Polyclonal anti-idiotypic opioid receptor antibodies generated by the monoclonal β-endorphin...

Vol. 132, No. 2, 1985

October 30, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 658-665

POLYcImAL AmI-ILxmYP1c OPIOID RECEPTDRAMlTBCDIESGENE%TED BY 'I'HE MCNCUN& E-ENEORPHIN ANJJIBcp>Y 3-E7

RWiger Schulz and Christian Gram&

Department of Neurophamacology, Max-Planck-Institut flir Psychiatric, Am Klopferspitz 18a, D-803.3 Planegg-Martinsried, Federal Republic of Germany

Received August 19, 1985

SUWARY: Anti-idiotypic antibodies were raised in rabbits against the mmo- clonal B-endor@in antibody 3-E7. ing to the 3-E7 antibody,

Thesg antibodies inhibit B-endorphin bind- bind& of H-diprenorphine to solubilized opioid

receptors and the binding of I-B-endorphin to rat brain mmbranes. EX- posure of NG-108CC15 hybrid cells to anti-idiotypic antibodies produces an opioid-like inhibition of FGE -stimulated cMP acmlation. These data suggest that the antibodies rai 's&d by the anti-idiotypic route both bind to and activate opioid receptors. 0 1985 Academic Press, In-=.

Antibodies have been successfully employ& for the study of the structure

and function of mmbrane-associated receptors (1). T%e majority of these

investigations ware conducted with idiotypic antibodies, that is, the re-

ceptors to be investigated were purified and this material served as inmuno-

gen. In the absence of receptor material an alternative method, the anti-

idiotypic route, has been proven useful for the generation of antibodies

against specific receptor types (2,3). The raising of anti-idiotypic anti-

bodies (anti-ID) requires an anti-hornme antibody as antigen. A resultant

anti-ID my behave as the "internal image" of the anti-hormone imnunoglobu-

lin. Consequently, the anti-ID may interact with the receptor with which the

hormone interferes (4).

We are reporting about the generation of polyclonal anti-ID[s directed

against the opioid receptor (a preliminary report (5) was given at the Inter-

national Narcotic Research Conference, 1984). As an jmnunogen, the mnoclonal

3-E7 antibody, which recognizes virtually all knm endqenous opioid

peptides, was employed (6). It has been suggested that this idiotypic anti-

body displays the specific requirewnts of an opioid receptor (7). It was

ass&, thus, that the 3-E7 antibody would be of use in the raising of

0006-291X/85 $1.50 Copyright 0 I985 by Academic Press, Inc. All rights of reproduction in any form reserved. 658

Vol. 132, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

opioid receptor anti-ID’s. Recently, the anti-idiotypic route of generating

antibodies to opioid receptors proved successful employing anti-morphine

antibodies as antigens (8,9).

METHCOS

Imnunization: The 3-E7 antibody was raised as described by Gramsch et al. (6), and material purified by PA-Sepharose was used for immnization. Male rabbits (New Zealand, 2.5 kg) were injected intrademally with the 3-E7 (1 rag/animal) in FYeund's canplete adjuvant. Booster inmunizations were per- formed at 4 W intervals. 10 days after the last imnmization, at the earliest, the animals were bled.

Isolation of anti-ID's: The individual antisera were submitted to immmoaf- finity chramtography (3-E7 antibcdy linked to Sepharose CL-4B). In order to increase the likelihrxd of separating an anti-ID directed against opioid receptors, leu-enkephalin (200 fl) was employed for elution. This opioid pentapeptide contains the message sequence of amino acids with which the 3-E7 interferes. Anti-ID’s directed to other structures of the 3-E7 are assuaed to be of no or little relevance for opioid receptors, and should not be eluted by leu-enkephalin. The eluate (20 ml.) was concentrated to 2 ml on a Amicon manbrane (cut off loo00 molecular weight) and subjected to gel filtration (Sephacryl S-300, 2.5 x 90 can, 0.1 M ammnim acetate, pH 7.4, 4OC) in the presence of 0.01% bovine serum albumine (BSA). 1Qnl fractions (flaw rate 0.25 ml/nun) were collected, lyophilized and examined for anti-ID activity enploy- ing a solid-phase radioimmnoassay (RIA).

Solid-phase PIA: Microtitre plates (Costar 2596, Cambridge, MA, USA) were coated with 3-E7 antibody (100 ~1 per ~11; 1 rrg/ml phosphate-buffered sali- ne, PBS) for 4h at 37'C. The plates were washed twice in PBS, containing 0.1% m 20, and saturated for 4h at 37OC with 3% BSA in PBS. The plates were @p washed in PBS/O.l% 'Ityeen 20 and distilled water. For the assay, 50 bl of

I-B-endozphin (loo00 cpn) was incubated with 100 ~1 of the material to be tested in buffer D (0.02 M phosphate buffer, pH 7.4, containing 0.15 M NaCl, O,Ol% thimerosal, 0.1% gelatine, 0.01% BSA, 0.1% Triton X-100) for 24h at 4 C. The plates wsre then washed three times in PBS/O.l% TWen 20 and distilled water. The wells were counted for radioactivity.

Opioid receptor binding: 1. Bat brain membranes: 2% corty hcmogenate (20 a~ Hepes-buffer, pH 7.4) was centrifuged (40 min, 35$CCg, 4 C). The pellet was dissolved in 50 ml soepes, containing 10 rrM di-K EIYTA. This hamgenate was kept for 15 min at 30 C and again centrifuged (15 min, 35ooOg). The pellet is taken up in Hepes (50 ml, pH 7.4), containing 3 nM MgSO,, 1COcrg BSA/ml and 5Qpg bacitracin/ml. For t$e test, 25~1 h-9 were incubated with 5&l of anti-ID's in Hepes at 30 C. After 1 h, 25),~1 I-B-eudorphin (250COcpn) was added and the incubation continued for a further 45 min. The tubes were centrifuged (clmin, Eppendorf centrifuge 5413) and the pellets counted for radioactivity.

2. Solubilized receptors, rat brain membranes: Receptor solubilization of brain (minus cerebellum) was carried out as described (10) at O°C by vigo- rously shak% for 300min in the presence of 2% digitonin. After ultracentri- fugation (10 g, lh, 4 C), the supematant was used for binding studies. The test consisted of 100111 so13ubilized receptor supernatant, la1 50 n@l Tris- buffer (pH 7.4) containing H-diprenorphine (25OCO cpn) and 50 ~1 Tr&s-buf$r containing the anti-ID. This mixture was incubated for 15 h at 4 C. 10 M cold diprenorphine was extpJoyed for calculation of the specific binding. Sepa- ration of bound fran free H-diprenorphine was performad by use of gel-*- tcg-raphy (Sephadex G50 sf; 90). The tritim activity per fraqion was counted following addition of "scintillation mix". The procedure for H-flu@trapezam bindingwasmdified:100 )~loflOg supernatantwas i.ncubatedatO~with 50 1.11 '&is-buffer, containing the anti-ID material. After 14 h 100 ~1 H-fluui-

659


trapszam (50X0 cpn) in Tris-buffer was added for a further 6Omin incubation. Separat3ion,of bound frcm f=r tritiated material was conducted as described for H-diprenorphine. 10 M cold flunitrapezam was employed for calculation of specific binding.

Determination of CAMP in intact NG108cC15 hybrid cells: Cells ware cultured as described by Hamprecht (11) -and harvested at the state of confluency. 2x10' cells were incubated at 37OC with the anti-ID material in LWEM mad&m containing 25 nM Hepes (final volum 80 ~1) at 37OC. After 1 h, the cells ware stimulated in the presence of Ho 2~T1724 (phosphodiesterase inhibitor)

medium.J&cpmure to the stable D-Ala ,D-Ieu-enkephalin (10 M) was conducted with PGE (final concentration each 10 M), each dissol? in 10~1 DEEM

10 mingrior to stimulation. The p &ductio% of cAMP was terminated by O.lN HCl (95 C) and CAMP was assayed by RIA (12).

Separation of anti-ID material by PA-Sepharose: 350 ~1 of PA-Gel in amwnim acetate (O.lM, pH 7.4) was packed in a cclumn (inner diameter W) and the buffer was sucked off. lC@l buffer containing the anti-ID material was pi- petted onto the gel. After lh at roan temperature, the dynamic phase was removed and tested for biologic activity. Control experimnts employed BSA- saturated Sepharose.

Isolated tissues: The guinea-pig ileum @I) (13) and the muse vas deferens (MVD) (14) were set up in 2 ml tissue bathes for electrical stimulation (60 V, 0.1 Hz, 0.5 msec). A naloxone-reversible inhibition of electrically ewked twitch tension is demonstrative for an opioid-like action.

Drugs and reagents: B-Endorphin (human), leucine-enkephalin, CD-Ala2,D- Leu5lEnkephalin (DADL) ms purchased fran Bachem (Switzerland). Protein A-Se- pharose (PA-Sepharose) and Sephadex G50 sf was from Pharmcia (Freiburg, Genmny).Dukc~'s WdifiedEagle Medium (DEEM) was franGibco (Karlsruhe, Germany). Other substrates ("reagent grade") were purchased either frm Sigma

Germany) or fran Calbiochem (Frankfurt, Germany). [N-mthyl- Ctyrosyl-3,5- HlEnkephali@l-leucine), 38

2ooO Ci/numl, and C Ityrosine-cAMP, 2ocO Ci/ ml, were frcan Amrsham Buchler (Brauuschweig, Germany).

Solid-phase RIA, screening: 12 rabbits were imnunized with the 3-E7 antibody

and screened for anti-ID, not earlier following the second booster injection.

The material eluted frm Sephacryl S-300 (fractions 6-26) was subjected to

the solid-phase HIA. All rabbit sera proved positive, that is, activity was

found at fractions 10 and 22, but mainly at fraction 18, where imnunoglobulin

IgG elutes. These positive reactions are not due to the presence of leu-enke-

phalin which was employed to elute the 3-E7 affinity column, since controls

with 3H-leu-enkephalin revealed its canplete separation by ultrafiltration.

In addition, enkephalin elutes frcnn Sephacryl S-300 beyond fraction 26. Sera

fran rabbits not immnized with 3-E7 displayed a very low activity in the

solid-phase FUA in the fractions indicated.

Vol. 132, No. 2. 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10 50

“fraction 18” I/l11

Figure 1: Inhibition of specifically bound 3 HiFiprenorPhine to solubilized rat brain opioid receptors by anti-ID antibodies ("fraction 18“). Sepharose t.reatnEntof "fraction 18" (0) did not affect the inhibitory capacity, whereas PA-Sepharose treated material (0) lost its biologic activity. t&ta are representative of ti expertits.

Fkxeptor binding studies, solubilized material: Senrm of rabbits repeatedly

boostered and bled were individually subjected to the solid-phase RIA. Frac-

tions 10, 18 and 22 were tested in the radio receptor assay by use of solu-

bilized brain material. This test revealed that only "fraction 18" of a

single rabbit interfered with the binding of diprenorphine. This material of

several bleedings was pooled and used in all further tests. Figure 1 displays

the percentage inhibition of 3H-diprenorphine binding to solubilized

receptors by "fraction 18" material, which was exposed to either Sepharose or

PA-Sepharose. The Sepharose treated "fraction 18" inhibited up to 60% of

3 H-diprenorphine binding, shckng a half-maxkl effect at 15 @l (1 ~1 of

"fraction 18" equals 100 ~1 sennn). In contrast, PA-Sepharose treated

material ccxnpletely lost its ability to interfere with opioid binding. The

lCXXK?Cg supematant also binds to 3 H-flunitrapezam. It was, thus, tested

&et&r "fraction 18" material (Sepharose treated and PA-Sepharose treated)

interferes with the binding of this non-opioid to solubilized receptors. Both

probes failed to affect flunitrapezam binding in anxxnts of up to 50~1.

Receptor binding studies, membrane fragments: Experiments were also con-

ducted with brain membranes. Incubation of membranes for 16 h at S°C caused a

loss of specific binding for 'H-diprenorphine, which displayed a specific

binding of only 20 to 40 %. In contrast, 90% binding is obtained at 35OC for

lh. Pre-incubation of membrane fragments with "fraction 18" (Sepharose

treated, lh, 3S°C), using identical concentrations as for solubilized recep-

661


“fraction 18” lI.rll

Figure 2: Inhibition of specifically bound 125 I-&endo*in to rat brain membranes by anti-ID antibodies ("fraction 18"). For further explanation see fiqre 1.

tom (figure 11, did not result in any inhibition of 3 H-diprenorphine bind-

ing. Since E-endorphin served as an imnunogen for the raising of the 3-E7

antibody, the receptor studies were extended to test the ability of "fraction

18" to interfere with 12' I-B-endorphin. Figure 2 demnstrates the potency of

"fraction 18" in inhibiting 1251-E-endorphin binding. 20 kl caused 90% inhi-

bition of specific binding and 6 (~1 was required for a half-maximal effect.

Again, PA-Sepharose treatment of "fraction 18" eliminated all binding capaci-

ty. Heat treatment (95OC, 30 min) of "fraction 18" also destroyed the opioid

receptor activity (data not shown).

NG108CYZ15 hybrid cells, CAMP content: The ability of "fraction 18" to acti-

vate opioid receptors was tested in hyhrid cells, employing inhibition of

CAMP accmulation as an index of opioid-like activity. Table 1 dmonstrates

that DADL inhibits CAMP synthesis and that this effect is blocked by

naloxone. A similar inhibitory effect was observed with "fraction 18" and the

material exposed to Sepharose. Again, PA-Sephamse ompletely eliminated the

inhibition. Attempts to antagonize the CAMP-inhibitory action of "fraction

18" by naloxone yielded equivocal results. In the presence of the narcotic

antagonist 10 ~1 of *'fraction 18" failed to depress the CAMP level to the

same degree as observed in the absence of naloxone.

Isolated tissues: WI and MVD are preparations containing highly sensitive

opioid receptors. Therefore, "fraction 18" was tested for its ability to

display opioid-like activity in these tissues. upto2OO(ll (finalbathm-

662


TAHLE1:cAMPccrNlwp IN NG108CC15 HYBRID =

material

EE2 1

Naloxone3 + DADL'

CAMP (fiml/2 x lo4 cells)

133 64

148

"fraction 18" 1 Pl 5 Bl 10 fil

t1 untreated , 135 85 58

I, I sepharose- treated 123 78 68

" t PA-Sepharo- se-treated 127 125 140

I, , Naloxone3 141 116 98

:w Hepes-buffer 3 lo-F 10 M, 10 min prior to the agonist

lume 2ml) proved ineffective in inhibiting electrically evoked twitches of

the GPI and the MVD (incubation up to 3h).

DISCUSSION

Inmunization of rabbits with the mnoclonal B-endorphin antibody 3-E7 ge-

nerates serum material which binds to the antigen (3-E7 antibody) linked

either to Sepharose (immnoaffinity gel) or to PVC-plates (solid-phase HIA).

This crude material elutes frcm gel chrcmatography in different positions,

including the fractions wherein IgG appears, that is fraction 18. An evalua-

tion of the opioid receptor binding (solubilized receptors) of all samples

positive in the solid-phase HIA reveals that only "fraction 18" from a single

rabbit interferes with receptor binding. The activity of "fraction 18" seems

to be confined to opioid receptors, since flunitrapezam binding was not af-

fected. These data suggest that "fraction 18" contains anti-ID to opioid

receptors.

The experiments with solubilized receptors contrast with data obtained frm

brain membranes in which anti-ID fails to interfere with the binding of the

rather small molecule diprenorphine, whereas the binding of the camparably

663


large B-endorphin molecule is blocked dose-dependently. This finding may

support the notion that diprenorphin and B-endorphin do not interact with the

receptor at identical sites. That is, these opioids may recognize different

receptor epitopes, though the occurence of sterichindrancebetmenthe anti-

ID and B-endorphin cannot be excluded. This interpretation may be in line

with our observations and a report by others (15) that B-endorphin fails to

bind to solubilized opioid receptors, whereas diprenorphine binds to both so-

lubilized receptors and receptors associated with mm&ranes. JQrthermore, the

antigen (3-E7) employed for the generation of anti-ID binds B-endorphin and

other endogenous opioid peptides (6) with high affinity, but does not bind

diprenorphine. In fact, these characteristics of the 3-E7 gave us confidence

in employing this antibody for raising an anti-ID to opioid receptors.

The supposed anti-ID has been shown not only to bind to opioid receptors,

but also to activate these in NG108CC15 cells. Since these hybrids carry

opioid receptors of the 6- and possibly e-type (16,17), the anti-ID may

interact either with each type or may only occupy a single type. Thus, a

specific preference of the anti-ID for one of the multiple opioid receptors

cannot be concluded from the present experiments. The action of naloxone in

antagonizing the action of the anti-ID may also favor the notion that the

antagonist and the antibody cmbine at different epitopes of the receptor

macrmlecule.

The experiments employing the isolated GPI and MVD did not reveal any

opioid-like activity of the anti-ID. It is conceivable that the imnunoglobu-

lin does not reach the receptors located inside these tissues. Whether the

inhibition of electrically evoked twitches of the MVD by an anti-ID, as re-

ported by Ng and Iscm (8), is due to an activation of opioid receptors re-

mainstobe demonstrated.

In sumnary, the data presented here support the original concept of Jerne

(4), and adopt& by Sage and Peterson (2), that the anti-idiotypic route of

generating antibodies to receptors is also applicable to opioid receptors.

The anti-ID material described here probably represents an IgG, as indicated

664


by its molecular size, and the ability of PA to bind the active principle. PA

has been shown to bind highly specific IgG (18). Though the exact nature of

the interaction betwaen the opioid receptor and the polyclonal anti-ID is not

kncwn, it appears that the antibody binds - and activates - different recep-

tor epitopes as cwpared to that to the opioids diprenorphine and naloxone.

The present results ccanplezent previous findings (19) on the generation of

monoclonal antibodies to opioid receptors using purified opioid receptors as

antigen.

We thank Dr. M.J. Millan for stylistic revision of the text. Supported by SFB 220.

1.

2. 3.

4. 5. 6.

7.

8. 9.

10.

11. 12.

13. 14.

15. 16.

17.

18.

19.

Rr

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Gramsch, c Inan, R., Hijllt, V., Weber, E. Hem, A. and Rieth- %iez;'G. (1983) P&. Natl. Acad. Sci. 80, 4084-4088. Ng, D.S. and Iscan, G.E. (1984) Europ. J. Pharm. 102, 187-190. Glasel, J.A. and Myers, W.E. (1985) Life Sci. 36, 2523-2529. Rtieqq, U.T., Hiller, J.M. and Siron, E.J. (1980) Europ. J. Pham. 64, 367-368. Hamprecht, B. (1977) Int. Rev. Cytol. 49, 99-170 . Brooker, G., Harper, J.F., Terasaki, W.L. and Moylan, R.D. (1979) A&. cycl. Nucl. Res. 10, 1-31. Schulz, R. and Goldstein, A. (1972) J. Pharnw. Exp. Ther. 183, 404-410. Hughes, J., Kosterlitz, H.W. and Leslie, F.M. (1975) Br. J. Phamacol. 53, 371-381. Hehnaste, D.M. and Li, C.H. (1985) Fed. Proc. 44, 421. Chang, K.J., Miller, R.J. and Cuatrecasas, P. (1978) Mol. Phann. 14, 961-970. -n&s, R.G., Ferrara, P. and Li, C.H. (1981) Prcc. Natl. Acad. Sci. 78, 2218-2220. Lindmark, R., Thor&-Tolling, K. and Sjijquist, J. (1983) J. -01. Meth. 62, 1-13. Bidlack, J.M., Denton, R.R. and H-11, L.W. (1983) Life Sci. 33, Suppl. I, 151-154

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Polyclonal anti-idiotypic opioid receptor antibodies generated by the monoclonal β-endorphin...

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