Applicationof a Sequence ... - Semantic Scholar

CLIN. CHEM. 27/4,

549-552

(1981)

Applicationof a Sequence-SpecificRadioimmunoassayfor the CarboxylTerminal Regionof Corticotropin Lawrence H. Lazarus,1 Richard P. DiAugustine,’ Masood N. Khan,2 Gloria 0. Jahnke,’ and Michael D. Erisman3 The carboxyl terminal region of corticotropin

(ACTH), (Ala34-Phe-Pro-Glu-Leu-Phe39), a region of the hormone conserved during evolution, served as an antigen for the production of a sequence-specific antiserum. In a radioimmunoassay, peptides that extend toward the amino terminal from Ala34, such as [Tyr,GlyJ-ACTH3439, ACm 1839, and ACTH, had greater affinity for the antibody, which suggests that the antiserum recognizes the peptide bond preceding the alanyl residue. The assay readily detects 30 to 50 pmol of ACTH per liter with an incubation of only 3 h, and the antiserum cross reacts with larger molecular

mass forms of the hormone.

The amount of

immunoreactive ACTH extracted by adsorption onto silicic acid from rat and human plasma was only 0.36 to 0.79 of that detected by a mid-region ACTH assay, which suggests proteolytic degradation at the carboxyl terminus of ACTH. AddItional Keyphrases:

corticotropin

in plasma and pituitary

peptide hormones Radioimmunoassays represent a diagnostic tool in the quantitation of polypeptide hormones isolated from biological tissues, including blood (1-3). Although the antisera used in these assays are commonly obtained by the use of the intact hormone as the immunogen, the technique may be used to produce antisera with different or multiple specificities. In the case of ACTH,4 the antibodies could cross-react with a-MSH and CLIP as well as other regions of the molecule that persist or vary evolutionarily. Thus, the heterogeneity of antibody specificities remains a major factor in the reproducible quantitation of hormones by different laboratories (3). One convenient method to alleviate this problem involves the use of small peptides coupled to larger immunogenic proteins (4,5); in this way, recognition of the hormone is potentially limited to one specific sequence. To obtain antibodies specific for the carboxyl terminal region of ACTH, we used the amino acid sequence of ACTH84-9, an evolutionarily conserved region of the hormone (6), in preparation of antiserum. We present data on the applicability of this antiserum for detecting ACTH in plasma of rats and humans for detecting larger forms of this hormone. Endocrinology Group, Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709. 2 Strathcona Medical Building, McGill University Clinic, Montreal, Quebec, Canada H3A 2B2. Department of Chemistry, Bloomsburg State College, Bloomsburg, PA 17815. Nonstandard abbreviations used: ACTH, corticotropin; CLIP, corticotropin-like intermediary peptide (ACTH’89); 13-LPH, f3-lipotropin; a- or $-MSH, a- or fi-melanotropin; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; ACTH’-1#{176} or ACTH’24, amino-terminal regions of ACTH; h-orb-, human or bovine source, respectively. Received Dec. 9, 1980; accepted Jan. 27, 1981. 1

Materials and Methods Peptides and Chemicals The synthetic ACTH sequence 1-24 (Cortrosyn) was donated by Organon, Inc., West Orange, NJ 07052; synthetic human ACTH was a gift of Ciba-Geigy Ltd., Base!, Switzerland; ACTH’’#{176}, ACTH4’1, ACTH3439, and [Tyr,GIyJACTH339 were from Bachem, Inc., Torrance, CA 90505. J. E. Rivier (The Salk Institute for Biological Sciences, San Diego, CA 92138) synthesized and supplied us with human (h) f3-MSH, bovine (b) i3-MSH, a-MSH, and hCLIP; C. H. Li (University of California, San Francisco, CA) generously provided b3-LPH. 1-Ethyl-3(3-dimethylaminopropyl) -carbodiimide, polyethylene glycol 6000, gamma-globulins, and BSA were from Sigma Chemical Co., St. Louis, MO 63178; Freund’s complete adjuvant from Calbiochem, San Diego, CA 92112; carrier-free Na1251 from New England Nuclear, Boston, MA 02118 or Amersham, Arlington Heights, IL 60005; and Sephadex LH-20 and G-75 from Pharmacia, Uppsala, Sweden. Polypropylene tubes, 12 X 75 mm, were used because ACTH avidly binds to both polystyrene and glass.

Biological

Samples

Blood from rats or humans was collected into polypropylene tubes surrounded by crushed ice and containing EDTA to give a concentration of 1 mg/mL (7). Plasma was obtained by centrifugation (1200 X g, 10 mm, 4 #{176}C). We added 60 mg of silicic acid (Bio-Sil A, 100-200 mesh; Bio-Rad Laboratories, Richmond, CA 94804) per milliliter of plasma, to adsorb ACTH (8), the mixture being allowed to stand for 30 mm at 4 #{176}C, then washed it with 5 mL of assay diluent and eluted with 5 mL of acetone/water/acetic acid (40/59/1 by vol). The acetone was evaporated in a stream of nitrogen and then either the samples were lyophilized or aliquots were diluted for immediate use in the assay. We extracted 1-g lots of powdered, lyophilized bovine pituitary glands by homogenization with a Polytron (Brinkmann Instruments, Westbury, NY 11590), using boiling 0.1 molfL acetic acid containing 5 g of 2-mercaptoethanol per liter, then boiling for an additional 10 mm according to Lee and Lee

(9). Procedures Production of antiserum. The peptide ACTH3439 was coupled to BSA by the carbodiimide method (10). The coupled peptide was dialyzed for 24 h, first against 4 L of 0.15 mol/L NaCl containing 28 mmol of 2-mercaptoethanol per liter (four changes), then against distilled water (two changes), as described previously (11). Thin-layer chromatography indicated that all the peptide was coupled to BSA. An equal volume of antigen was emulsified with adjuvant, and a total of 100 zg of coupled peptide was injected subcutaneously over 10 sites on the back of New Zealand White rabbits at four- to seven-week intervals. Jodincition. We iodinated the peptide [Tyr,Gly]-ACTH33#{176} by using Chloramine T as the oxidant (12) at a molar ratio of CLINICAL CHEMISTRY,

Vol. 27, No. 4, 1981

549

0 0 ‘C 0

m

N m

io’

io

Polypeptude,

pmol/L

Fig. 1. Specificity of the carboxyl-terminal region ACTH antiserum: cross reactivity of ACTH39 (a), [Tyr,Giy]-ACTH339 (#{149}), ACTH18-39(CLIP) (A), and ACTH (0) with the antiserum (1000-fold final dilution), as determined with use of 1251-Iabeled [Tyr,Gly] -ACTH34-39 peptide/iodide/Chloramine T of 1/2/50 and purified it on 0.7 X 4 cm Sephadex LH-20 columns in n-butanol/acetic acid/ water (10/2/1 by vol) (13). About 80% of the radiolabeled peptide could be precipitated with an excess of antiserum. ACTH1-24 was iodinated under similar conditions and purified by adsorption on silicic acid (14). Radioimmunoassay. Assays were done in duplicate or triplicate samples in a total reaction volume of 100 L. The final concentration of constituents of the assay diluent were, per liter, 50 mmol of potassium phosphate (pH 7.6), 25 mmol of NaC1, 30 mmol of 2-mercaptoethanol, 400 mega-int. units of aprotinin (an enzyme inhibitor), 2 g of BSA, and 1 g of gamma-globulins. To the 100-zL mixture we added 10 000 cpm of 1251-labeled [Tyr,Gly]-ACTH3439 and the carboxylterminal region antiserum (B-XII-161-3, 1000-fold final dilution). Standard peptides (ACTH and [Tyr,GlyJACTH3439), plasma, or extracts were diluted with this assay diluent. The tubes were incubated either at 22 #{176}C for 3 h or overnight (16 to 18 h) at 4 #{176}C, and the reaction was terminated by adding 0.1 mL of out-dated human plasma followed by 1 mL of a 156 g/L solution of polyethylene glycol in phosphate buffer (50 mmol/L, pH 7.6). We thoroughly mixed and centrifuged (4000 X g, 10 mm, 4 #{176}C) the samples, aspirated the supernate, and counted the radioactivity in the pellet. In assaying for the mid-region of ACTH (specific for residues 19 through 24) we used antiserum A-7 (40 000-fold final dilution) under similar conditions (Khan and DiAugustine, unpublished data). Column chromatography. Sephadex G-75 columns, 1.5 X 98 cm, were equilibrated in and eluted with 0.1 mol/L acetic acid containing 1 mmol of EDTA per liter. A 1.0-mL solution of extract clarified by centrifugation (9000 X g, 5 mm, 4 #{176}C) was applied to the column, and 0.96-mL fractions were collected at a flow rate of 30 mL/h. These fractions were lyophilized and redissolved in diluent before assay.

Results Specificity

of the Radioimmunoassay

The absence of a tyrosyl residue in ACTH34-39 prompted us to use the analog [Tyr,GlyJ-ACTH339 as the radioligand. In contrast to iodinated ACTH, this peptide did not appreciably adsorb onto glass or plastic surfaces; mean nonspecific radioactivity in 24 samples without antiserum or antiserum in the presence of 100 ng of peptide was 2.67% (SD 0.48%) of 550

CLINICAL

CHEMISTRY,

Vol. 27, No. 4, 1981

the total radioactivity. With peptide more than a year old, this background increased to 5.53% (SD 0.84%) (n = 11). As shown in Figure 1, a series of parallel displacement curves was obtained with ACTH, CLIP, [Tyr,Gly]-ACTH3439, and ACTH339, with 50% displacement for each peptide at 80,80, 160, and 600 pmol/L, respectively. No other ACTH-related peptides lacking the carboxyl-terminal hexapeptide (including a-MSH, fl-MSH, and 13-LPH at concentrations up to 10 smol/L) competed with the antibody in this assay. The least amount of ACTH detected with the antibody was I to 3 pg per tube. The within- and between-assay coefficients of variation (n = 7) for ACTH were 11 and 16% at 23 pg, and 8 and 12% at 41 pg, respectively. A proteolytic inhibitor, such as aprotinin (Trasylol), is needed in the diluent because of the relatively low titer of the antiserum. However, if other inhibitors (e.g., bacitracin, sodium azide, and thimerosal) are simultaneously present in the diluent, the binding of peptide to antibody is greatly reduced. Within three to six months we obtained large quantities of antiserum for use at 1000-fold final dilution. Continued reimmunization of the seven rabbits during six to 10 months failed to increase the titer of the antiserum, but rather led to an overall decrease in titer and sensitivity (i.e., the 50% displacement value) in all antisera.

Quantitation

of Plasma ACTH

Table 1 summarizes the results of the immunoassay of ACTH in rat and human plasma with use of antisera to the carboxyl-terminal region and antisera to the mid-region of ACTH. The difference in the amount of ACTH found by the two assays cannot be attributed to loss of ACTH during extraction from plasma; the recovery of hACTH extraneously added to outdated human plasma or to plasma from hypophysectomized rats was the same by both radioimmunoassays. Even in the case of ether- or methoxyflurane-exposed rats or adrenalectomized animals, in which plasma ACTH concentrations are significantly greater than in the controls, the carboxyl-terminal region assay detected only 0.36 to 0.79 as much as the mid-region assay. This also was true for assay results for four plasma samples from the authors, taken between 1500 and 1600 hours: the ratio between the two ACTH immunoassays ranged from 0.68 to 0.72. A 36-year-old woman (M.C.) who had undergone bilateral adrenalectomy for treatment of Cushing’s disease and who was on glucocorticoid replacement therapy (30 mg of hydrocortisone and 0.05 mg of 9-a-fluorocortisol per day) had above-normal ACTH concentrations by both assays, but in this case the ratio between carboxy-terminal and mid-region assays was 1.3. A male patient (K.M.) with small-cell carcinoma of the lung and Cushing’s syndrome exhibited about a fivefold increase in the mid-region assay, while the value as determined by the carboxyl-terminal region assay remained within the normal range (20-50 ng/L). These findings agree with a previous report of similar endocrine disorders in which concentrations of immunoreactive ACTH in individual plasma samples varied considerably with different region-specific assays (3).

Fractionation The carboxyl-terminal region assay cross reacts with ACTH from bovine pituitary (Figure 2). An immunoreactive peak representing material of relative molecular mass of 31 000 to 34 000, probably the pro-ACTH//3-LPH precursor (15), was readily detected in the column eluates. Furthermore, the pooled peak areas I (fractions 63-69), II (fractions 90-97), and III (fractions 111-120)-representing the pro-hormone, an

intermediate spectively-all

fragment

of the pro-hormone,

cross reacted

in a manner

and ACTH, re-

parallel

to the cross

Table 1. Quantitation of ACTH in Rat and Human Plasma ACTH, ng/L, mean (and SD) MId-regIon (MR) assay

Plasma8

Rats (n = 3) Normal Etherized Methoxyflurane Adrenalectomized

Sham adrenalectomized Humans Normal (n

=

COCH-t.rmlnal region (CT) assay

Ratio (CT/MR)

38.3 223 135 1364 49.3

(5.3) (61.1) (25.4) (203) (13.7)

19.3 177 64.3 495 26.7

(4.8) (85.6) (16.4) (88.8) (6.2)

0.50 0.79

48.8

(8.5)

34.4 226 30

(6.0)

0.70 1.31 0.11

0.48

0.36 0.54

4)

Patients M.C. (Cushing’s disease)

173

KM. (Cushing’s syndrome)

278

Analytical recovery of synthetic human ACTH (100 ng/L) added to out-dated human plasma or plasma from hypophysectomized rats ranged from 69 to 81% and 72 to 79%, respectIvely, by either immunoassay. Loss of ACTH reflects a variation in its adsorption and elution from the silicic acid and its adhesion even to plastic pipettes and tubes.

reactivity of [Tyr,Gly]-ACTH3439 (Figure tract also exhibited parallel displacement.

3). The crude

ex-

Discussion Accurate quantitation of the total concentration of a peptide hormone in biological fluids or tissues by use of a sequence-specific radioimmunoassay requires that the antibody cross react with the intact hormone, a subunit, a fragment containing that sequence, and the precursor form of the hormone. Our immunoassay meets all these criteria. Thus, it is evident that (a) only the specific sequence ACTH34-39 cross reacts with the antiserum, and (b) peptides with sequences which extend from the amino-terminal residue Ala34 of that peptide exhibit enhanced displacement of the label. Because the peptide is coupled to BSA through the free amino group of Ala34, the antibody probably recognizes the peptide bond between Ala34 and a carboxyl group in BSA. If the antibody

recognized a peptide coupled through the carboxy terminus Phe39, we would not observe the enhancement in displacing the radioligand by ACTH or [Tyr,Gly] -ACTH.t439 The range of values we found for immunoreactive ACTH in plasma from normal subjects is in accord with those from other clinical or laboratory studies (2,3, 7, 14, 16). However, in plasma from patients with Cushing’s syndrome (ectopic ACTH syndrome), Orth et al. (3) found that their “NH2-terminal” ACTH assay (with a specificity similar to our midregion assay) gave lower values for ACTH when compared with their assay of the “extreme” carboxyl terminal (of unknown specificity, but estimated to be somewhere in the region of residues 25 to 39). Thus, a comparison between the values quantitated for ACTH among different laboratories in which the precise specificity of the antisera is suspect must be interpreted with caution. This is particularly important in evaluating the degradation of ACTH in blood or other tissues because major proteolytic cleavage sites seem to be both the amino- and carboxyl-termini (17). Sample volu!ne(ML) 10

0I

C

tO

0 (.1

2 9c C

81

5’

>

7C

SC

V

0 5 I-

0

-03C

C

E

-

E

2( IC

Fraction

FIg. 2. Chromatogram of Sephadex 0-75 of extracts of bovine pituitary gland extracts The 1.5 X 98cm column was eluted wIth 0.1 mol/L acetic acid containing EDTA (1 mmol/L). The extract (1.0 mL) was applIed and 0.96-mL fractions were collected at a flow rate of 30 mL/h. The arrows indIcate relative molecular mass markers: 1, Blue Dextran (marks void volume); 2, ovalbumin (M, 45 000); 3, cytochrome c (M, 12 400); 4, bovine Insulin (M, 6612); and 5, vItamin B12 (M, 1355)

10

i05 Peptide (p9)

10’

Fig. 3. Cross reactivity of the extracts of bovine pituitary and regions from the Sephadex 0-75 column fractionation (Fig. 2) lmmisoreactive peptides: 0, the original acetic acid extract V. pooled area I (cokiwu fractIons 63-69, representIng the Pro-ACTH/f3.UH regIon); V, pooled area II (column fractions 90-97 are the Intermediate14 fragments of the proACTH/9-t.PH molecule); , pooled area Ill (coiumn fractIons 111-120 are ACTH); and#{149} [Tyr,Giy)-AcTH39 standard CLINICAL CHEMISTRY,

Vol. 27. No. 4, 1981

551

Our sequence-specific

investigator

and clinician

radioimmunoassay

the knowledge

offers

both

the

that the material

recognized in the assay is strictly limited to the 34-39 region of ACTH. Furthermore, the relatively short time required for this assay (3 h) makes practicable its use in routine clinical analyses of plasma samples for diagnosis of pituitary function or ectopic ACTH-producing tumors. We thank the following individuals for their technical assistance: J. Cohen, S. Ellison, R. Khan, T. Masagee, M. Schwartz, and C. Soldato. We also thank Dr. D. Ontjes (University of North Carolina School of Medicine, Chapel Hill, NC 27514) for the samples of blood from patients with Cushing’s disease and syndrome.

References 1. Yalow, R. S., Glick, S. M., Roth, J., and Berson, S. A., Radioimof human plasma ACTH. J. Clin. Endocrinol. Metab. 24, 1219-1225 (1964). 2. Marton, J., Stark, E., Meretey, K., and Schuister, D., Evaluation of a radioimmunoassay for plasma adrenocorticotrophin. J. Endocrinol. 78, 309-319 (1978). 3. Orth, D. N., Nicholson, W. E., Mitchell, W. M., eta!., Biologic and immunologic characterization and physical separation of ACTH and ACTH fragments in the ectopic ACTH syndrome. J. Clin. Invest. 52, munoassay

1756-1769

(1973).

4. Orth, D. N., General considerations for radioimmunoassay of peptide hormones. Methods Enzymol. 37, 22-38 (1975). 5. Segre, G. V., Tregear, G. W., and Potts, J. T., Jr., Development and application of sequence specific radioimmunoassays for analysis of the metabolism of parathyroid hormone. Methods Enzymol. 37,38-66 (1975).

6. Orth, D. N., Adrenocorticotropic hormone and melanocyte stimulating hormone (ACTH and MSH). In Methods of Hormone Radioimmunoassay, B. M. Jaffe and H. R. Behrman, Eds., Academic Press, New York, NY, 1974, pp 125-159.

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7. Krieger, D. T., Liotta, A. S., Suda, T., et al., Human plasma immunoreactive lipotropin and adrenocorticotropin in normal subjects and in patients with pituitary-adrenal disease. J. Clin. Endocrinol. Metab. 58, 566-571 (1979). 8. Vaudry, H., Oliver, C., Ustegui, R., et al., Adsorption of polypeptide hormones to silicate powders: Development of reproducible methods for extracting ACTH and MSH from plasma. Anal. Biochem. 76, 281-291 (1976). 9. Lee, T. H., and Lee, M. S., Purification and characterization of high-molecular-weight forms of adrenocorticotropic hormone of ovine pituitary glands. Biochemistry IS, 2824-2829 (1977). 10. Goodfriend, T. L., Levine, L., and Fasman, G. D., Antibodies to bradykinin and angiotensin: A use of carbodiimides in immunology. Science 144, 1344-1346 (1964). 1!. Lazarus, L. H., and DiAugustine, R. P., Radioimmunoassay for the tachykinin peptide physalaemin: Detection of a physalaemin-like substance in rabbit stomach. Anal. Biochem. 107, 350-357 (1980). 12. Hunter, W. M., and Greenwood, F. C., Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature 194, 495-496 (1962). 13. Lazarus, L. H., Perrin, M. H., and Brown, M. R., Mast cell binding of neurotensin: I. lodination of neurotensin and characterization of the interaction of neurotensin with mast cell receptor site, J. Biol. Chem. 252,7174-7179 (1977). 14. Rees, L. H., Cook, D. M., Kendall, J. W., et al., A radioimmunoassay for rat plasma ACTH. Endocrinology 89, 254-261 (1971). 15. Eipper, B. A., and Mains, R. E., Structure and biosynthesis of pro-adrenocorticotropin/endorphin and related peptides. Endocr. Rev. 1, 1-27 (1980). 16. Lefkowitz, R. J., Roth, J., and Pastan, I., Radioreceptor assay of adrenocorticotropic hormone: New approach to assay of polypeptide hormones in plasma. Science 170, 633-635 (1970). 17. Bennett, H. P., and McMartin, C., Peptide hormones and their analogues: Distribution, clearance from the circulation, and inactivation in vivo. Pharrnacol. Rev. 30, 247-292 (1979).