- Email: [email protected]
Radioimmunoassays for β-endorphin: Comparison of properties of two antisera
Life Sciences, Vol . 22, pp . 1123-1130 Printed in +:he U .S .A .
rergamon Press
OF PROPERTIES OF RADIOIMMUNOASSAYS FOR ß-ENDORPHIN : COMPARISON * TWO ANTISERA Maureen Ross, Vertan Ghazarossian, B .M . Cox and Avram Goldstein Addiction Research Foundation, and Department of Pharmacology, Stanford University, Palo Alto, California 94304
The opioid activity of ß-endorphin depends upon a free amino-terminal Tyr residue . Larger precursor peptides are inactive, as is the metabolic product from which Tyrl has been cleaved . Immunoassays have the advantage of great sensitivity but the disadvantage of ambiguity. Ambiguity arises when the antigenic determinant is a sequence not required for biological activity . Two antisera are described with antigenic determinants not hitherto reported . One of these is suitable for assays of ß-endorphin in human plasma . The other contains an antibody that recognizes ß-endorphin only if amino-terminal Tyr is présent, thus achieving a coincidence of immunoreactivity'and opioid activity . Development of reliable methodology for the detection and quantitation of small concentrations of potent biologically active compounds must necessarily precede their discovery and isolation . Unquestionably, the development of two bioassay systems for the opioid drugs, the myenteric plexus longitudinal muscle preparation (1) and the mouse vas deferens (2), and the development of the opiate receptor binding assay (3,4,5) were of paramount importance in the identification of the endogenous ligands for the opioid receptor . It was apparent even in the very early work on endorphins that there was more than one endogenous ligand for the opiate receptor . Concurrent with the determination of the structure of two brain opioid peptides, methionine (Met)-enkephalin and leucine (Leu)-enkephalin (6), were reports of a much larger opioid peptide in pituitary (7,8) . Opioid activity in brain was subsequently shown to be associated with much larger molecular weight species as well as the enkephalins (9) . *Presentéd at a symposium in honor of Otto Krayer, Tucson, Arizona, March 11, 1978 .
0300-9653/78/0410-1123$02 .00/0
Copyright © 1978 Pergamon Press
1124
Radioimmunoassays for ß-Endorphin
Vol . 22, No .s 13-15, 1978
Met-enkephalin is containéd within the pituitary hormone ßlipotropin (ß-LPH) as residues 61-65 (Fig . 1) . Realization of this fact quickly led to the discovery that other fragments of ß-LPH which possessed residue 61 as the amino-terminal were also active in the opioid assays with ß-endorphin (ß-LPH-(61-91)) being most potent . ß-LPH, which lacks the free amino group on Tyr 6 i, is devoid of opioid activity (10) and is presumed to represent an inactive precursor from which the endorphins are derived by limited proteolysis . More recently, ß-LPH itself has been shown to be present as the C-terminal amino acid sequence of a 31000 dalton precursor which also contains ACTH (11,12) . a-endorphin Net-enkephalin H - Tyr - Gly - Gly - Phe - lYt - Thr - fier - Glu - Lye - Sez - Gln - Thr 1 Z 5 9 10 Pro
(Ile)~t
13
Aen - Lys - Val - Ils - Ale - Aen - Lye - PM - Lev - Thr - Val - Uu/ / ZS Z3 20 16 15 Ala \His - Lye - Lys - Cly - Gln - OH Z7 30 31 (7Yr)t
(Glu - 09)t
FIG . 1 Structure of porcine ßp -endorphin . Precursor peptide, ß-LPH, contains 91 amino acids ; its carboxy terminal sequence of 31 residues is ß-endorphin, shown here . Thus Tyr61 of ß-LPH is Tyr l of ß-endorphin . Sequence 1-5 is Met-enkephalin ; sequence 1-16 is a-endorphin . * a substitution at indicated position in sheep (ß o-) or camel (ß c-) endorphin . t ~ substitutions at indicated positions in human ßh-endorphin . Hairpin-loop secondary structure ie represented symbolically ; precise structure is unknown .
While the bioassays and receptor binding assay possess the overriding advantage of measuring only biologically active species they cannot qualitatively distinguish one substance with opioid activity from another . Increasingly, radioimmunoassay is being used to measure both the enkephalins and ß-endorphin in tissue extracts and biological fluids . Ideâlly, the inherent specificity of antibodies could permit the unique determination of a particular endorphin in a single assay system . For peptide hormones, the reliability of radioimmunoassay for the measurement of a single substance has become increasingly uncertain as the generality of the pre-hormone ~ prohormone i hormone synthetic mechanism has beçome better established . Difficulties arise in the interpretation of data based solely on immunoreactivity, especially when antisera are used for which the antigenic determinants are distinct from the parts of the molecule needed for biological activity . For example, a widely used antibody, which recognizes the C-terminal region of ß-endorphin (13), reacts with the active ß-endorphin, the inactive ß -LPH, and the inactive 31K precursor . It would also recognize des-Tyrß-endorphin (i .e . ß-endorphin-(2-31)), which is inactive, but it fails to recognize a.-endorphin or the enkephalins, which are active .
Vol . 22, No .s 13-15, 1978
Radioi~unoassays for ß-Endorphin
1125
We have developed antibodies both to the intact ß-endorphin molecule and to its amino-terminal fragment, a-endorphin(ß-endorphin-(1-16)) . Use of a-endorphin as the-immunizing agent not only increases the probability that the antiserum developed will respond to an antigenic determinant that encompasses the biologically essential part of the ß-endorphin structure, but also eliminates the problem of species variations . All known species variants of ß-endorphin contain substitutions only in the C-terminal half of the molecule . METHODS Peptide Conjugation and Immunization Procedure To elicit antibody production, a-endorphin was conjugated to bovine thyroglobulin with 1-ethyl-3(3-dimethyl-amino-propyl)carbodiimide using molar ratios of peptide :carrier :conjugating agent of 118 :1 :200 as described by Skowsky and Fisher (14) . ßendorphin was coupled to m-aminobenzyloxymethyl cellulose by a modification of the procedure of Moudgal et al (15) . 1 mg of ß-endorphin was used per 6 .5 mg of m-aminobenzyloxymethyl cellulose . The peptide conjugates in isotonic saline were homogenized with an equal volume of Freund's complete adjuvant . Adult male New Zealand White rabbits were injected intradermally with 100 200 ug of the conjugated peptide as described by Vaitukaitis et al . (16) . After an initial series of three immunizations at fourweek intervals, animals received booster injections at three-month intervals . Radiolabelled peptides izs izs I-ß-endorphin were prepared by a l-a-endorphin and Iomodification of the procedure of Hunter and Greenwood (17) . dinated a-endorphin was purified by gel filtration on a Sephadex G-15 column (24 x 1 .2 cm) eluted with 50 mM sodium phosphate bufIodinfer containing 0 .18 bovine serum albumin and 50 mM RC1 . ated ß-endorphin was purified in a similar manner using Sephadex G-25 . Radioimmunoassay for a-endorphin Test substances or a-endorphin in 100 ul of 50 mM sodium phosphate buffer containing 0 .18 bovine serum albumin and 0 .98 NaCl, were incubated with 100 u 1 of antiserum of the appropriate dilution . The initial incubation was carried out at 4° C for a minimum of 48 hours, at which time 100 ul of iodinated a-endorand a second phin (1000-5000 cpm) in the same buffer was added 1zs Antibody-bound l-a-endorphin incubation (24 hours) commenced . was separated from free peptide by the addition of polyethylene glycol to final concentration of 12 .58 (18) with20u1 of normal rabbit plasma as carrier . Samples were centrifuged at 4° C and the supernatants aspirated off . The precipitated izs I-a-endorphin was counted at an efficiency of 708 in a Searle Model 1190 gamma counter . Under these conditions the antiserum to a-endorphin is usable at a final dilution of 1/20,000 with about 408 of the labelled peptide bound in the absence of competing ligand .
a
112 6
Radioi~unoassays for ß-Endorphin
Vol . 22, No .s 13-15, 1978
Radioimmunoassay fo r ß-endorphin . The assay was performed as described for a-endorphin except for the use of 125 2-ß_endorphin as radiolabelled peptide . Antiserum was usable at a final dilution of 1/1500 with about 408 of the labelled peptide bound . Crossreactivity studies . Percent crossreactivity is defined as 100 times the ratio of the IC50 (molar concentration required to inhibit 508 of the antibody binding of labelled peptide) of a-endorphin or ß-endor phin to the IC50 of the test substance . MATERIALS a-endorphin used for antibody production was the generous gift of Dr . Roger Guillemin . All fragments of porcine ß-endorphin (ß p-endorphin) used for crossreactivity studies were prepared by solid state peptide synthesis and were the gift of Dr . ßp -endorphin used to produce antibodies was isoNicholas Ling . lated from Armour porcine MSH concentrate, as described by Gentleman et al (19) . Human ß-lipotropin (ßh-LPH), ovine ß-lipotropin (ß o -LPH), bovine melanocyte stimulating hormone (ßbMSH) and leucines-ßh-endorphin were donated by Dr . C .H . Li . a-endorphi n and camel ß-endorphin (ß c -endorphin) used as standards in the radioimmunoassays were purchased from .Pierce Chemical Company, Rockford, 211 . ßh-endorphin was obtained from Peninsula Laboratories, San Carlos, Calif . Met(O)s-ßh-endorphin (sulfoxide) was prepared and donated by Dr . J .K . Chang of Peninsula Laboratories . a-melanocyte stimulating hormone (aMSH) was obtained from Calbiochem, La Jolla, Calif. Opiate drugs were obtained from the following sources : naloxone hydrochloride, Endo Laboratories ; morphine sulphate and methadone hydrochloride, S .B . Penick ; R-a-acetyl-methadol and naltrexone hydrochlorides, National Institute on Drug Abuse ; ~.-a-acetyl-N-normethadol and k-a-acetyl-N,N-normethadol hydrochlorides, Applied Science Laboratories, Inc . Bovine albumin (fraction V), 1-ethyl-3(3-dimethyl-aminopropyl) carbodümide and bovine thyroglobulin were obtained from Sigma Chemical Co ., St . Louis, Mo . ; polyethylene glycol 6000 was purchased from J .T . Baker Chemical Co ., Phillipsburg, N .J . ; m-aminobenzyloxymethyl cellulose was obtained from Miles-Veda Ltd ., Kankakee, I11 . RESULTS AND DISCUSSION Antibody to a-endorphin . The antibodies raised to the thyroglobulin conjugate of aendorphin are of very high affinity, being capable of measuring that peptide over the range 10-100 pM . Half maximal displacement of 125 1-a-endorphin is obtained at 30 pM nonradioactive a-endorphin (Fig . 2) . This is a concentration of 50 pg/ml or a total of 5 pg in the 100 ul volume used in the assay . ßendorphin, which contains the entire sequence of a-endorphin, crossreacts only 188 with this antiserum . Even this degree
Vol. 22, No .s 13-15, 1978
Radioitmnunoassays for ß-Endorphin
1127
FIG . 2 0
z o ~ m i
S z m x o_ rc S z W
= >K ö M
to
too
tooo
CONCENTRATION OF PEPTIDE - pN
toooo
Displacement of 1251-aendorphin antibody binding by aerial dilutions of a-endorphin (a-E), ßcendorphin (ß-E), ßo-LPH, and tnethionine-enkephalin (MET-ENR) . Results are expressed as percent of total 125 I-a-endorphin binding in the absence of cotapeting ligand .
of crossreactivity permits measurement of ß-endorphin over the range 80-800 pM . The IC50 of the radioimmunoassay with ßc -endorphin as the unlabelled competing ligand is 180 pM (630 pg/ml) (Fig . 2) . The antiserum discriminates between a-endorphin and shorter opioid peptides such~as Met-enkephalin and Leu-enkephalin, which fail to compete for antibody binding at concentrations as high as 1 uM (Table 1) . Biologically inactive fragments of ß-endorphin lacking the entire enkephalin nucleus [ß-endorphin-(6-31, 8-31, 10-31 and 12-31)] are also inactive in the radioimmtmoassay . However, removal of only Tyrl from ßh-endorphin, which destroys all biological activity, does not impair immtmological activity . ß-LPH crossreacts only 5$ with this antiserum. TABLE 1 Crossreactivities with a-Endorphin Antiserum
Substance a-endorphin [ß-endorphin-(1-16)] ß -endorphin ßc-endorphin Mét-enkephalin Leu-enkephalin ß -LPH LPH ßh ß -endorphin-(2-31) ßp-endorphin-(6-31) ßP-endorphin-(8-31) ßp-endorphin-(10-31) ßp-endorphin-(12-31) M~t(0) 5 endorphin -ßh
Percent Cros sreactivity 100 18 14 <0 .004 <0 .004 6 4 18 <.O1 < .O1 < .O1 <.O1 46
Substance
Percent Crossreactivity
Met(0) 5-a-endorphin 80 Leu -ßh-endorphin 1 < .0005 ß -MSH < .0005 abMSH < .0001 Morphine sulfate < .0001 Heroin AC1 < .0001 Methadone HC1 R-a-acetyl methadol HC1 < .0001 k-a-acetyl-Nnormethadol HC1 < .0001 lt-a-acetyl-N,Nnormethadol HC1 <,0001 < .0001 Naloxone HC1 < .0001 Naltrexone HC1
112 8
Radioimmunoassays for ß-Endorphin
Vol . 22, No .s 13-15, 1978
The large discrepancy between the degree of crossreactivity observed with ß-endorphin -(2-31) and -(6-31) is noteworthy, indicating that the antigenic determinant is in the amino-terminal portion of the peptide . Since Met-enkephalin (ß-endorphin-(1-5)) does not crossreact, it follows that the antigenic determinant must include at least residues 5 and 6 (Met-Thr) . In agreement with this interpretation, substitution of Leu for MetS in ßh-endorphin reduces the potency of the peptide 14-fold . Given that the antigenic determinant is localized in the amino-terminal region of a-endorphin, it is interesting that the crossreactivity of ß-endorphin is only 18$ . This failure of ß endorphin to react identically to a-endorphin provides further evidence for a hairpin loop secondary structure in ß-endorphin (20) . Interference from the carboxy-terminal portion of ß-endorphin must impair the recognition of this peptide by the antibody . Existence of such secondary structure has also been suggested by the relative resistance of ß-endorphin to enzymatic degradation as compared with a-endorphin (21) . Surprisingly, oxidation of MetS in ß-endorphin results in a three-fold increase in immunoreactivity . A similar modification of a-endorphin does not enhance the antibody affinity for that ligand . This implies that the MetS residue is important for the maintenance of the ßendorphin secondary structure . The high sensitivity of the a-endorphin antiserum and its recognition of human ß-endorphin should make it useful for routine determinations of circulating ß-endorphin levels in human glasma . Such an assay has been developed and will be described elsewhere . The complete lack of crossreactivity of opiate agonists and antagçnists permits this assay to be used in opiate addicts at various stages of their addiction and during maintenance treatment with methadone, LRAM, and naltrexone . Antibody to B-endorphin . With the antiserum raised to the aminocellulose conjugate of ß -endorphin, 508 displacement of radiolabelled ß-endorphin is a~hieved with 1 .1 nM (3 .8 ng/ml) nonradioactive ßc -endorphin (Fig . 3) . Approximately six-fold loss of sensitivity is incurred in the measurement of ß-endorphin by the use of this antiserum
FIG . 3
Competition of ß~-endorphin (ß-E), ß p -endorphin(2-31), ßp-endorphin-(1031) and ß o -LPH with 125Iß-endorphin for binding to the antiserum raised to ßp-endorphin . .v
~
io
ioo
CDNCENTRATlDN OF PEPTIDE-nM
vol .
22, No .s 13-15,
1978
Radioimmunoassays for ß-Endorphin
1129
TABLE 2 Croasreactivities with ß-Endorphin Antiserum
Substance ß -endorphin ßc-LPH Mét-enkephalin Leu-enlcephalin a-endorphin [ß-endorphin-(1-16)] ß -endorphin-(1-27) ßp-endorphin-(1-19) ßp-endorphin-(2-31) ßp-endorphin-(6-31)
Percent Croasreactivity 100 8 <0 .001 <0 .001 <0 .04 75 <0 .02 75 70
Substance ß -endorphin-(10-31) ßp -endorphin-(14-31) ßp -endorphin-(18-31)
ßp-MSH abMSH Morphine sulfate Methadone HC1 Naloxone HC1 Naltrexone HC1
Percent Crossreactivity 45 .2 <.02 < .004 <,004 < .0001 <,0001 < .0001 < .0001
rather than the antiserum developed to a-endorphin . Crossreactivity studies indicate that this antiserum has entirely different antigenic determinants from the a-endorphin antibody (Table 2) . Fragments derived from the amino-terminal portion of ß-endorphin such as Met-enkephalin and a-endorphin fail to interact . Similarly, carboxy-terminal fragments of ß-endorphin such as ß endorphin-(14-31) and -(18-31) display little crossreactivity . Immunoreactivity is retained only by fragments that contain both a contribution from residues 10-13 and another from the region of residues 20-27 (Table 2) . The data are insufficient to distinguish between two separate antigenic determinants in these regions and a continuous antigenic determinant extending between them, such as residues 13-20 . A number of fragments of ß-endorphin that crossreact with this antiserum cannot compete with iodinated ß-endorphin at some of the antibody binding sites (Fig . 3) . Fragments consisting of residues 2-31 and 10-31 are capable of displacing only 70$ of the A similar pattern of crossreactivity bound iss l-ß_endorphin . (not shown) is observed with ß-endorphin-(6-31) . ß o -LPH, which is only 8$ as effective as ß-endorphin in competing for antibody binding, also shows this anomalous behavior . These findings imply that the antiserum consists of a heterogenous population of antibodies . In addition to the antibodies previously described, there appears to be a second population of different specificity, requiring the presence of amino-terminal Tyrl of ß-endorphin . Blockage of the free amino group by the addition of residues to the left, as in the case of ß-LPH, prevents recognition by this class of antibodies . The presence of this second class of antibody binding sites greatly extends the usefulness and reliability of this radioimmunoassay . A free amino group on Tyrl is an absolute requirement both for biological activity and for the complete displacement of labelled ß-endorphin
1130
Radioimmuaoseeays for ß-Endorphin
Vol . 22, No .s 13-15,
1978
from the antibody . Insurance that a substance being measured in the radioimmunoassay is in fact biologically active can be inferred from its ability to inhibit 100$ of the radiolabelled peptïde binding . Neither inactive metabolites of ß-endorphin nor inactive precursors can satisfy this criterion . ACKNOWLEDGEMENTS We thank Keiko Otsu for expert technical assistance . Dr . J .D . Fisher (Armour Pharmaceuticals, Kankakee, I11.) provided generous gifts of porcine pituitary extracts . We thank Drs . Nicholas Ling, Roger Guillemin, C.H . Li and J . K. Chang for the donation of peptides . We are especially grateful to Dr . Laughton E .M . Miles for his invaluable counsel during the development of these radioimmunoassays . This work was supported by research grant DA01199 and training grant DA07063 from National Institute on Drug Abuse. REFERENCES 1. A.W . KOSTERLITZ and A.J . WATT, Brit . J. Pharmacol . _33, 266-276 (1968) . 2 . J . HUGHES, H .W . KOSTERLITZ and F .M . LESLIE, Brit . J. Pharmacol . _53, 371381 (1975) . 3 . A. GOLDSTEIN, L .I . LOWNEY and B.K . PAL, Proc . Natl . Acad . Sci. U.S .A . _68, 1742-1747 (1971) . 4 . C .B, PERT and S .H . SNYDER, Science _179, 1011-1014 (1973) . 5 . E.J . SIMON, J .M . HILLER and'I . EDELMAN, Proc . Natl . Acad . Sci. U .S .A . _70, 1947-1949 (1973) . 6 . J. HUGHES, T .W . SMITH, H.W . KOSTERLITZ, L.A . FOTHERGILL and B.A . MORGAN, Nature _258, 577-579 (1975) . 7 . H. TESCIÛ;LIACHER, R.E . OPAEIM, B .M . COX and A. GOLDSTEIN, Life Sci . _16, 1771-1776 (1975) . 8 . B.M . COX, .R .E . OPHEIM, H. TESCAEMACHER and A . GOLDSTEIN, Life Sci . _16, 1777-1782 (1975) . 9 . M. ROSS, T.-P. SU, B .M . COX and A. GOLDSTEIN, Opiates and Endogenous Opioid Peptides , p . 35-40, North Holland Publishing Co ., Amsterdam (1976) . 10 . B.M . COX, A. GOLDSTEIN and C .A . LI, Proc . Natl . Acad . Sci . U .S .A. ._73, 18211823 (1976) . 11 . R.E . MAINS, B .A . EIPPER and N . LING, Proc . Natl . Acad . Sci. U.S .A . _74, 3014-3018 (1977) . 12 . J.L . ROBERTS and E . HERBERT, Proc . Natl . Acad . Sci . U .S .A . _74, 4826-4830 (1977) . 13 . R. GUILLEMIN, N. LING and T .M . VARGO, Biochem. Biophys . Res . Commun . 77, 361-366 (1977) . 14 . W.R . SROWSKY and D.A . FISHER, J. Lab . Clin . Med . 80, 131-144 (1972) .
15 . N.R . MOUDGAL and R.R . PORTER, Biochem . Biophys . Acta _71, 185-187 (1963) . 16 . J . VAITUKAITIS, J .B . ROBBINS, E . NIESCHLAG and G .T . ROSS, J . Clin . Endocr . 33, 988-991 (1971) . 17 . W.M . HUNTER and F .C . GREENWOOD, Nature _194, 495-496 (1962) . 18 . B. DESBUQUOIS and G.D . AURBACH, J . Clin . Endocr . _33, 732-738 (1971) . 19 . S . GENTLEMAN, L.I . LOWNEY, B.M . COX and A. GOLDSTEIN, J . Chromat . (in press, 1978) . 20 . M. HOLLÔSI, M . KAJTl,R and L. GRl~F, FEBS Letter _74, 185-189 (1977) . 21 . B .M . HUSTEN and D.G . SMITH, Biochem. Biophys . Res . Commun. 77, 86-94 (1977) .
rergamon Press
OF PROPERTIES OF RADIOIMMUNOASSAYS FOR ß-ENDORPHIN : COMPARISON * TWO ANTISERA Maureen Ross, Vertan Ghazarossian, B .M . Cox and Avram Goldstein Addiction Research Foundation, and Department of Pharmacology, Stanford University, Palo Alto, California 94304
The opioid activity of ß-endorphin depends upon a free amino-terminal Tyr residue . Larger precursor peptides are inactive, as is the metabolic product from which Tyrl has been cleaved . Immunoassays have the advantage of great sensitivity but the disadvantage of ambiguity. Ambiguity arises when the antigenic determinant is a sequence not required for biological activity . Two antisera are described with antigenic determinants not hitherto reported . One of these is suitable for assays of ß-endorphin in human plasma . The other contains an antibody that recognizes ß-endorphin only if amino-terminal Tyr is présent, thus achieving a coincidence of immunoreactivity'and opioid activity . Development of reliable methodology for the detection and quantitation of small concentrations of potent biologically active compounds must necessarily precede their discovery and isolation . Unquestionably, the development of two bioassay systems for the opioid drugs, the myenteric plexus longitudinal muscle preparation (1) and the mouse vas deferens (2), and the development of the opiate receptor binding assay (3,4,5) were of paramount importance in the identification of the endogenous ligands for the opioid receptor . It was apparent even in the very early work on endorphins that there was more than one endogenous ligand for the opiate receptor . Concurrent with the determination of the structure of two brain opioid peptides, methionine (Met)-enkephalin and leucine (Leu)-enkephalin (6), were reports of a much larger opioid peptide in pituitary (7,8) . Opioid activity in brain was subsequently shown to be associated with much larger molecular weight species as well as the enkephalins (9) . *Presentéd at a symposium in honor of Otto Krayer, Tucson, Arizona, March 11, 1978 .
0300-9653/78/0410-1123$02 .00/0
Copyright © 1978 Pergamon Press
1124
Radioimmunoassays for ß-Endorphin
Vol . 22, No .s 13-15, 1978
Met-enkephalin is containéd within the pituitary hormone ßlipotropin (ß-LPH) as residues 61-65 (Fig . 1) . Realization of this fact quickly led to the discovery that other fragments of ß-LPH which possessed residue 61 as the amino-terminal were also active in the opioid assays with ß-endorphin (ß-LPH-(61-91)) being most potent . ß-LPH, which lacks the free amino group on Tyr 6 i, is devoid of opioid activity (10) and is presumed to represent an inactive precursor from which the endorphins are derived by limited proteolysis . More recently, ß-LPH itself has been shown to be present as the C-terminal amino acid sequence of a 31000 dalton precursor which also contains ACTH (11,12) . a-endorphin Net-enkephalin H - Tyr - Gly - Gly - Phe - lYt - Thr - fier - Glu - Lye - Sez - Gln - Thr 1 Z 5 9 10 Pro
(Ile)~t
13
Aen - Lys - Val - Ils - Ale - Aen - Lye - PM - Lev - Thr - Val - Uu/ / ZS Z3 20 16 15 Ala \His - Lye - Lys - Cly - Gln - OH Z7 30 31 (7Yr)t
(Glu - 09)t
FIG . 1 Structure of porcine ßp -endorphin . Precursor peptide, ß-LPH, contains 91 amino acids ; its carboxy terminal sequence of 31 residues is ß-endorphin, shown here . Thus Tyr61 of ß-LPH is Tyr l of ß-endorphin . Sequence 1-5 is Met-enkephalin ; sequence 1-16 is a-endorphin . * a substitution at indicated position in sheep (ß o-) or camel (ß c-) endorphin . t ~ substitutions at indicated positions in human ßh-endorphin . Hairpin-loop secondary structure ie represented symbolically ; precise structure is unknown .
While the bioassays and receptor binding assay possess the overriding advantage of measuring only biologically active species they cannot qualitatively distinguish one substance with opioid activity from another . Increasingly, radioimmunoassay is being used to measure both the enkephalins and ß-endorphin in tissue extracts and biological fluids . Ideâlly, the inherent specificity of antibodies could permit the unique determination of a particular endorphin in a single assay system . For peptide hormones, the reliability of radioimmunoassay for the measurement of a single substance has become increasingly uncertain as the generality of the pre-hormone ~ prohormone i hormone synthetic mechanism has beçome better established . Difficulties arise in the interpretation of data based solely on immunoreactivity, especially when antisera are used for which the antigenic determinants are distinct from the parts of the molecule needed for biological activity . For example, a widely used antibody, which recognizes the C-terminal region of ß-endorphin (13), reacts with the active ß-endorphin, the inactive ß -LPH, and the inactive 31K precursor . It would also recognize des-Tyrß-endorphin (i .e . ß-endorphin-(2-31)), which is inactive, but it fails to recognize a.-endorphin or the enkephalins, which are active .
Vol . 22, No .s 13-15, 1978
Radioi~unoassays for ß-Endorphin
1125
We have developed antibodies both to the intact ß-endorphin molecule and to its amino-terminal fragment, a-endorphin(ß-endorphin-(1-16)) . Use of a-endorphin as the-immunizing agent not only increases the probability that the antiserum developed will respond to an antigenic determinant that encompasses the biologically essential part of the ß-endorphin structure, but also eliminates the problem of species variations . All known species variants of ß-endorphin contain substitutions only in the C-terminal half of the molecule . METHODS Peptide Conjugation and Immunization Procedure To elicit antibody production, a-endorphin was conjugated to bovine thyroglobulin with 1-ethyl-3(3-dimethyl-amino-propyl)carbodiimide using molar ratios of peptide :carrier :conjugating agent of 118 :1 :200 as described by Skowsky and Fisher (14) . ßendorphin was coupled to m-aminobenzyloxymethyl cellulose by a modification of the procedure of Moudgal et al (15) . 1 mg of ß-endorphin was used per 6 .5 mg of m-aminobenzyloxymethyl cellulose . The peptide conjugates in isotonic saline were homogenized with an equal volume of Freund's complete adjuvant . Adult male New Zealand White rabbits were injected intradermally with 100 200 ug of the conjugated peptide as described by Vaitukaitis et al . (16) . After an initial series of three immunizations at fourweek intervals, animals received booster injections at three-month intervals . Radiolabelled peptides izs izs I-ß-endorphin were prepared by a l-a-endorphin and Iomodification of the procedure of Hunter and Greenwood (17) . dinated a-endorphin was purified by gel filtration on a Sephadex G-15 column (24 x 1 .2 cm) eluted with 50 mM sodium phosphate bufIodinfer containing 0 .18 bovine serum albumin and 50 mM RC1 . ated ß-endorphin was purified in a similar manner using Sephadex G-25 . Radioimmunoassay for a-endorphin Test substances or a-endorphin in 100 ul of 50 mM sodium phosphate buffer containing 0 .18 bovine serum albumin and 0 .98 NaCl, were incubated with 100 u 1 of antiserum of the appropriate dilution . The initial incubation was carried out at 4° C for a minimum of 48 hours, at which time 100 ul of iodinated a-endorand a second phin (1000-5000 cpm) in the same buffer was added 1zs Antibody-bound l-a-endorphin incubation (24 hours) commenced . was separated from free peptide by the addition of polyethylene glycol to final concentration of 12 .58 (18) with20u1 of normal rabbit plasma as carrier . Samples were centrifuged at 4° C and the supernatants aspirated off . The precipitated izs I-a-endorphin was counted at an efficiency of 708 in a Searle Model 1190 gamma counter . Under these conditions the antiserum to a-endorphin is usable at a final dilution of 1/20,000 with about 408 of the labelled peptide bound in the absence of competing ligand .
a
112 6
Radioi~unoassays for ß-Endorphin
Vol . 22, No .s 13-15, 1978
Radioimmunoassay fo r ß-endorphin . The assay was performed as described for a-endorphin except for the use of 125 2-ß_endorphin as radiolabelled peptide . Antiserum was usable at a final dilution of 1/1500 with about 408 of the labelled peptide bound . Crossreactivity studies . Percent crossreactivity is defined as 100 times the ratio of the IC50 (molar concentration required to inhibit 508 of the antibody binding of labelled peptide) of a-endorphin or ß-endor phin to the IC50 of the test substance . MATERIALS a-endorphin used for antibody production was the generous gift of Dr . Roger Guillemin . All fragments of porcine ß-endorphin (ß p-endorphin) used for crossreactivity studies were prepared by solid state peptide synthesis and were the gift of Dr . ßp -endorphin used to produce antibodies was isoNicholas Ling . lated from Armour porcine MSH concentrate, as described by Gentleman et al (19) . Human ß-lipotropin (ßh-LPH), ovine ß-lipotropin (ß o -LPH), bovine melanocyte stimulating hormone (ßbMSH) and leucines-ßh-endorphin were donated by Dr . C .H . Li . a-endorphi n and camel ß-endorphin (ß c -endorphin) used as standards in the radioimmunoassays were purchased from .Pierce Chemical Company, Rockford, 211 . ßh-endorphin was obtained from Peninsula Laboratories, San Carlos, Calif . Met(O)s-ßh-endorphin (sulfoxide) was prepared and donated by Dr . J .K . Chang of Peninsula Laboratories . a-melanocyte stimulating hormone (aMSH) was obtained from Calbiochem, La Jolla, Calif. Opiate drugs were obtained from the following sources : naloxone hydrochloride, Endo Laboratories ; morphine sulphate and methadone hydrochloride, S .B . Penick ; R-a-acetyl-methadol and naltrexone hydrochlorides, National Institute on Drug Abuse ; ~.-a-acetyl-N-normethadol and k-a-acetyl-N,N-normethadol hydrochlorides, Applied Science Laboratories, Inc . Bovine albumin (fraction V), 1-ethyl-3(3-dimethyl-aminopropyl) carbodümide and bovine thyroglobulin were obtained from Sigma Chemical Co ., St . Louis, Mo . ; polyethylene glycol 6000 was purchased from J .T . Baker Chemical Co ., Phillipsburg, N .J . ; m-aminobenzyloxymethyl cellulose was obtained from Miles-Veda Ltd ., Kankakee, I11 . RESULTS AND DISCUSSION Antibody to a-endorphin . The antibodies raised to the thyroglobulin conjugate of aendorphin are of very high affinity, being capable of measuring that peptide over the range 10-100 pM . Half maximal displacement of 125 1-a-endorphin is obtained at 30 pM nonradioactive a-endorphin (Fig . 2) . This is a concentration of 50 pg/ml or a total of 5 pg in the 100 ul volume used in the assay . ßendorphin, which contains the entire sequence of a-endorphin, crossreacts only 188 with this antiserum . Even this degree
Vol. 22, No .s 13-15, 1978
Radioitmnunoassays for ß-Endorphin
1127
FIG . 2 0
z o ~ m i
S z m x o_ rc S z W
= >K ö M
to
too
tooo
CONCENTRATION OF PEPTIDE - pN
toooo
Displacement of 1251-aendorphin antibody binding by aerial dilutions of a-endorphin (a-E), ßcendorphin (ß-E), ßo-LPH, and tnethionine-enkephalin (MET-ENR) . Results are expressed as percent of total 125 I-a-endorphin binding in the absence of cotapeting ligand .
of crossreactivity permits measurement of ß-endorphin over the range 80-800 pM . The IC50 of the radioimmunoassay with ßc -endorphin as the unlabelled competing ligand is 180 pM (630 pg/ml) (Fig . 2) . The antiserum discriminates between a-endorphin and shorter opioid peptides such~as Met-enkephalin and Leu-enkephalin, which fail to compete for antibody binding at concentrations as high as 1 uM (Table 1) . Biologically inactive fragments of ß-endorphin lacking the entire enkephalin nucleus [ß-endorphin-(6-31, 8-31, 10-31 and 12-31)] are also inactive in the radioimmtmoassay . However, removal of only Tyrl from ßh-endorphin, which destroys all biological activity, does not impair immtmological activity . ß-LPH crossreacts only 5$ with this antiserum. TABLE 1 Crossreactivities with a-Endorphin Antiserum
Substance a-endorphin [ß-endorphin-(1-16)] ß -endorphin ßc-endorphin Mét-enkephalin Leu-enkephalin ß -LPH LPH ßh ß -endorphin-(2-31) ßp-endorphin-(6-31) ßP-endorphin-(8-31) ßp-endorphin-(10-31) ßp-endorphin-(12-31) M~t(0) 5 endorphin -ßh
Percent Cros sreactivity 100 18 14 <0 .004 <0 .004 6 4 18 <.O1 < .O1 < .O1 <.O1 46
Substance
Percent Crossreactivity
Met(0) 5-a-endorphin 80 Leu -ßh-endorphin 1 < .0005 ß -MSH < .0005 abMSH < .0001 Morphine sulfate < .0001 Heroin AC1 < .0001 Methadone HC1 R-a-acetyl methadol HC1 < .0001 k-a-acetyl-Nnormethadol HC1 < .0001 lt-a-acetyl-N,Nnormethadol HC1 <,0001 < .0001 Naloxone HC1 < .0001 Naltrexone HC1
112 8
Radioimmunoassays for ß-Endorphin
Vol . 22, No .s 13-15, 1978
The large discrepancy between the degree of crossreactivity observed with ß-endorphin -(2-31) and -(6-31) is noteworthy, indicating that the antigenic determinant is in the amino-terminal portion of the peptide . Since Met-enkephalin (ß-endorphin-(1-5)) does not crossreact, it follows that the antigenic determinant must include at least residues 5 and 6 (Met-Thr) . In agreement with this interpretation, substitution of Leu for MetS in ßh-endorphin reduces the potency of the peptide 14-fold . Given that the antigenic determinant is localized in the amino-terminal region of a-endorphin, it is interesting that the crossreactivity of ß-endorphin is only 18$ . This failure of ß endorphin to react identically to a-endorphin provides further evidence for a hairpin loop secondary structure in ß-endorphin (20) . Interference from the carboxy-terminal portion of ß-endorphin must impair the recognition of this peptide by the antibody . Existence of such secondary structure has also been suggested by the relative resistance of ß-endorphin to enzymatic degradation as compared with a-endorphin (21) . Surprisingly, oxidation of MetS in ß-endorphin results in a three-fold increase in immunoreactivity . A similar modification of a-endorphin does not enhance the antibody affinity for that ligand . This implies that the MetS residue is important for the maintenance of the ßendorphin secondary structure . The high sensitivity of the a-endorphin antiserum and its recognition of human ß-endorphin should make it useful for routine determinations of circulating ß-endorphin levels in human glasma . Such an assay has been developed and will be described elsewhere . The complete lack of crossreactivity of opiate agonists and antagçnists permits this assay to be used in opiate addicts at various stages of their addiction and during maintenance treatment with methadone, LRAM, and naltrexone . Antibody to B-endorphin . With the antiserum raised to the aminocellulose conjugate of ß -endorphin, 508 displacement of radiolabelled ß-endorphin is a~hieved with 1 .1 nM (3 .8 ng/ml) nonradioactive ßc -endorphin (Fig . 3) . Approximately six-fold loss of sensitivity is incurred in the measurement of ß-endorphin by the use of this antiserum
FIG . 3
Competition of ß~-endorphin (ß-E), ß p -endorphin(2-31), ßp-endorphin-(1031) and ß o -LPH with 125Iß-endorphin for binding to the antiserum raised to ßp-endorphin . .v
~
io
ioo
CDNCENTRATlDN OF PEPTIDE-nM
vol .
22, No .s 13-15,
1978
Radioimmunoassays for ß-Endorphin
1129
TABLE 2 Croasreactivities with ß-Endorphin Antiserum
Substance ß -endorphin ßc-LPH Mét-enkephalin Leu-enlcephalin a-endorphin [ß-endorphin-(1-16)] ß -endorphin-(1-27) ßp-endorphin-(1-19) ßp-endorphin-(2-31) ßp-endorphin-(6-31)
Percent Croasreactivity 100 8 <0 .001 <0 .001 <0 .04 75 <0 .02 75 70
Substance ß -endorphin-(10-31) ßp -endorphin-(14-31) ßp -endorphin-(18-31)
ßp-MSH abMSH Morphine sulfate Methadone HC1 Naloxone HC1 Naltrexone HC1
Percent Crossreactivity 45 .2 <.02 < .004 <,004 < .0001 <,0001 < .0001 < .0001
rather than the antiserum developed to a-endorphin . Crossreactivity studies indicate that this antiserum has entirely different antigenic determinants from the a-endorphin antibody (Table 2) . Fragments derived from the amino-terminal portion of ß-endorphin such as Met-enkephalin and a-endorphin fail to interact . Similarly, carboxy-terminal fragments of ß-endorphin such as ß endorphin-(14-31) and -(18-31) display little crossreactivity . Immunoreactivity is retained only by fragments that contain both a contribution from residues 10-13 and another from the region of residues 20-27 (Table 2) . The data are insufficient to distinguish between two separate antigenic determinants in these regions and a continuous antigenic determinant extending between them, such as residues 13-20 . A number of fragments of ß-endorphin that crossreact with this antiserum cannot compete with iodinated ß-endorphin at some of the antibody binding sites (Fig . 3) . Fragments consisting of residues 2-31 and 10-31 are capable of displacing only 70$ of the A similar pattern of crossreactivity bound iss l-ß_endorphin . (not shown) is observed with ß-endorphin-(6-31) . ß o -LPH, which is only 8$ as effective as ß-endorphin in competing for antibody binding, also shows this anomalous behavior . These findings imply that the antiserum consists of a heterogenous population of antibodies . In addition to the antibodies previously described, there appears to be a second population of different specificity, requiring the presence of amino-terminal Tyrl of ß-endorphin . Blockage of the free amino group by the addition of residues to the left, as in the case of ß-LPH, prevents recognition by this class of antibodies . The presence of this second class of antibody binding sites greatly extends the usefulness and reliability of this radioimmunoassay . A free amino group on Tyrl is an absolute requirement both for biological activity and for the complete displacement of labelled ß-endorphin
1130
Radioimmuaoseeays for ß-Endorphin
Vol . 22, No .s 13-15,
1978
from the antibody . Insurance that a substance being measured in the radioimmunoassay is in fact biologically active can be inferred from its ability to inhibit 100$ of the radiolabelled peptïde binding . Neither inactive metabolites of ß-endorphin nor inactive precursors can satisfy this criterion . ACKNOWLEDGEMENTS We thank Keiko Otsu for expert technical assistance . Dr . J .D . Fisher (Armour Pharmaceuticals, Kankakee, I11.) provided generous gifts of porcine pituitary extracts . We thank Drs . Nicholas Ling, Roger Guillemin, C.H . Li and J . K. Chang for the donation of peptides . We are especially grateful to Dr . Laughton E .M . Miles for his invaluable counsel during the development of these radioimmunoassays . This work was supported by research grant DA01199 and training grant DA07063 from National Institute on Drug Abuse. REFERENCES 1. A.W . KOSTERLITZ and A.J . WATT, Brit . J. Pharmacol . _33, 266-276 (1968) . 2 . J . HUGHES, H .W . KOSTERLITZ and F .M . LESLIE, Brit . J. Pharmacol . _53, 371381 (1975) . 3 . A. GOLDSTEIN, L .I . LOWNEY and B.K . PAL, Proc . Natl . Acad . Sci. U.S .A . _68, 1742-1747 (1971) . 4 . C .B, PERT and S .H . SNYDER, Science _179, 1011-1014 (1973) . 5 . E.J . SIMON, J .M . HILLER and'I . EDELMAN, Proc . Natl . Acad . Sci. U .S .A . _70, 1947-1949 (1973) . 6 . J. HUGHES, T .W . SMITH, H.W . KOSTERLITZ, L.A . FOTHERGILL and B.A . MORGAN, Nature _258, 577-579 (1975) . 7 . H. TESCIÛ;LIACHER, R.E . OPAEIM, B .M . COX and A. GOLDSTEIN, Life Sci . _16, 1771-1776 (1975) . 8 . B.M . COX, .R .E . OPHEIM, H. TESCAEMACHER and A . GOLDSTEIN, Life Sci . _16, 1777-1782 (1975) . 9 . M. ROSS, T.-P. SU, B .M . COX and A. GOLDSTEIN, Opiates and Endogenous Opioid Peptides , p . 35-40, North Holland Publishing Co ., Amsterdam (1976) . 10 . B.M . COX, A. GOLDSTEIN and C .A . LI, Proc . Natl . Acad . Sci . U .S .A. ._73, 18211823 (1976) . 11 . R.E . MAINS, B .A . EIPPER and N . LING, Proc . Natl . Acad . Sci. U.S .A . _74, 3014-3018 (1977) . 12 . J.L . ROBERTS and E . HERBERT, Proc . Natl . Acad . Sci . U .S .A . _74, 4826-4830 (1977) . 13 . R. GUILLEMIN, N. LING and T .M . VARGO, Biochem. Biophys . Res . Commun . 77, 361-366 (1977) . 14 . W.R . SROWSKY and D.A . FISHER, J. Lab . Clin . Med . 80, 131-144 (1972) .
15 . N.R . MOUDGAL and R.R . PORTER, Biochem . Biophys . Acta _71, 185-187 (1963) . 16 . J . VAITUKAITIS, J .B . ROBBINS, E . NIESCHLAG and G .T . ROSS, J . Clin . Endocr . 33, 988-991 (1971) . 17 . W.M . HUNTER and F .C . GREENWOOD, Nature _194, 495-496 (1962) . 18 . B. DESBUQUOIS and G.D . AURBACH, J . Clin . Endocr . _33, 732-738 (1971) . 19 . S . GENTLEMAN, L.I . LOWNEY, B.M . COX and A. GOLDSTEIN, J . Chromat . (in press, 1978) . 20 . M. HOLLÔSI, M . KAJTl,R and L. GRl~F, FEBS Letter _74, 185-189 (1977) . 21 . B .M . HUSTEN and D.G . SMITH, Biochem. Biophys . Res . Commun. 77, 86-94 (1977) .