Preparation and assessment of antisera to ACTH

Molecular and Cellular Endocrinology, 8 (1977) 213-223 0 Elsevier/North-Holland Scientific Publishers Ltd.

PREPARATION

AND ASSESSMENT OF ANTISERA

TO ACTH

J. MARTON *t, Katalin MERETEY, E. STARK and D. SCHULSTER Institute of Experimental Medicine, Hungarian Academy of Sciences and National Institute for Rheumatism, Budapest, Hungary, and Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton BNI 9QC, U.K.

Received 9 March 1977; accepted 13 May 1977

Antisera to ACTH were produced in rabbits injected repeatedly at multiple intradermal sites with synthetic [Aspz5, Ala26, Gly2’]~h-corticotropin-(l-28)-octacosapeptide-bovinegamma globulin conjugate (octacosapeptide is a sequence analogue of a#,-Za-ACTH). Antibodies to extracted human or porcine ACTH were detected in all of the sera 1 month after immunization. A considerable proportion of the antisera obtained from a single final bleeding 5 months after the primary immunization were suitable for sensitive radioimmunoassay. The antisera were shown to neutralize the steroidogenic activity of ACTH in an isolated rat adrenal cell bioassay system. Titres estimated from antiserum dilution curves and relative avidities from the standard curves were compared. It was possible to detect picogram amounts of ACTH in plasma-free medium with the best antisera. The method described is an effective means of producing antisera to the weakly immunogenic N-terminal fragment of the ACTH molecule. Keywords:

antisera to ACTH; radioimmunoassay.

It is generally accepted that the assay of adrenocorticotrophic hormone (ACTH) is one of the most difficult among radioimmunoassays for polypeptide hormones. There have been several reports on radioimmunoassays for ACTH (Felber, 1963; Yalow et al., 1964; Demura et al., 1966; Berson and Yalow, 1968; Landon and Greenwood, 1968; Orth et al., 1968; Rees et al., 1971; Matsukura et al., 1971; Galskov, 1972) and on the production and characterization of antisera to ACTH for use in radioimmunoassay (McGuire et al., 1965; Imura et al., 1965, 1967; Felber et al., 1966; Fleischer et al., 1966; Gelzer, 1968; Felber and Aubert, 1969; Aubert and Felber, 1969; Voigt et al., 1971; Rose and Newsome, 1972; Proeschel et al., 1974). The raising of suitable antisera to ACTH is still a considerable problem, inasmuch as the low circulating level and rapid degradation of the homone call for high avidity antisera to a weak immunogen. * IAEA fellow for 1975-1976 at Sussex University, where part of this work was carried out. t To whom requests for reprints should be directed. Present address: II Department of Anatomy, Semmelweis University Medical School, Tiizolto u. 58, 1094 Budapest, Hungary. 213

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et al.

Discrepancy between estimates of ACTH content of biological samples obtained by radioimmunoassay and bioassay (Yalow et al., 1964; Fleischer et al., 1965; Sparks et al., 1965; Besser et al., 1971) and systematic comparison of the bioactivity of ACTH fragments and their immunorea~ti~ty with different antisera (Imura et al., 1965, 1967; Fleischer et al., 1965, 1966; Felber et al., 1966; Aubert and Felber, 1969) have led to the conclusion that the structural requirements for the immunological reactivity are not necessarily identical with those for the biological activity of ACTH. Since the refationship between the chemical structure and biological activity of the ACTH molecule has been well defined (Ney et al., 1964; Lebovitz and Engel, 1964) and the N-terminal 24 amino acid sequence is known to possess the complete steroidogenic activity of the whole molecule, dissociation of the biological activity and immunoreactivity would be expected to be minimized with antisera directed against the bioactive N-terminal portion of the molecule. Though antisera have been raised by immunizing with an N-terminal fragment, it is generally accepted that the cr-corticotropin-(l-24)tetracosapeptide (o’-24-ACTH *) and ~Y’-‘~-ACTH, while having full steroidogenic activity, appear to be distinctly less immunogenic than the natural ACTH (Axelrod et al., 1963; Felber et al., 1966; Gelzer, 1968; Proeschel et al,, 1974). Almost all attempts to improve the immunogenicity of the ACTH by coupling to carrier proteins, or by adsorbing the polypeptide to charcoal (McGuire et al., 1965; Gelzer, 1968; Reichlin et al., 1968; Landon and Greenwood, 1968; Rayyis and Bethune, 1969; Voigt et al., 1971; Rose and Newsome, 1972; Proeschel et al., 1974) were either done with the whole ACTH molecule or were unsuccessful. In this study the preparation and assessment of antisera to [Asp2’, Ala26, Gly27]~~~2s-ACTH bovine gamma globulin conjugate with high titre, avidity and specificity is described.

MATERIAL

AND METHODS

Chloramine T, bovine gamma globulin, lactoperoxidase, 1-ethyl-3-(3-diethylaminopropyl~arbodiimide hydrochloride (CDI) were purchased from Sigma Chemical Co. Ltd., London. Na’2SI (Radiochemical Centre, Amersham, Bucks., U.K.), Ala26, Gly27] XX+, ‘-*‘-ACTH (Gedeon Richter Ltd., Budapest, Hungary), [AspZS, collagenase (P-L Biochemicals Inc., Milwaukee, Wis., U.S.A.), Vycor glass granules No. 7930 (Corning Glass Works, Corning, N.Y.), Metyrapone (CIBA-Geigy, Horsham, U.K.) and Freund’s complete adjuvant (Calbiochem Ltd., Hereford, U.K.) were used. on-ACTH, International Working Standard for Corticotrophin (3rd IWS), * The rules of the IUPAC-IUB Commission on Biochemical Nomenclature for naming synthetic modifications of natural peptides (cf. Biochemistry 6, 362-364 (1967)) were followed; however, for the sake of brevity the nomenclature of Li (1959) is employed in this paper.

Antisera to ACTH for radioimmunoassay

215

was obtained from the WHO International

Laboratory for Biological Standards, London. (u,-ACTH (Dr. P.J. Lowry, St. Bartholomew’s Hospital, London), c$,-““ACTH ai-‘4-ACTH (Y;‘-~~-ACTH, @-MSH (Dr. K. Medzihradsky, Eotviis University, Budapest, Hungary) and the Bordetella pertzwis vaccine (Dr. Z. Csiszer, Natl. Inst. Serobact. Prod. Res., HUMAN, Budapest, Hungary) were generous gifts. ~oducti~n ofantisera to ACTH Synthetic [ASHES, Ala26, GIy27]~~-28 -ACTH was coupled to bovine gamma globulin with CDL according to the procedure of Goodfriend et al. (1964), with minor modification. [Asp*‘, Ala26, Giy27]-cu~‘2s-ACTH (6 pmol in 0.3 ml of 0.15 M NaCl, pH 4) was mixed with bovine gamma globulin (0.13 Fmol in 0.3 ml of 0.05 M phosphate buffer, pH 7.4, and 0.15 M NaCl) in a siliconized glass tube. The tube was protected from light by aluminium foil and the reaction was started by the addition of CD1 (1 mmol in 0.4 ml of distilled water) and was left to proceed for 1 h at room temperature with occasional gentle agitation. The reaction mixture was then diluted to 5 ml with distilled water and dialysed at 4°C for 24 h. In a pilot study using [ ‘2sI]ACTH to follow the coupling reaction, about 70% of the radioactivity was found in the conjugate after dialysis. The dialysed conjugate was used shortly after preparation or was stored in aliquots at -20°C. The injection procedure was similar to that described by Meretey and Kocsar (1974). The conjugate was diluted with isotonic saline, Metyrapone was added and thoroughly emulsified with equal volumes of complete Freund’s adjuvant. 17 New Zealand white rabbits were injected at multiple intradermal sites on the back, each with 2 ml emulsion containing approx. 0.7 mg [Asp2’, Ala26, Gly27]-o.~-2S-ACTH as bovine gamma globulin conjugate and 100 mg of Metyrapone. Bordetella pertussis vaccine (2 X 10’ bacteria) was given intravenously at the same time. Two booster injections of the same immunogen were given at multiple intradermal sites at 2 month intervals. The antisera were obtained 4 weeks after the final booster, when the animals were anaesthetized with pentobarbital and killed by exsanguination. A group of untreated rabbits of the same age, sex and body weight were killed and served as controls. The adrenal and pituitary glands were excised and weighed and serial sections were studied with routine histology. Merthiolate (0.01%) was added to the sera which were stored in aliquots at -20°C. Repeated thawing and freezing was avoided. Iodination of ACTH and radioimmunoassay 2 pg of synthetic o/r-39-ACTH or highly purified natural ACTH in 10 ~10.005 N HCl was iodinated with Na12’ I (1 mCi) using the chloramine T (Greenwood et al., 1963; Berson and Yalow, 1968) or the lactoperoxidase method (McIlhinney and Schulster, 1974). The [l*’ I]ACTH was separated from the free iodide and damaged components by selective adsorption to Vycor glass granules (Ratcliffe and Edwards, 1971; Rees et al., 1971). The steps of purification were followed by chromatoelectrophoresis on Whatman 3 MM paper (Berson and Yalow, 1968), and the purified

J. Mwton

216

et al.

labelled hormone eluted from the adsorbent was stored at 4°C and could be used for up to 2 weeks. The level of substitution was about 0.8-0.9 atom of r*‘l per molecule of ACTH. The mean specific radioactivity of the tracer was 432 * 35 LtCi/ @g and the fractional distribution of radioactivity between [‘251]ACTH, damaged labelled hormone and free ‘251 assessed by chromatoelectrophoresis was 0.79 i: 0.06, 0.06 It 0.02 and 0.15 + 0.07 (mean + SD) on 10 consecutive iodinations. Details of the radioimmunoassay procedure are reported elsewhere (Marton et al., 1977). The method of Rodbard and Lewafd (1970) was used for the analysis of rad~oimmunoassay data.

The method is based on the measurement lagenase disaggregated isolated rat adrenal (~chardson and Schulster, 1972).

of corticosterone production by colcells and has been described earlier

CORTICOSTERONE w/ml/hour

vi5 I.U.

ACTH

f

,a” ml

ADRENAL

1;;” CELL

9;’ SUSPENSION

Pig. 1. Steroidogenic activity of ACTH (3rd IWS) on isolated rat adrenal cells after preincubaor an anti-ACTH (o-- - - - -0) tion of the hormone for 24 h at 4°C with a normal (0~) rabbit serum. Aliquots of the preincubates were incubated with IO5 cells for 4.5 min at 37°C and corticosterone production was measured. Preincubation was carried out with 100X final dilution of the sera.

Antisera to A CTH for ~ad~airnrn~n~ssa~

217

RESULTS 11 final serum samples were obtained, as 6 of the 17 animals given the primary immunization died during the course of the studies. The adrenal weight of the immunized rabbits (552 +- 50 mg) was significantly higher (I’< 0.001) than that of nonimmunized controls (324 + 22 mg) of the same body weight. This increase in adrenal weight is due to the hypertrophy of the cortex; the width of the cortex (2613 t 109 pm) or zona fasciculata (1653 +-74 p) at the largest cross-section of the gland from the immunized animals was signi~cantly Iarger (P
r”

1/rsoooo

855 857 647.

1/500000

840 853 842 1 654‘

846

l/35000

a51

850

1/30000

043

1 1/100000

I-

I

I

I

I

l/25000

1 / 3000

I

1/20000 1/12000

i-

,-

I-

t-

I

I

10-b

10-5

RECIPROCAL

I

10-4 OF THE DILUTION

I

10-3

1

10 T

FACTOR

Fig. 2. Antiserum dilution curves and extrapolated titres of the antisera. Increasing dilutions of a normal rabbit serum (NS) and different antisera (842...857) were incubated with 10 pg [ t2%]ACTH at 4°C for 24 h in a total volume of 250 ~1. The bound-over total radioactivity is plotted against the reciprocat of the dilution factor. Titre: reciprocal of the dilution factor, at which 50% of the added standard amount of tracer is bound.

218

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Marton

et al.

vious macroscopic or histological difference was observed between the immunized and control groups. Antibodies to ACTH were present in all the 17 rabbit sera 4 weeks after the primary immunization, and no binding of the labelled hormone was detected using normal rabbit sera. The steroidogeni~ activity of uniabelled ACTH was neutralized by the immune sera. In fig. 1 the corticosterone production of isolated adrenal cells is shown. The response to increasing doses of ACTH preincubated with an antiserum was significantly less than in the controls, where the hormone was preincubated with a normal rabbit serum. The inhibition of corticosterone production indicates.the loss of steroidogenic activity on the formation of the ACTH-antibody complex. Screening and selection of the antisera was performed according to titre, avidity and specificity of the ACTH-antibody binding. Antiserum dilution curves are shown in fig. 2. 10 pg of [ ‘*‘I] ACTH was incubated for 24 h with increasing dilutions of a normal serum and different antisera. The titre of the antiserum as usually defined is the reciprocal of the dilution factor at which 50% of the added constant amount of labelled hormone is bound. The titres of the antisera varied from l/3000

0.6 -

651

650

ANT,-[ASP*~,

647

a4a

ALA26,

a55 GLV*‘]

64 -cx;-*~-ACTW

RABBIT

SERUM

Fig. 3. Comparison of relative avidity of the different antisera. The effect of 250 pg/tube 3rd IWS ACTH on the binding of [ 12SI]ACTH (B*so/Bo; mean t SD) is taken as an index of avidity. An appropriate dilution of each antiserum resulting in about a 50% binding of the total radioactivity (10 pg[ 1251]ACTH) added in the absence of the standard was used (incubation volume 250 gl, 48 h at 4°C).

Antisera to ACTHJor ra~ioi~~~~loassa~

219

to l/l 50,000. All of the antisera bound the [‘251]ACTH at a 1 in 3000 or higher dilution and the binding varied as a function of the dilution. No dilution-dependent binding was seen with the normal rabbit serum. The avidity of different antisera is compared in fig. 3. Displacement of 10 pg [‘251]ACTH by 250 pg standard ACTH is compared at a dilution of the different antisera giving 50% binding of the tracer in the absence of standard ACTH. Displacement under these conditions varies from about 55% to 10% and the antisera can be divided broadly into two groups, those with higher and those with lower avidity. A similar distribution of the avidity of the antisera can be seen in fig. 4, where in a similar experiment complete standard curves were compared. The detection limit of the assay with the more sensitive antisera is about 10 pg while that of the less sensitive ones is about 90 pg. The sensitivity of the assay is defined as the least amount of standard hormone which yields a response which can be significantly distinguished from the zero or blank response. The most avid antisera seem to be suitable for the measurement of resting plasma ACTH levels after preliminary extraction of the hormone, which - apart from a few exceptions (Berson and Yalow, 1968; Matsukura et al., 1971; Galskov, 1972) - is required in most ACTH

10

100

1000

pg

ACTH

10000

/TUBE

Fig. 4. Comparison of the relative avidity of different antisera. Standard curves of bound radioactivity/bound radioactivity in the absence of standard ACTH (B/Be) vs. log ACTH concentration (10 pg [ 1251]ACTH, 250 ~1 incubation volume, 48 h at 4°C dilution of antisera; cf. fig. 3). Inset: Iogit-log plot of the same data.

J. Marton et al.

220 1

I

I

I

I

1.0 -

or, 1

1

I

I

I

10

100

1000

10000

fm&a

POLY PEPTIDE

I

1 100000

TIJSE

Fig. 5. Antigen specificity of a selected antiser~ltn (847). Binding of porcine ACTH (3rd 1WS) and related polypeptide fragments to the anti-[Asp25, Ala26, Giy27]*~-28-ACTH serum. B/B0 (see legend to fig. 3) vs. log polypeptide concentration (10 pg [ t2%fACTH, l/60,000 final dilution of the antiserum), 250 ~1 incubation volume, 48 h at 4°C). The residual variance for the weighted regression indicating the ‘goodness of fit’ was between 0.5 and 2.0, i.e. within the acceptable range (Rodbard and Lewald, 1970).

radioimmunoassay techniques (Yalow et al., 1964; Demura et al., 1966; Orth et al., 1968; Landon and Greenwood, 1968; Rees et al., 1971; Genazzani et al., 1974; Horgan and Riley, 1974; Voigt et al., 1974; Usategui et al., 1976). Fig. 5 shows the antigen specificity of one selected antiserum. Neither P-MSH nor Lyi5-39-ACTH cross-reacted, even at high concentrations. ~ompa~ng equimolar concentrations parallel in~bition curves were seen - in decreasing order of potency - with the [Asp2’, Ala26, Gly27]ly~~28-ACTH, porcine 3rd IWS ACTH and ~r’-‘~ACTH polypeptides.

DlSCUSSlON Antibodies to ACTH were demonstrated in all of the rabbits within 4 weeks after the primary immunization with [ASPIC, Ala26, Gly27]~~~2s-ACTH-bovine gamma globulin conjugate. Antisera of considerable avidity and high titre were obtamed in 6 out of 17 animals. This is an acceptable proportion and a relatively short period for producing suitable antisera for radioimmunoassay. Data on the immuno-

Antisera to ACTH for radioimmunoassay

221

genicity of the different ACTH-protein conjugates are controversial. While or,ACTH-rabbit serum albumin (McCuire et al., 196.5) an-ACTH-human gamma globulin (Rose and Newsome, 1972) and cY’-24-ACTH-bovine serum albumin (Orth, 1974) have been described as potent, others including cY1-24-ACTH-bovine serum albumin (Proeschel et al., 1974) have poor immunogenicity. The finding of adrenocortical hypertrophy in the immunized rabbits at autopsy may reflect sustained higher plasma ACTH levels and most probably shows that the circulating antibodies cannot effectively neutralize the excess hormone in the circulation. As there was no obvious relationship between the degree of hypertrophy and quality of the antiserum produced, the only relevant inference may be that a concomitant sustained higher glucocorticoid secretion may have an adverse influence on the antibody production. Metyrapone treatment has been used by Berson and Yalow (1968) and Galskov (1972) to prevent the stimulation of corticoid secretion during immunization with ACTH. The failure of Axelrod et al. (1963) to raise antibodies to (Y’-*~-ACTH and corresponding ACTH analogues of similar amino acid sequence, but lacking the steroidogenic activity, may reflect the importance of the inhibition of glucocorticoid hypersecretion during the immune response. Demonstration in vitro of the neutralization of the steroidogenic activity of extracted natural porcine ACTH in the isolated adrenocortical cell bioassay system serum is interpreted as by the anti-[Asp*‘, Ala26, Gly*‘]+~~~*~ -ACTH-immune evidence that the antibody combines with the N-terminal region of the molecule, i.e. the sequence required for steroidogenic activity. Previous studies with different antisera (Fleischer et al., 1965, 1966; McGuire et al., 1965; Galskov, 1972) have also shown neutralization of steroidogenic or lipolytic activity of the hormone. In the screening and selection of antisera the traditional approach was used. An approximate value of serum titre - a dilution suitable for assay - was obtained from the antiserum dilution curves. Using this dilution and a constant amount of unlabelled standard ACTH the relative avidity of a number of antisera could be compared in a single experiment. The reliability of the approach as a practical criterion for selection is indicated by a similar order of relative avidities when complete standard curves set up with the different antisera on different days were compared. No significant correlation was found between titre and avidity of the antisera. Comparison of equilibrium constants obtained by Scatchard (1949) analysis was adopted by Proeschel et al. (1974) as an alternative approach to the selection of anti-ACTH sera. The underlying assumption of the analysis in all binding studies is that the labelled and unlabelled hormone have the same affinities for the binding sites. We have provided experimental evidence (Marton and Cooper, 1977) that there is a considerable difference between the equilibrium constant of [‘*‘I]ACTH and unlabelled ACTH for several of our antisera. As the simple experimental approach outlined above for obtaining valid estimates of the binding parameters is not applicable in this case (Chang et al., 1975; Marton and Cooper, 1977) for practical purposes the equally reliable comparison of relative avidities was chosen.

222

J. Marton

et al.

Previous failure in raising antisera by immunizing with the N-terminal fragment (Axelrod et al., 1963) may probably not be due to the lack of immunogenicity of the peptide, but perhaps to other conditions of the immunization protocol. It seems, indeed, that if an N-terminal or C-terminal specific antiserum to ACTH is needed, immunization using that fragment would increase the chance of obtaining the specific antiserum more often and also perhaps its affinity to that particular fragment. The method described appears to be an effective means of producing avid antisera to the small, weakly immunogenic N-terminal fragment of the ACTH molecule.

ACKNOWLEDGEMENTS We are grateful to Dr. M. Palkovits for the histological investigation Mrs. M. Vallent and Mr. J. Horvath for excellent technical assistance.

and thank

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