the influence of heat treatment
on ACTH antigenicity, two standard curves had been established in human plasma medium: one with unheated given ACTH amounts, the other with the same ACTH concentrations treated by heat (60°C 40 min). Both curves are very alike (Fig. 7). Therefore, plasma heat processing does not considerably modify the antigenic properties of ACTH. (5) Standard Curve
Drawn from nine reference points, the standard curve is regarded as satisfactory under the following conditions: (1) The ACTH damage in the control tubes remains below 12%; (2) the B/T ratio, after deducting the damage rate, is over 30% ; the initial B/T ratio falls by over 70%, after adding 50 pg standard hormone.
318
VAUDRY
ET AL.
AS 228 1/500.000
lo-‘= M Pepttde
5. Assessment of 22 B serum specificity. Inhibition curves obtained with p ACTH antiserum and different ACTH-related peptides. Only the whole p ACTH molecule and the 24 (N terminal) amino acid sequence involve an important displacement of the bound radioactivity while a and /3 MSH do not crossreact. W-m, B p MSH; C-O, (Y MSH. V-V’, ‘+‘p ACTH; A-A, B ‘-**ACTH; + -+, ‘r=p ACTH; 0-0, ‘-16ACTH. FIG.
FIG. 7. Effects of heat treatment and plasma addition on the standard curve. Comparison between standard curves established with fresh ACTH in buffer medium (*), fresh ACTH in frog, ACTH-free, plasma (O), fresh ACTH in human, ACTH-free, plasma (O), heat-treated ACTH in human, ACTH-free plasma (0).
AS 22 0 1/500.000 ‘?I
L
,
4
20
40
60
hACTH 419
I
100
*
200 PB ACCTR
FIG. 6. Comparison of immunological reactivity of human ACTH (A-A), porcine ACTH (*-*), and frog pituitary extract (0-O). There is a close similarity between human and frog ACTH and an important cross-reaction between porcine and frog ACTH.
Under these conditions, the assay sensitivity threshold reaches 4 pg ACTH and the ACTH rate assessment is correct between 0 and 60 pg (Fig. 8). The ACTH rate was determined with the heterologous system indicated above (tracer, 1311 h ACTH; antibody, 22 B serum anti-p ACTH; standard, p ACTH). The results are recorded in Table 1. There is no statistically significant difference (P = 0.05) between males and females. The distribution histogram (Fig. 9) points out that two plasma pools in three have an ACTH rate ranging from 30 to 50 pglml. Only one pool shows a figure above 100 pg/mI (130 pg/ml). Two samples have nondetectable ACTH.
RADIOIMMUNOASSAY
OF
ACTH
319
IN FROG
TABLE PLASMA
Sex d 0
3 and 0
ACTH
LEVELS
1 IN NORMAL
FROGS
Number of plasma pools”
ACTH* Cpg/ml)
6 6 32
44.4 + 9.3 41.0 + 11.7 38.8’ k 7.8
‘Each pool of plasma corresponds to four or five animals. b Mean and confidence interval (P = 0.05). ’ This value is the mean of 32 pools of plasma issued from 130 animals.
DISCUSSION
100
.
200 PO l ACTW
FIG. 8. Standard curve. Displacement by unlabeled synthetic porcine ACTH of lzlI human ACTH. Antiserum dilution, 1500,000; amount of labeled hormone, 2 pg.
NUMBER OF POOLS IN EACH CLASS t
ACTH RATE pg/ml FIG. 9. Distribution histogram of plasma ACTH rate in 32 pools of frog plasma. Two samples have nondetectable ACTH (ND); two samples in three have 30-50 pg/ml ACTH rate; one sample has more than 100 pglml ACTH.
Even though it is theoretically possible to measure very small amounts of ACTH with the recent biological techniques (Kitabchi et al., 1970; Sayers er al., 1971), a radioimmunoassay alone makes it currently possible to carry out a series of simultaneous measures. Moreover, its very low sensitivity threshold admits of tests on very small plasma volumes (100-500 ~1). It is consequently especially appropriate for the measure of the ACTH rate in small-size low vertebrates. However, no plasma ACTH assay has yet been performed in the Poi’kilotherms. As a consequence, the frog ACTH rates can only be compared to those recorded for the mammals (Donald et a/., 1968; Matsuyama ef al., 1971; Oliver, 1971; Proeschel et al., 1974; Boddy et al., 1974). Whatever the species under study, these figures, in resting subjects, generally range from 0 to 100 pg/ml and are often below 50 pglml. Our findings on the plasma ACTH rate in the normal frogs are thus comparable to those already recorded for the rat, the sheep, the pig, and man. These figures also tally with those determined by biological means in man (Vance et al., 1962; Espiner et al., 1963; Girard et al., 1966; Kitabschi et al., 1971), in the hedgehog (Hoo-Paris, 1971) and in the rat (Mialhe et al., 1973). Yet, these results can meet with criticism.
320
VAUDRY
ET AL.
Allowing for the small quantities of rate, to multiply the recorded figures by a blood which can be drawn from a subject, factor K so that all the assays have been carried out on KJ$ plasma pools coming from different anK > 1, Y imals. Therefore, the assays do not take into consideration the individual variations where K, is the association constant which have been clearly shown in man between the serum and the standard (por(Berson ef al., 1968; Oliver, 197 1; Gelzer, cine ACTH) and K, is the association con1972). On the other hand, we have proved stant between the serum and frog plasma ACTH. that the ACTH rate is not significantly Moreover, the synthetic ACTH strucinfluenced by the sex of the animals. These tures (porcine standard and human tracer) findings agree with those of Binoux et al. (1965) and Oliver (1971) in man. determined by Shepherd et al. (1956, p ACTH) and Lee et al. (1961, h ACTH) A current systematic study of the daily and used in our studies are erroneous. Rinand seasonal ACTH plasma secretion iker er al. (1972) have found four anomrhythms in the frog during the hibernation or sexually active periods may point out alies in the 25-31 sequence of these polyimportant variations. For, in man, the peptides. Though the new molecule (h plasma ACTH rate varies throughout the ACTH) synthetized by Sieber et ul. ( 1972) has the same biological properties as that day (Binoux er al., 1965; Berson et al., used as a tracer in our study (Tesser et ul., 1968; Oliver, 1971), during the menstrual 1973; Schenkel-Hulliger et al., 1974), its cycle and pregnancy (Genazzani et al., properties may notably be 1973). Nycthemeral variations of the immunological ACTH rate have also been shown in the different. Nevertheless, the development of the rat (Matsuyama et al., 1971; Rees et al., radioimmunoassay has already 1971). In the same way, there is a cir- ACTH cadian rhythm of ACTH rate among the made possible the assessment, in the frog, of the hormone concentration in the whole hibernating wild animals (Hoo-Paris, adenohypophysis, in the pars distalis and 1971). in the pars intermedia (Vaudry et al., Moreover, an amphibian ACTH ra1974). dioimmunoassay in a heterologous system The assessment of the plasma ACTH may bring two types of errors. rate will shortly be useful when following We have noticed that the ACTH plasma the evolution of the corticotropic function concentrations do not vary linearly with under various experimental conditions the plasma dilutions in the incubation (stress, interrenalectomy, metyrapone intubes. According to Yalow et al. (1971), jections, total or partial hypophysectomy). this phenomenon is inherent to hetThus, the direct assessment of the erologous immunoassays. Yet, the presACTH rate, will give valuable indications ence of antigenic fragments in the samples which will supplement the numerous cytounder study or a structural heterogeneity logical and ultrastructural studies available in the plasma ACTH may also bring about on the amphibian pituitaries. the same variations. The plasma ACTH rate assessed during our tests cannot be taken as accurate figACKNOWLEDGMENTS ures, because the avidity of 22 B serum is We are indebted to Dr. R. Depieds, Laboratoires lower for frog ACTH than for porcine d’Endocrinologie, Facultk des Sciences Nord, MarACTH. It would, therefore, be desirable, seille, France, for his generous gift of serum 22 B, to to get an exact assessment of the ACTH Dr. P. A. Desaulles and Dr. W. Rittel, CIBA Lab-
RADIOIMMUNOASSAY oratoires, Base], Switzerland, and Dr. G. Fekete, Richter Laboratoires, Budapest, Hungary for their supplies of polypeptide hormones, to Dr. A. Capet and M. C. Simon for constructive technical assistance, and to J. Lecourt for secretarial assistance. The present studies were supported by grants from the Direction GCnerale de la Recherche Scientifique et Technique (73.7-1853).
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terrenalectomie. Etude cytoimmuno-chimique et ultrastructurale. Z. Zellforsch. 132, 334-346. DOERR-SCHOTT, J., AND DUBOIS, M. P. (1972b). Identification par immunofluorescence des cellules corticotropes et melanotropes de I’hypophyse des Amphibiens. Z. Zelljorsch. 132, 323-331. DONALD, R. A., MURPHY, S. S., AND NABARRO, J. D. N. (1968). The plasma corticotrophin response to insulin hypogiycoemia, lysinevasopressin and metyrapone in pigs. J. Endocrkol. 41, 509-5 18. ESPINER, E. A., BEAVEN, D. W., AND HART, D. S. (1963). Bioassay of corticotrophin using the sheep’s transplanted adrenal gland. ./. Endocrinol. 27, 267-268. GALSKOV, A. (1972). Radioimmunochemical corticotropin determination. Acta Endocrinol. 69 (Suppl.), 162 pp. GENAZZANI, A. R., FRAIOILI, F., FELBER, J. P., CONTI, C., AND FIORETTI, P. (1973). ACTH radioimmunoassay: Methodological problems and clinical applications. In “Symposium on Radioimmunoassay and Related Procedures in Clinical Medicine and Research,” (International Atomic Energy Agency, Eds.) Istanbul. GIRARD, F., BINOUX, M., AND PHAM-HUU-TRUNG (1966). MCthode d’estimation de I’activite corticotrope du plasma. Rev. Franc. C/in. Biol. 11, 732-742, HOO-PARIS, R. (1971). Hibernation et secretion corticotrope hypophysaire du Htrisson, Erinaceus europaeus L. Ann. Endocrinol. Paris 32, 743-752. ICHIKAWA, Y., NISHIKAI, M., KAWAGOE, M., YoSHIDA, K., AND HOMMA, M. (1972). Plasma corticotropin, cortisol and growth hormone responses to hypoglycemia in the morning and evening. J. C/in. Endocrinol. 34, 895-898. D’ISTRIA, M., DELRIO, G., AND CHIEFFI, G. (1973). Radioimmunoassay of testosterone estradiol 17 and estrone in plasma of males and females of Rana esclrlentu during reproductive cycle. In “7e Congr. Eur. Endocrinol. Comp.” (J. Szentagothai et F. Hajos, Eds.), p. 241. KITABCHI, A. E., SHARMA, A. K., AND WEST, W. H. ( 197 1). A sensitive bioassay method for ACTH using isolated rat adrenal cells. Horm. Metab. Res. 3, 133-134. LEE, T. H., LERNER, A. B., AND BUETTNERJANUSCH, V. (1961). On the structure of human cotticotropin. J. Biol. Chem. 236, 2970-2974. MATSUYAMA, H., RUHMANN-WENNHOLD, A., AND NELSON, D. H. (1971). Radioimmunoassay of plasma ACTH in intact rats. Endocrinology 88, 692-695. MCKEOWN, B. A. (1972). Prolactin and growth hor-
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mone concentration in the plasma of the toad Bufo bufo following ectopic transplantation of the pars distalis. Gen. Comp. Endocrinol. 19, 167-174. MIALHE, C., KOCH, B., BUCHER, B., AND BRIAUD, B. (1973). Etude de la secrktion corticotrope au tours de l’hypertrophie compensatrice de la surrknale. C. R. Acad. Sci. Paris 276, 589-592. OLIVER, C. (197 1). Le dosage radioimmunologique de l’ACTH plasmatique. Thkse, Marseille. PATENT, G. J., AND FOA, P. P. (1971). Radioimmunoassay of insulin in fishes, experiments in vivo and in vitro. Gen. Comp. Endocrinol. 16, 41-46. PROESCHEL, M. F., COURVALIN, J. C., DONNADIEU, M., BINOUX, M., AND GIRARD, F. (1974). Preparation and evaluation of ACTH antibodies. Acta Endocrinol.
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S. S., AND BETHUNE, J. E. (1969). Radioimmunoassayable ACTH in dexamethasone nonsuppressible Cushing’s syndrome. J. C/in. Endocrinol. Metab. 29, 1231-1237. REES, L. H., RATCLIFFE, J. G., KENDALL, J. W., KNIGHT, R. A., ALLEN, C., AND COOK, D. M. (1971). A radioimmunoassay for rat plasma corticotrophin. J. Endocrinol. 51, 3-4. ROSSELIN, G., ASSAN, R., YALLOW, R. S., AND BERSON, S. A. (1966). Separation of antibody bound and unbound peptide hormones labelled with iodine- 13 1 by talcum powder and precipitated silica. Nature (London) 212, 355-358. RINIKER, B., SIEBER, P., RITTEL, W., AND ZUBBER, H. (1972). Revised amino-acid sequences for porcine and human adrenocorticotrophic hormone. Nature New Biol. 235, 114-115. SAYERS, G., SWALLOW, R. L., AND GIORDANO, N. D. (1971). An improved technique for the preparation of isolated rat adrenal cells: a sensitive accurate and specific method for the assay of ACTH. Endocrinology 88, 1063-1068. SCHENKEL-HULLIGER, L., MAIER, R., BARTHE, P. L., DESAULLES, P. A., JARRET, A., RINIKER, B., RITTEL, W., AND SIEBER, P. (1974). Biological activity of synthetic human corticotrophin with RAYYIS,
ET AL. revised
amino
acid
sequence.
Acta
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