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Characterization and application of a radioimmunoassay for β-endorphin using an antiserum with negligible cross-reactivity against β-lipotropin
Regulatory Peptides, 5 (1982) 65-75
65
Elsevier Biomedical Press
Characterization and application of a radioimmunoassay for fl-endorphin using an antiserum with negligible cross-reactivity against fl-lipotropin M a r g a r e t a B r a m n e r t , R o l f E k m a n , I n g v a r L a r s s o n a n d J a n I. T h o r e l l Department of Endocrinology, and Department of Nuclear Medicine, Lund University, MalmiJ General Hospital, Malmo, Sweden (Received 28 June 1982; accepted for publication 22 September 1982)
Summary High avidity antisera against fl-endorphin (flh-EP) were obtained in two of five rabbits immunized with unconjugated synthetic human flh-EP. One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with fl-lipotropin (flh-LPH) and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/1. The cross-reaction with methionine-enkephalin (flh-LPH 61-65) was 9%, while that with a-endorphin (flh-LPH 61-76) was 69%. This implied that the specific recognition site was in the amino-terminal region of flh-EP. Although this sequence is present in flh-LPH it was poorly recognized by the antiserum, suggesting that the free anainoterminal is essential. This interpretation was supported by the finding that a-Nacetyl-flh-EP was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/1, flh-EP was not detectable ( < 3 pmol/l) in 26 of 27 extracted plasma samples in healthy blood donors, in one it was 5 pmol/1. In five of six patients with an enlarged sella turcica, but without clinical and laboratory evidence of pituitary dysfunction, flh-EP was detectable (5 + 3 pmol/l; mean _+ S.D.) after metyrapone, flh-EP was elevated in Addison's disease (23, 54 and 76 pmol/l), Nelson's syndrome (37, 39 and 109 pmol/1), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushing's disease (7 pmol/1). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human flh-EP. The specificity of the antiserum K-7762 was such that the flh-EP concentration in plasma
Address for correspondence: Dr. Margareta Bramnert, Department of Endocrinology, Lund University Clinics, Malm8 General Hospital, S-214 01 MaimS, Sweden. Telephone 46 40 332329. 0167-0115/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
66
extracts could be reliably estimated by radioimmunoassay without prior chromatography. fl-endorphin; fl-lipotropin; antisera; plasma extracts; cross-reactivity
Introduction
fl-Endorphin (flh-EP), the carboxy-terminal 31 amino acid sequence of fl-lipotropin (flh-LPH), is generated by cleavage from the pro-opiomelanocortin precursor with flh-LPH as an anticipated intermediate [1,2]. Most antisera against flh-EP cross-react with flh-LPH [3-13]. With one exception [4] flh-EP antisera have been raised against flh-EP conjugated to carrier proteins. The present report characterizes antisera raised against unconjugated flh-EP. One of these antisera had negligible cross-reactivity with flh-LPH. The specificity of this antiserum permitted the determination of flh-EP in plasma extracts without prior chromatography.
Materials and Methods
Peptides Synthetic human Bh-EP, human a-N-acetyl-flh-EP, flh-LPH 61-69, flh-LPH 61-87, flh-LPH 88-91 and flh-LPH 66-91 were purchased from Peninsula, San Carlos, CA, U.S.A.; human flh-LPH from Bioflex, Ozone, NY, U.S.A.; synthetic c~-endorphin (flh-LPH 61-76), methionine-enkephalin (fl h-LPH 61-65) and leucine-enkephalin (flh-LPH 61-64-1eucine) from Peptide Institute, Mino-ski, Osaka, Japan.
Immunization procedure Synthetic human flh-EP, dissolved in isotonic saline (100 #g/ml), was emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, MI, U.S.A.). The mixture was injected intradermally at multiple sites in the back of five rabbits [14]. Each rabbit was initially given a total amount of 100/tg of flh-EP. After 18 and 29 weeks each rabbit was boosted with another 20 ~tg of flh-EP in the same solution. Antisera were collected 2-3 weeks after the second booster dose and coded K7762-K-7766.
Preparation of ~:5I-labelled fla-EP Human flh-EP (2.5 /zg) was iodinated with 0.25 mCi Na125I (The Radiochemical Centre, Amersham, England) by the lactoperoxidase technique [15]. The reaction was terminated after 10 s by the addition of 0.5 rnl 0.1 M acetate buffer, pH 5.0, containing 2% (w/v) human serum albumin, HSA (Behring, Marburg, F.R.G.). The iodinated flh-EP was purified on a Sephadex G-50 (Pharmacia, Uppsala, Sweden) column (1.1 × 20 cm) pretreated with the same acetate-HSA buffer as above. The tracer was eluted with 0.1 M acetate buffer, pH 5.0, and collected in tubes contain-
67 ing 1 ml acetate-HSA buffer and 500 KIU aprotinin (Trasylol ®, Bayer, Leverkusen, F.R.G.). Iodinated /3h-EP stored at - 2 0 ° C was stable with unchanged antibody binding for 3 weeks. The calculated specific activity ranged from 8.3 to 11.4 MBq/nmol.
Radioimmunoassay (RIA ) 100 /~1 of flh-EP standard solutions (22-2285 pmol/1) in 0.05 M phosphate buffer, pH 7.4, containing 0.9% (w/v) saline, 1% (w/v) human serum albumin (PBS-HSA), 0.5% (v/v) mercaptoethanol (Sigma, St. Louis, MO, U.S.A.) and 500 KIU Trasylol ® per ml were incubated with 200 ~1 antiserum (final dilution 1 : 12 500) for 24 h at +4°C. 200/~1 of iodinated J3h-EP (about 10000 cpm) was added and the incubation continued for another 24 h. Antibody-bound tracer was separated from free tracer by the addition of 500/LI 1% (w/v) charcoal (Merck, Darmstadt, F.R.G.) in PBS-HSA containing 0.1% (w/v) Dextran T 70 (Pharmacia, Uppsala, Sweden). After 10 rain at 4°C the samples were centrifuged at 2400 × g. The radioactivity of the supernatants was counted in a Nuclear Enterprise gamma counter (NE 1600) with a counting efficiency for 1251 of about 80%.
Characterization of the antisera The specificity of the antisera was tested in the radioimmunoassay using a wide concentration range of/3h-LPH, a-N-acetyl-flh-EP, a-endorphin, Met- and Leu-enkephalin and various flh-LPH fragments. Only antisera with an acceptable titer and avidity were tested (K-7762 and K-7763). Cross-reactivity was expressed as the ratio of the molar concentration of flh-EP to that of the tested peptide, causing 50% inhibition of tracer binding. The ratio was given in per cent. The association constant was calculated from a Scatchard plot [16] of a standard curve incubated under equilibrium conditions.
Normal subjects and patients Blood was collected under basal conditions from 27 subjectively healthy blood donors and from patients with diseases known to be associated with abnormal A C T H secretion (Table I). Plasma ACTH levels were determined by a radioimmunoassay [17]. Six patients, admitted because of accidentally discovered enlarged sella turcica without any clinical or laboratory evidence of hormonal abnormality, underwent the metyrapone test to evaluate the pituitary-adrenal axis.
Metyrapone test Metyrapone was administered orally in a dose of 750 mg every times beginning at 8 a.m. on day 1. The effect on steroid hormone evaluated by the determination of plasma 1 l-deoxycortisol using a [ 18,19]. Blood samples were collected at 8 a.m. (before the first dose and then at 4 p.m. on day 1 and at 8 a.m. on day 2.
fourth hour six production was RIA procedure of metyrapone)
Collection and preparation of blood samples Blood samples were drawn from an antecubital vein directly into glass tubes
M/61 F/42 M/47 M/57 F/21 F/31
M/18
F/49
M/37 M/57 M/32 M/44
Sex/Age
Untreated Untreated Untreated Adrenalectomized 8 years earlier because of Cushing's disease. Adrenalectomized 9 years earlier because of Cushing's disease. Continuing ACTH secretion in spite of pituitary irradiation 3 years ago. Adrenalectomized 6 years earlier because of Cushing's disease. Pulmonary malignancy Pulmonary malignancy Pancreatic malignancy Untreated Untreated Untreated
Remarks
27 59 76 < 3 < 3 7
109
39
< 3 in 26, 5 in one 23 54 76 37
flh-EP ( p m o l / l )
> 110 ** 260 640 70 * 26 26
340
37
91 > 200 ** > 200 ** 75
n.d.
ACTH ( p m o l / l )
Reference values at 8 a.m. for plasma ACTH for the patient marked * were 55-95 p m o l / I and for the others 5-30 pmol/I. ** Plasma not diluted for exact ACTH determination. n.d., not determined.
Cushing's disease
Ectopic ACTH production
Nelson's syndrome
Healthy blood donors (n = 27) Addison's disease
Subjects
Plasma flh-EP and ACTH in human subjects studied under basal conditions
TABLE I
69
K-7762 B x 100 Bo
100'
50"-
i
O. 4 F - - ,
--i
10 ~
,
10 2
T4
103
,
10s
10
--
~0
~
K-7763
100-
: ~h.............~
~
~
~=
~
50-
%
........~ ......... 0
. ..................
t
:......... <>
101
102
103 Concentration
104
10 s
106
pmoI/I
Fig. 1. The inhibition of 125I-labelled flh-EP binding to the antisera K-7762 and K-7763, respectively, by flh-EP, flh-LPH and related peptides, flh-lipotropin, flh-LPH (1-91) © . . . . . . O; flh-endorphin, flh-LPH (61-91) • e; ah-endorphin, flh-LPH (61-76) • • ; Met-enkephalin, flh-LPH (61-65) [2 D; Leu-enkephalin, flh-LPH (61-64-1eucine) zx zx. TABLE II Cross-reactivity of the two flh-EP antisera (as percent on a molar basis) Peptide
flh-Endorphin a-N-Acetyl-flh-endorphin fls-Lipotropin an-Endorphin Met-Enkephalin Leu-Enkephalin C'-Fragment Other ~ flh-Endorphin j Fragments
Corresponding amino acid sequence in flh-LPH
61-91 61-91 (a-N-acetyl) 1-91 61-76 61-65 61-64-Leu 61-87 61-69 66-91 88-91
Antiserum K-7762
K-7763
100 1.2 1.5 69 9 0.04 43 67 2 0
100 373 102 0 0 0 75 0 185 0
70 containing Trasylol ~ (500 K I U / m l ) and EDTA (1 mg/ml). Plasma was immediately separated by centrifugation at 1000 × g for 15 rain at 4°C and stored at - 2 0 ° C until assayed. After thawing the plasma at room temperature, flh-EP was extracted by a modification of the silicic acid absorption method described by Nakai et al. [10]. 3 ml of plasma diluted 1:4 with distilled water were extracted with 75 mg silicic acid (100 mesh, Mallinckrodt, Inc., St. Louis, MO, U.S.A.). After centrifugation and decantation of the plasma, the silicic acid was washed with 10 ml of distilled water. followed by 5 ml of diethyl ether./3h-EP was eluted with 4.0 ml 40% (v/v) acetone in 1% (v/v) acetic acid. The eluates were lyophilized after evaporation of the solvent under air. The lyophilized eluates were reconstituted in 300 ~1 of the RIA buffer. The recovery in the extraction procedure was tested by the addition of synthetic flh-EP.
Preparation of hog pituitary extracts Freshly collected hog pituitaries were separated into anterior and neurointermediate lobes and stored at - 2 0 ° C . The thawed tissues were homogenized with a Potter-Elvehjem glass homogenizer in 5 ml cold 1 M acetic acid containing 20 mM HC1, 0.01% (v/v) phenylmethylsulfonyl fluoride and Trasylol ® (100 K I U / m l ) . The homogenates were centrifuged at 4°C and 5000 × g for 20 min and the supernatants lyophilized. The lyophilized material was redissolved in 0.5-1 ml of 4 M guanidine-HC1 and analyzed by gel chromatography on a 0.9 × 60 cm column of Bio-Gel P-100 (Biorad Laboratories, Richmond, CA, U.S.A.) equilibrated with 4 M guanidine-HC1 and eluted at room temperature with the same solvent at 1.7 m l / h . Fractions of 0.8 ml were collected and diluted (at least 1 : 50) before assay. The column was calibrated with Dextran blue (void volume = Vo), flh-LPH, flh-EP and Na125I (salt peak = Vt).
Results
Characterization of the ~h-EP RIA Two of five rabbits generated antibodies with high avidity. The association constants were calculated to be 1.2 and 1.7 × 101° 1/mol for K-7762 and K-7763 respectively. Both antisera could be used at a final dilution of 1:12500, giving 30-35% binding of the tracer (bound/total). The two antisera displayed different specificities (Fig. 1). Their cross-reactivity with different flh-LPH related peptides was summarized in Table II. One antiserum, K-7762, displayed only 1.5% cross-reactivity with flh-LPH. It did not recognize flh-LPH 88-91, but all amino-terminal fragments were detected to a varying degree, but a-N-acetyl-flh-EP only to a very low extent. It cross-reacted with flh-LPH 61-76 (ah-EP) to 69%, flh-LPH 61-69 to 67%, flh-LPH 61-87 to 43% and flh-LPH 61-65 (Met-enkephalin) to 9%, but with Leu-enkephalin less than 0.4% and with flh-LPH 66-91 to 2%. The other antiserum, K-7763, cross-reacted with/3h-LPH to 100%, with flh-LPH 66-91 to > 100% and with flh-LPH 61-87 to 75%. It recognized a-N-acetyl-
71
~ KIO0 30.
2(3
10-
°O
so
16o
1so
fmol/tube
(9
500 1000 pmol/L
1500
Fig. 2. Standard curve for flh-EP RIA using the antiserum K-7762. B: bound tracer; T: total amount of tracer.
flh-EP but neither the shorter amino-terminal fragments 61-69, 61-67 and 61-65 of flh-LPH, nor Leu-enkephalin, nor the carboxy-terminal peptide flh-LPH 88-91. For specific flh-EP RIA the antiserum K-7762 was used. The standard curve was optimal between 25 and 225 pmol flh-EP per liter. Half maximal displacement of the tracer was obtained with 180 pmol/1 of non-radioactive/3h-EP (Fig. 2). The sensitivity of the assay calculated from the variation of the response metameter at zero flh-EP concentration was 1.9 fmol/tube. The interassay and intraassay coefficients of variation were 7.2% (n = 21) and 7.1% (n = 52), respectively. The mean recovery of flh-EP was 72 + 9% (mean + S.D.; n = 28). The sensitivity of the assay was calculated to be 3 pmol/1, corrected for 70% recovery.
T A B L E 1II Effect of metyrapone on plasma 1 l-deoxycortisol and flh-EP as studied in six patients with an enlarged sella turcica on X-ray. but without evidence of disturbed pituitary function Day 1 8 a.m. 11 -Deoxycortisol ( n m o l / 1 ) Mean _+S.D. Range /3h-EP (pmol/1) Mean _+S.D. Range
24__+8 10-34 <3
Day 2 8 a.m. 4 p.m.
262_+ 110 130-395
714_+205 476-1056
<3
5+3 < 3-8.5
72
Vo 307 -
B-LPH
B-END
Vt
-
o~
.,> fi/il ti
,,,.,. 2 0 Z
,=
o
.
.
.
.
.
.
.
.
.
.
.
.
.
ATi
10-
E
_E
O-
2'o
go
40
Effluent volume lmll
Vo I
30
B-LPH
B-END
Vt
T-v
==
=:o PI Z tu
2O
o
P
o
¢1
10
E
_E
-//
2'o
3'o Effluent
volume
40 (ml)
Fig. 3. Gel chromatography (Bio-Gel P-100) elution profiles of extracts of hog pituitary, (A) anterior lobe. (B) neurointermediate lobe. Immunoreactivity was determined by RIA employing the two antisera K-7762 (O . . . . . . e) and K-7763 (O ©). Two markers were used, flh-LPH and flh-EP. 1~,,= void volume, 1/t = total elution volume.
Plasma concentrations of flh-EP Twenty-six of the 27 healthy blood donors had no detectable flh-EP in plasma (Table I), but one had 5 pmol/1. In the patients with Addison's disease flh-EP in
73 plasma was 23, 54 and 76 pmol/1, with Nelson's syndrome 37, 39 and 109 pmol/1 and with ectopic ACTH production 27, 59 and 76 pmol/1 (Table I). In the patients with Cushing's disease flh-EP was undetectable in 2 of 3 and in the third it was 7 pmol/1. In the six patients with enlarged sella turcica all demonstrated a clear increase in plasma 11-deoxycortisol after metyrapone. Five of them increased plasma flh-EP from undetectable to detectable levels (range 3.5-8.5 pmol/L), while in one flh-EP was still undetectable after metyrapone (Table III). Pituitary extracts
The chromatographic elution profiles of extracts of anterior and neurointermediate lobes of hog pituitaries were illustrated in Figure 3. Using antiserum K-7762 only one single peak of flh-EP-like immunoreactivity was found in the extract of the anterior pituitary. It co-eluted with synthetic human /gh-EP. On the other hand, K-7763 revealed two immunoreactive peaks co-eluting with flh-LPH and flh-EP, respectively. In the extracts of the neurointermediate lobe the two antisera, K-7762 and K-7763, revealed only one single immunoreactive peak co-eluting with flh-EP.
Discussion
Our antisera K-7762 and K-7763 raised against unconjugated flh-EP were comparable to those obtained by others using conjugated flh-EP in terms of avidity and titer [3,4,11,13]. In this respect our antisera differed from an earlier report [4], where unconjugated flh-EP elicited an antiserum with a low titer and showed a complete cross-reactivity with flh-LPH. Antisera against flh-EP usually cross-react with flh-LPH to a varying degree [3-13]. Antiserum K-7762 showed negligible cross-reactivity with flh-LPH 1-91 and recognized flh-LPH 61-69, flh-LPH 61-76 and flh-LPH 61-87. The cross-reactivity with flh-LPH 61-65 (Met-enkephalin) was low. Apparently, the intact amino-terminal is important for recognition. This was demonstrated by the almost complete loss of immunoreactivity, when the amino-terminal tyrosine was acetylated or if the methionine group in position 5 was substituted by leucine (as in Leu-enkephalin), and the low cross-reactivity with flh-LPH 66-91, in which the amino-terminal pentapeptide is missing. Antiserum K-7763 cross-reacted fully with flh-LPH and seemed to recognize the carboxy-terminal end of flh-EP. It appears that antisera directed against the carboxy-terminal portion of flh-EP recognize also flh-LPH, while antisera directed against the amino-terminal portion recognize flh-EP, an-EP (flhLPH 61-76) and C'-fragment (flh-LPH 61-87), but not flh-LPH because of the requirement for a free amino-terminal in position 61. Recent studies indicate, that a major portion of fl-EP in the intermediate lobe of rat pituitaries is N-acetylated [20,21[. This derivative of flh-EP was recognized by antiserum K-7763, but not by K-7762. So far, N-acetylated flh-EP in man has been identified only in an ectopic ACTH producing tumour and not in normal pituitary or pituitary adenomas [22].
74 There is no evidence for conversion of Bh-LPH to fih-EP in plasma [23,24]. The plasma /~h-EP concentration measured with the antiserum K-7762 was undetectable ( < 3 pmol/l) in all but one of the healthy subjects. This is partially in agreement with previous studies, in which /~h-EP was undetectable in 50-100% of plasmas from normal individuals under basal conditions [11,12,24-26], with one exception [13]. In our study, plasma /3h-EP levels increased following metyrapone, but the increase was of a smaller magnitude than earlier reported [11,13]. Diseases with increased plasma A C T H levels are known to be associated with increased plasma/~h-EP levels [10,12,20,22]; this was confirmed in the present study. With antiserum K-7762, the gel chromatographic analysis of extracts of the anterior lobe of hog pituitaries revealed a single immunoreactive peak co-eluting with synthetic human /~h-EP, while K-7763 demonstrated two peaks corresponding to ]3h-LPH and ,Sh-EP, respectively. The different specificities of the two antisera have been corroborated by immunohistochemistry (R. HS.kanson et al., personal communication). With both antisera the immunostaining of the ACTH cells of formaldehyde-fixed hog pituitaries was blocked by preabsorption with/3u-EP, while /3h-LPH blocked the immunoreaction of antiserum K-7763, but not that of antiserum K-7762. In conclusion, a ]3h-EP antiserum with negligible cross-reactivity against flh-LPH was generated by immunization of rabbits using unconjugated flh-EP. This antiserum permitted the direct determination of/?h-EP in plasma extracts without prior chromatography.
Acknowledgements This work was supported by grants from the Swedish Medical Research Council (03X-05917), Thorsten and Elsa Segerfalk's Foundation, Bergvall's Foundation, and Greta and Johan Kock's Foundation.
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75 8 Li, C.H., Rao, A.J., Donee, B.A. and Yamashiro, D., ,~-Endorphin: Lack of correlation between opiate activity and immunoreactivity by radioimmunoassay, Biochem. Biophys. Res. Commun., 75 (1977) 576-580. 9 Liotta, A.S., Suda, T. and Kreiger, D.T., fl-Lipotropin is the major opioid-like peptide of human pituitary and rat pars distalis: Lack of significant /3-endorphin. Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2950-2954. 10 Nakai, Y., Nakao, K., Shoga, O., Imura, H. and Li, C.H., Presence of immunoreactive B-endorphin in plasma of patients with Nelson's syndrome and Addison's disease, Life Sci., 23 (1978) 2293-2298. 11 Wardlaw, S.L. and Frantz, A.G., Measurements of fl-endorphin in human plasma, J. Clin. Endocrinol. Metab., 48 (1979) 176-180. 12 Wiedemann, E., Saito, T., Linfoot, J. and Li, C.H., Specific radioimmunoassay of human B-endorphin in unextracted plasma, J. Clin. Endocrinol. Metab., 49 (1979) 478-480. 13 Wilkes, M.M., Stewart, R.D., Bruni, J.F., Quigley, M.E., Yen, S.S.C., Ling, N. and Chr&tien, M., A specific homologous radioimmunoassay for human fl-endorphin: Direct measurement in biological fluids, J. Clin. Endocrinol. Metab., 50 (1980) 309-315. 14 Vaitukaitis, J., Robbins, J.B., Nieschlag, E. and Ross, G.T., A method for producing specific antisera with small doses of immunogen, J. Clin. Endocrinol. Metab., 33 (1971) 988-991. 15 Thorell, J.l. and Johansson, B.G., Enzymatic iodination of polypeptides with 1251 to high specific activity, Biochim. Biophys. Acta, 251 (1971) 363-369. 16 Scatchard, G., The attraction of proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660-672. 17 Hummer, L., Radioimmmunoassay of plasma corticotropin and related procedures in medicine. I.A.E.A. Syrup. Vienna, 2 (1978) 391-403. 18 Kao, M., Voina, S., Nicolas, A. and Horton, R., Parallel radioimmunoassay for plasma cortisol and 11-deoxycortisol, Clin. Chem., 21 (1975) 1644-1647. 19 Thorell, J.l. and Larson, S.M., Radioimmunoassay and related assays, The C.V. Mosby Company, St. Louis, MO, 1978, p. 47. 20 Seizinger, B.T. and H611t, V., In vitro biosynthesis and N-acetylation of fl-endorphin in pars intermedia of rat pituitary, Biochem. Biophys. Res. Commun., 96 (1980) 535-615. 21 Smyth, D.G. and Zakarian, S., Selective processing of fl-endorphin in regions of porcine pituitary, Nature, 288 (1980) 613-615. 22 Suda, T., Tozawa, F., Yamaguchi, H., Shibasaki, T., Demura, H. and Shizume, K., Multiple forms of immunoreactive/~-endorphin are present in an ectopic adrenocorticotropin-producing tumor but not in normal pituitary or pituitary adenomas, J. Clin. Endocrinol. Metab., 54 (1982) 167-171. 23 Gilles, G., Ratter, S., Grossman, A., Gaillard, R., Lowry, P.J., Besser, G.M. and Rees, L.H. Secretion of ACTH, LPH and ,8-endorphin from human pituitary tumours in vitro, Clin. Endocrinol., 13 (1980) 197-205. 24 Suda, T., Liotta, A.S. and Krieger, D.T.,/~-Endorphin is not detectable in plasma from normal human subjects, Science, 202 (1978) 221-223. 25 Yamaguchi, H., Liotta, A.S. and Krieger, D.T., Simultaneous determination of human plasma immunoreactive/3-1ipotropin, y-lip0tropin, and ~8-endorphin using immunoaffinity chromatography, J. Clin. Endocrinol. Metab., 51 (1980) 1002-1007. 26 Nakao, K., Nakai, Y., Jingami, H., Oki, S., Fukata, J. and lmura, H., Substantial rise of plasma ,B-endorphin levels after insulin-induced hypoglycemia in human subjects, J. Clin. Endocrinol. Metab., 49 (1979) 838-841.
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Elsevier Biomedical Press
Characterization and application of a radioimmunoassay for fl-endorphin using an antiserum with negligible cross-reactivity against fl-lipotropin M a r g a r e t a B r a m n e r t , R o l f E k m a n , I n g v a r L a r s s o n a n d J a n I. T h o r e l l Department of Endocrinology, and Department of Nuclear Medicine, Lund University, MalmiJ General Hospital, Malmo, Sweden (Received 28 June 1982; accepted for publication 22 September 1982)
Summary High avidity antisera against fl-endorphin (flh-EP) were obtained in two of five rabbits immunized with unconjugated synthetic human flh-EP. One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with fl-lipotropin (flh-LPH) and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/1. The cross-reaction with methionine-enkephalin (flh-LPH 61-65) was 9%, while that with a-endorphin (flh-LPH 61-76) was 69%. This implied that the specific recognition site was in the amino-terminal region of flh-EP. Although this sequence is present in flh-LPH it was poorly recognized by the antiserum, suggesting that the free anainoterminal is essential. This interpretation was supported by the finding that a-Nacetyl-flh-EP was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/1, flh-EP was not detectable ( < 3 pmol/l) in 26 of 27 extracted plasma samples in healthy blood donors, in one it was 5 pmol/1. In five of six patients with an enlarged sella turcica, but without clinical and laboratory evidence of pituitary dysfunction, flh-EP was detectable (5 + 3 pmol/l; mean _+ S.D.) after metyrapone, flh-EP was elevated in Addison's disease (23, 54 and 76 pmol/l), Nelson's syndrome (37, 39 and 109 pmol/1), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushing's disease (7 pmol/1). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human flh-EP. The specificity of the antiserum K-7762 was such that the flh-EP concentration in plasma
Address for correspondence: Dr. Margareta Bramnert, Department of Endocrinology, Lund University Clinics, Malm8 General Hospital, S-214 01 MaimS, Sweden. Telephone 46 40 332329. 0167-0115/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
66
extracts could be reliably estimated by radioimmunoassay without prior chromatography. fl-endorphin; fl-lipotropin; antisera; plasma extracts; cross-reactivity
Introduction
fl-Endorphin (flh-EP), the carboxy-terminal 31 amino acid sequence of fl-lipotropin (flh-LPH), is generated by cleavage from the pro-opiomelanocortin precursor with flh-LPH as an anticipated intermediate [1,2]. Most antisera against flh-EP cross-react with flh-LPH [3-13]. With one exception [4] flh-EP antisera have been raised against flh-EP conjugated to carrier proteins. The present report characterizes antisera raised against unconjugated flh-EP. One of these antisera had negligible cross-reactivity with flh-LPH. The specificity of this antiserum permitted the determination of flh-EP in plasma extracts without prior chromatography.
Materials and Methods
Peptides Synthetic human Bh-EP, human a-N-acetyl-flh-EP, flh-LPH 61-69, flh-LPH 61-87, flh-LPH 88-91 and flh-LPH 66-91 were purchased from Peninsula, San Carlos, CA, U.S.A.; human flh-LPH from Bioflex, Ozone, NY, U.S.A.; synthetic c~-endorphin (flh-LPH 61-76), methionine-enkephalin (fl h-LPH 61-65) and leucine-enkephalin (flh-LPH 61-64-1eucine) from Peptide Institute, Mino-ski, Osaka, Japan.
Immunization procedure Synthetic human flh-EP, dissolved in isotonic saline (100 #g/ml), was emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, MI, U.S.A.). The mixture was injected intradermally at multiple sites in the back of five rabbits [14]. Each rabbit was initially given a total amount of 100/tg of flh-EP. After 18 and 29 weeks each rabbit was boosted with another 20 ~tg of flh-EP in the same solution. Antisera were collected 2-3 weeks after the second booster dose and coded K7762-K-7766.
Preparation of ~:5I-labelled fla-EP Human flh-EP (2.5 /zg) was iodinated with 0.25 mCi Na125I (The Radiochemical Centre, Amersham, England) by the lactoperoxidase technique [15]. The reaction was terminated after 10 s by the addition of 0.5 rnl 0.1 M acetate buffer, pH 5.0, containing 2% (w/v) human serum albumin, HSA (Behring, Marburg, F.R.G.). The iodinated flh-EP was purified on a Sephadex G-50 (Pharmacia, Uppsala, Sweden) column (1.1 × 20 cm) pretreated with the same acetate-HSA buffer as above. The tracer was eluted with 0.1 M acetate buffer, pH 5.0, and collected in tubes contain-
67 ing 1 ml acetate-HSA buffer and 500 KIU aprotinin (Trasylol ®, Bayer, Leverkusen, F.R.G.). Iodinated /3h-EP stored at - 2 0 ° C was stable with unchanged antibody binding for 3 weeks. The calculated specific activity ranged from 8.3 to 11.4 MBq/nmol.
Radioimmunoassay (RIA ) 100 /~1 of flh-EP standard solutions (22-2285 pmol/1) in 0.05 M phosphate buffer, pH 7.4, containing 0.9% (w/v) saline, 1% (w/v) human serum albumin (PBS-HSA), 0.5% (v/v) mercaptoethanol (Sigma, St. Louis, MO, U.S.A.) and 500 KIU Trasylol ® per ml were incubated with 200 ~1 antiserum (final dilution 1 : 12 500) for 24 h at +4°C. 200/~1 of iodinated J3h-EP (about 10000 cpm) was added and the incubation continued for another 24 h. Antibody-bound tracer was separated from free tracer by the addition of 500/LI 1% (w/v) charcoal (Merck, Darmstadt, F.R.G.) in PBS-HSA containing 0.1% (w/v) Dextran T 70 (Pharmacia, Uppsala, Sweden). After 10 rain at 4°C the samples were centrifuged at 2400 × g. The radioactivity of the supernatants was counted in a Nuclear Enterprise gamma counter (NE 1600) with a counting efficiency for 1251 of about 80%.
Characterization of the antisera The specificity of the antisera was tested in the radioimmunoassay using a wide concentration range of/3h-LPH, a-N-acetyl-flh-EP, a-endorphin, Met- and Leu-enkephalin and various flh-LPH fragments. Only antisera with an acceptable titer and avidity were tested (K-7762 and K-7763). Cross-reactivity was expressed as the ratio of the molar concentration of flh-EP to that of the tested peptide, causing 50% inhibition of tracer binding. The ratio was given in per cent. The association constant was calculated from a Scatchard plot [16] of a standard curve incubated under equilibrium conditions.
Normal subjects and patients Blood was collected under basal conditions from 27 subjectively healthy blood donors and from patients with diseases known to be associated with abnormal A C T H secretion (Table I). Plasma ACTH levels were determined by a radioimmunoassay [17]. Six patients, admitted because of accidentally discovered enlarged sella turcica without any clinical or laboratory evidence of hormonal abnormality, underwent the metyrapone test to evaluate the pituitary-adrenal axis.
Metyrapone test Metyrapone was administered orally in a dose of 750 mg every times beginning at 8 a.m. on day 1. The effect on steroid hormone evaluated by the determination of plasma 1 l-deoxycortisol using a [ 18,19]. Blood samples were collected at 8 a.m. (before the first dose and then at 4 p.m. on day 1 and at 8 a.m. on day 2.
fourth hour six production was RIA procedure of metyrapone)
Collection and preparation of blood samples Blood samples were drawn from an antecubital vein directly into glass tubes
M/61 F/42 M/47 M/57 F/21 F/31
M/18
F/49
M/37 M/57 M/32 M/44
Sex/Age
Untreated Untreated Untreated Adrenalectomized 8 years earlier because of Cushing's disease. Adrenalectomized 9 years earlier because of Cushing's disease. Continuing ACTH secretion in spite of pituitary irradiation 3 years ago. Adrenalectomized 6 years earlier because of Cushing's disease. Pulmonary malignancy Pulmonary malignancy Pancreatic malignancy Untreated Untreated Untreated
Remarks
27 59 76 < 3 < 3 7
109
39
< 3 in 26, 5 in one 23 54 76 37
flh-EP ( p m o l / l )
> 110 ** 260 640 70 * 26 26
340
37
91 > 200 ** > 200 ** 75
n.d.
ACTH ( p m o l / l )
Reference values at 8 a.m. for plasma ACTH for the patient marked * were 55-95 p m o l / I and for the others 5-30 pmol/I. ** Plasma not diluted for exact ACTH determination. n.d., not determined.
Cushing's disease
Ectopic ACTH production
Nelson's syndrome
Healthy blood donors (n = 27) Addison's disease
Subjects
Plasma flh-EP and ACTH in human subjects studied under basal conditions
TABLE I
69
K-7762 B x 100 Bo
100'
50"-
i
O. 4 F - - ,
--i
10 ~
,
10 2
T4
103
,
10s
10
--
~0
~
K-7763
100-
: ~h.............~
~
~
~=
~
50-
%
........~ ......... 0
. ..................
t
:......... <>
101
102
103 Concentration
104
10 s
106
pmoI/I
Fig. 1. The inhibition of 125I-labelled flh-EP binding to the antisera K-7762 and K-7763, respectively, by flh-EP, flh-LPH and related peptides, flh-lipotropin, flh-LPH (1-91) © . . . . . . O; flh-endorphin, flh-LPH (61-91) • e; ah-endorphin, flh-LPH (61-76) • • ; Met-enkephalin, flh-LPH (61-65) [2 D; Leu-enkephalin, flh-LPH (61-64-1eucine) zx zx. TABLE II Cross-reactivity of the two flh-EP antisera (as percent on a molar basis) Peptide
flh-Endorphin a-N-Acetyl-flh-endorphin fls-Lipotropin an-Endorphin Met-Enkephalin Leu-Enkephalin C'-Fragment Other ~ flh-Endorphin j Fragments
Corresponding amino acid sequence in flh-LPH
61-91 61-91 (a-N-acetyl) 1-91 61-76 61-65 61-64-Leu 61-87 61-69 66-91 88-91
Antiserum K-7762
K-7763
100 1.2 1.5 69 9 0.04 43 67 2 0
100 373 102 0 0 0 75 0 185 0
70 containing Trasylol ~ (500 K I U / m l ) and EDTA (1 mg/ml). Plasma was immediately separated by centrifugation at 1000 × g for 15 rain at 4°C and stored at - 2 0 ° C until assayed. After thawing the plasma at room temperature, flh-EP was extracted by a modification of the silicic acid absorption method described by Nakai et al. [10]. 3 ml of plasma diluted 1:4 with distilled water were extracted with 75 mg silicic acid (100 mesh, Mallinckrodt, Inc., St. Louis, MO, U.S.A.). After centrifugation and decantation of the plasma, the silicic acid was washed with 10 ml of distilled water. followed by 5 ml of diethyl ether./3h-EP was eluted with 4.0 ml 40% (v/v) acetone in 1% (v/v) acetic acid. The eluates were lyophilized after evaporation of the solvent under air. The lyophilized eluates were reconstituted in 300 ~1 of the RIA buffer. The recovery in the extraction procedure was tested by the addition of synthetic flh-EP.
Preparation of hog pituitary extracts Freshly collected hog pituitaries were separated into anterior and neurointermediate lobes and stored at - 2 0 ° C . The thawed tissues were homogenized with a Potter-Elvehjem glass homogenizer in 5 ml cold 1 M acetic acid containing 20 mM HC1, 0.01% (v/v) phenylmethylsulfonyl fluoride and Trasylol ® (100 K I U / m l ) . The homogenates were centrifuged at 4°C and 5000 × g for 20 min and the supernatants lyophilized. The lyophilized material was redissolved in 0.5-1 ml of 4 M guanidine-HC1 and analyzed by gel chromatography on a 0.9 × 60 cm column of Bio-Gel P-100 (Biorad Laboratories, Richmond, CA, U.S.A.) equilibrated with 4 M guanidine-HC1 and eluted at room temperature with the same solvent at 1.7 m l / h . Fractions of 0.8 ml were collected and diluted (at least 1 : 50) before assay. The column was calibrated with Dextran blue (void volume = Vo), flh-LPH, flh-EP and Na125I (salt peak = Vt).
Results
Characterization of the ~h-EP RIA Two of five rabbits generated antibodies with high avidity. The association constants were calculated to be 1.2 and 1.7 × 101° 1/mol for K-7762 and K-7763 respectively. Both antisera could be used at a final dilution of 1:12500, giving 30-35% binding of the tracer (bound/total). The two antisera displayed different specificities (Fig. 1). Their cross-reactivity with different flh-LPH related peptides was summarized in Table II. One antiserum, K-7762, displayed only 1.5% cross-reactivity with flh-LPH. It did not recognize flh-LPH 88-91, but all amino-terminal fragments were detected to a varying degree, but a-N-acetyl-flh-EP only to a very low extent. It cross-reacted with flh-LPH 61-76 (ah-EP) to 69%, flh-LPH 61-69 to 67%, flh-LPH 61-87 to 43% and flh-LPH 61-65 (Met-enkephalin) to 9%, but with Leu-enkephalin less than 0.4% and with flh-LPH 66-91 to 2%. The other antiserum, K-7763, cross-reacted with/3h-LPH to 100%, with flh-LPH 66-91 to > 100% and with flh-LPH 61-87 to 75%. It recognized a-N-acetyl-
71
~ KIO0 30.
2(3
10-
°O
so
16o
1so
fmol/tube
(9
500 1000 pmol/L
1500
Fig. 2. Standard curve for flh-EP RIA using the antiserum K-7762. B: bound tracer; T: total amount of tracer.
flh-EP but neither the shorter amino-terminal fragments 61-69, 61-67 and 61-65 of flh-LPH, nor Leu-enkephalin, nor the carboxy-terminal peptide flh-LPH 88-91. For specific flh-EP RIA the antiserum K-7762 was used. The standard curve was optimal between 25 and 225 pmol flh-EP per liter. Half maximal displacement of the tracer was obtained with 180 pmol/1 of non-radioactive/3h-EP (Fig. 2). The sensitivity of the assay calculated from the variation of the response metameter at zero flh-EP concentration was 1.9 fmol/tube. The interassay and intraassay coefficients of variation were 7.2% (n = 21) and 7.1% (n = 52), respectively. The mean recovery of flh-EP was 72 + 9% (mean + S.D.; n = 28). The sensitivity of the assay was calculated to be 3 pmol/1, corrected for 70% recovery.
T A B L E 1II Effect of metyrapone on plasma 1 l-deoxycortisol and flh-EP as studied in six patients with an enlarged sella turcica on X-ray. but without evidence of disturbed pituitary function Day 1 8 a.m. 11 -Deoxycortisol ( n m o l / 1 ) Mean _+S.D. Range /3h-EP (pmol/1) Mean _+S.D. Range
24__+8 10-34 <3
Day 2 8 a.m. 4 p.m.
262_+ 110 130-395
714_+205 476-1056
<3
5+3 < 3-8.5
72
Vo 307 -
B-LPH
B-END
Vt
-
o~
.,> fi/il ti
,,,.,. 2 0 Z
,=
o
.
.
.
.
.
.
.
.
.
.
.
.
.
ATi
10-
E
_E
O-
2'o
go
40
Effluent volume lmll
Vo I
30
B-LPH
B-END
Vt
T-v
==
=:o PI Z tu
2O
o
P
o
¢1
10
E
_E
-//
2'o
3'o Effluent
volume
40 (ml)
Fig. 3. Gel chromatography (Bio-Gel P-100) elution profiles of extracts of hog pituitary, (A) anterior lobe. (B) neurointermediate lobe. Immunoreactivity was determined by RIA employing the two antisera K-7762 (O . . . . . . e) and K-7763 (O ©). Two markers were used, flh-LPH and flh-EP. 1~,,= void volume, 1/t = total elution volume.
Plasma concentrations of flh-EP Twenty-six of the 27 healthy blood donors had no detectable flh-EP in plasma (Table I), but one had 5 pmol/1. In the patients with Addison's disease flh-EP in
73 plasma was 23, 54 and 76 pmol/1, with Nelson's syndrome 37, 39 and 109 pmol/1 and with ectopic ACTH production 27, 59 and 76 pmol/1 (Table I). In the patients with Cushing's disease flh-EP was undetectable in 2 of 3 and in the third it was 7 pmol/1. In the six patients with enlarged sella turcica all demonstrated a clear increase in plasma 11-deoxycortisol after metyrapone. Five of them increased plasma flh-EP from undetectable to detectable levels (range 3.5-8.5 pmol/L), while in one flh-EP was still undetectable after metyrapone (Table III). Pituitary extracts
The chromatographic elution profiles of extracts of anterior and neurointermediate lobes of hog pituitaries were illustrated in Figure 3. Using antiserum K-7762 only one single peak of flh-EP-like immunoreactivity was found in the extract of the anterior pituitary. It co-eluted with synthetic human /gh-EP. On the other hand, K-7763 revealed two immunoreactive peaks co-eluting with flh-LPH and flh-EP, respectively. In the extracts of the neurointermediate lobe the two antisera, K-7762 and K-7763, revealed only one single immunoreactive peak co-eluting with flh-EP.
Discussion
Our antisera K-7762 and K-7763 raised against unconjugated flh-EP were comparable to those obtained by others using conjugated flh-EP in terms of avidity and titer [3,4,11,13]. In this respect our antisera differed from an earlier report [4], where unconjugated flh-EP elicited an antiserum with a low titer and showed a complete cross-reactivity with flh-LPH. Antisera against flh-EP usually cross-react with flh-LPH to a varying degree [3-13]. Antiserum K-7762 showed negligible cross-reactivity with flh-LPH 1-91 and recognized flh-LPH 61-69, flh-LPH 61-76 and flh-LPH 61-87. The cross-reactivity with flh-LPH 61-65 (Met-enkephalin) was low. Apparently, the intact amino-terminal is important for recognition. This was demonstrated by the almost complete loss of immunoreactivity, when the amino-terminal tyrosine was acetylated or if the methionine group in position 5 was substituted by leucine (as in Leu-enkephalin), and the low cross-reactivity with flh-LPH 66-91, in which the amino-terminal pentapeptide is missing. Antiserum K-7763 cross-reacted fully with flh-LPH and seemed to recognize the carboxy-terminal end of flh-EP. It appears that antisera directed against the carboxy-terminal portion of flh-EP recognize also flh-LPH, while antisera directed against the amino-terminal portion recognize flh-EP, an-EP (flhLPH 61-76) and C'-fragment (flh-LPH 61-87), but not flh-LPH because of the requirement for a free amino-terminal in position 61. Recent studies indicate, that a major portion of fl-EP in the intermediate lobe of rat pituitaries is N-acetylated [20,21[. This derivative of flh-EP was recognized by antiserum K-7763, but not by K-7762. So far, N-acetylated flh-EP in man has been identified only in an ectopic ACTH producing tumour and not in normal pituitary or pituitary adenomas [22].
74 There is no evidence for conversion of Bh-LPH to fih-EP in plasma [23,24]. The plasma /~h-EP concentration measured with the antiserum K-7762 was undetectable ( < 3 pmol/l) in all but one of the healthy subjects. This is partially in agreement with previous studies, in which /~h-EP was undetectable in 50-100% of plasmas from normal individuals under basal conditions [11,12,24-26], with one exception [13]. In our study, plasma /3h-EP levels increased following metyrapone, but the increase was of a smaller magnitude than earlier reported [11,13]. Diseases with increased plasma A C T H levels are known to be associated with increased plasma/~h-EP levels [10,12,20,22]; this was confirmed in the present study. With antiserum K-7762, the gel chromatographic analysis of extracts of the anterior lobe of hog pituitaries revealed a single immunoreactive peak co-eluting with synthetic human /~h-EP, while K-7763 demonstrated two peaks corresponding to ]3h-LPH and ,Sh-EP, respectively. The different specificities of the two antisera have been corroborated by immunohistochemistry (R. HS.kanson et al., personal communication). With both antisera the immunostaining of the ACTH cells of formaldehyde-fixed hog pituitaries was blocked by preabsorption with/3u-EP, while /3h-LPH blocked the immunoreaction of antiserum K-7763, but not that of antiserum K-7762. In conclusion, a ]3h-EP antiserum with negligible cross-reactivity against flh-LPH was generated by immunization of rabbits using unconjugated flh-EP. This antiserum permitted the direct determination of/?h-EP in plasma extracts without prior chromatography.
Acknowledgements This work was supported by grants from the Swedish Medical Research Council (03X-05917), Thorsten and Elsa Segerfalk's Foundation, Bergvall's Foundation, and Greta and Johan Kock's Foundation.
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