Journal of Neurochemisiry Raven Press, Ltd., New York 0 199 1 International Society for Neurochemistrj

A Monoclonal Antibody Recognizing K - but Not p- and 6-Opioid Receptors Katalin Maderspach, KlBra Nkmeth, J6zsef Simon, Shndor Benyhe, MBria Szucs, and MBria Wollemann Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

Abstract: A monoclonal antibody (mAb), KA8, that interacts with the K-opioid receptor binding site was generated. BALB/ c female mice were immunized with a partially purified Kopioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/OAG8 myeloma cells. The antibodyproducing hybridomas were screened for competition with opioid ligands in a modified enzyme-linked immunosorbent assay. The cell line KA8 secretes an IgGl (K-light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A-Sepharose C U B was able to precipitate the antigen from a solubilized and affinity-purified frog brain K-opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration-dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when pand 6-opioid binding was suppressed by 100 nM [DAla2,NMe-Phe4,Gly-oll-enkephalin (DAGO) and 100 nM

[~Ala~,~-Leu’]-enkephalin (DADLE), respectively, and to 100% when p-, 6-, and K2-siteswere blocked by 5 pLMDADLE. However, the pspecific 13H]DAG0 and the 6-prefemng [3H]DADLEbinding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the K- but not the p- and &subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKCbinding to guinea pig cerebellar membranes. These findings suggest homologies in the K-opioid binding site of frog brain and rat brain as well as chick neurons, but the Kopioid receptor subtype in the guinea pig cerebellum may be different. Key words: K-Opioid receptor-Monoclonal antibody-Brain (frog, chick, rat, guinea pig). Maderspach K. et al. A monoclonal antibody recognizing K - but not p- and 6opioid receptors. J. Neurochem. 56, 1897- 1904 ( 1 99 1).

Monoclonal antibodies (mAbs) directed against receptor proteins are versatile tools for receptor analysis. This is of special interest at the opioid receptors because many difficulties have been encountered in the purification and determination of the primary structure of the receptor protein. Among these are the very low receptor density and protein instability (Simon et al., 1984, 1986). The heterogeneity of opioid receptors, based on pharmacological and ligand binding data, is now generally accepted. There are at least three major subtypes of the receptor, designated p, 6, and K (Martin et al., 1976;Paterson et al., 1983;Holaday and Tortella, 1984; Zukin and Zukin, 1984; Goldstein, 1987; Goldstein and Naidu, 1989). However, the molecular structure

of these receptor subtypes-whether they are distinct molecular entities or reside on the same macromolecule-is still not well understood. Application of monospecific antibodies has already provided some information. The mAb OR-689.2.4, prepared by Bidlack and coworkers (Bidlack and Denton, 1985; Bidlack and O’Malley, 1986; Bidlack et al., 1988),recognizes a molecular epitope common in p- and &receptors by inhibiting P-endorphin, Pen-enkephalin and [D-Pen*,DPen5]-enkephalin(DPDPE) binding. This antibody was raised against purified rat brain opioid receptors and precipitates a 35-kDa protein. The polyclonal antibody prepared by Roy et al. (1988) against p-opioid binding protein purified from bovine brain is able to precipitate

Received October 18, 1990; accepted November 20, 1990. Address correspondence and reprints requests to Dr. K. Maderspach at Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary. The present address of Dr. J. Simon is MRC Molecular Neurobiology Unit, MRC Centre, Cambridge CB2 2QH, U.K.

Abbreviations used: DADLE, [D-AlaZ,~-LeuS]-enkephalin; DAGO, [~-Ala~,NMe-Phe~,Gly-ol]-enkephalin; DPDPE, [~-Pen~,~-Pen~]enkephalin; EKC, ethylketocyclazocine;ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.

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a 58-kDa protein of rat brain and a 45-kDa protein of NG 108-15 cells, a finding suggesting antigenic similarities in p- and 6-opioid binding sites. These antibodies are not interacting with K-Opioid receptors. The mAb 3B4F11 introduced by Bero et al. ( 1988)was generated against the same bovine brain p-opioid binding protein as was used by Roy et al. ( 1988). This antibody interacted with p- and K- receptor subtypes of rat brain, an observation suggestingsome molecular homologies between these receptors as well. Another strategy for preparation of opioid receptorspecific mAbs is the antiidiotype technique. Myers and Glasel(1986) described an antimorphine antiidiotype immunoglobulin with preceptor and some 6-receptor specificity on rat neural and NG 108-15 cell membranes. The anti-p-endorphin antibody of Gramsch et al. (1988) proved to be equally potent in displacing pand 6- ligands but not K-ligands in rat brain membranes. At present no results are available for mAbs or polyclonal antibodies recognizing primarily the K-subtype of opioid receptors. Recent data show that amphibian and avian brains are enriched in the proportion of the K-SUbtype of opioid receptors (Ruegg et al., 1981; Puget et al., 1983; Simon et al., 1984; bander, 1988;Csillag et al., 1989). Previously we reported that in the frog (Rana esculenta) brain >70% of the total opioid binding sites belong to the K-SUbtype (Simon et al., 1984; Benyhe et al., 1989). We also described that active opioid receptors can be solubilized from frog as well as rat brain using digitonin as a detergent (Simon et al., 1984, 1986). Gel filtration of this solubilized material on Sepharose 6B resulted in complete separation of the K- from the p- and &-sites (Simon et al., 1985). More recently, we succeeded in purifying the K-opioid receptor subtype to apparent homogeneity by affinity chromatography (Simon et al., 1987, 1990). The purified material is, however, available only in small quantities, which hardly could be used for immunization of animals to raise either polyclonal antibodies or mAbs. To overcome this problem we applied the Sepharose 6B fraction of frog brain membrane proteins enriched in K- and free from pand &binding sites as antigen. The mAbs were generated by the hybridoma technology. We describe here a monoclonal IgG1 (K- light chain) immunoglobulin produced by the cell clone KA8 that recognizes the ligand binding site of the K-opioid receptor subtype in frog and rat brain as well as chick cultured neurons. MATERIALS AND METHODS

Biological Research Center of the Hungarian Academy of Sciences (Szeged, Hungary) (T6th et al., 1982; Benyhe et al., 1985). Peroxidase-labeled goat anti-mouse IgG was purchased from Heintel (Austria). Other immunochemicals and hybridoma reagents as well as proteinase inhibitors were from Sigma. Materials for cell cultivation were from GIBCO. Protein A-Sepharose CL4B and Sepharose 6B were products of Pharmacia. The low-molecular-weight protein standard kit for sodium dodecyl sulfate-polyacrylamidegel electrophoresis was purchased from Bio-Rad. All other chemicals were analytical grade from Reanal (Hungary). The mouse mAb isotyping kit was a product of Amersham.

Antigen Frog ( R . esculenta) brain membranes were prepared and solubilized with 1% digitonin (Simon et al., 1984). The solubilized material was subjected to gel permeation chromatography on a Sepharose 6B column (Simon et al., 1985).

Generation of mAbs BALB/c female mice (6-8 weeks old) were immunized with 50-100 pg of protein injected subcutaneously three to five times at intervals of 2-3 weeks. The first injection was given with Freund's complete adjuvant, and the subsequent ones were with Freund's incomplete adjuvant. Antibody production was checked in the serum of the animals after the second injection by enzyme-linked immunosorbent assay (ELISA) using peroxidase-conjugated goat anti-mouse IgG as a second antibody and o-phenylenediamine plus H202 as substrates (Campbell, 1984). A booster injection was given in phosphate-buffered saline, without adjuvant. After 2 days the spleen cells were hybridized with SP2/OAG8 myeloma cells according to the method given originally by Kohler and Milstein (1975). Conditioned medium of growing hybrid colonies was screened for antibody production by ELISA using the K-receptor-enriched protein fraction from a Sepharose 6B column, frog brain membrane particulate preparation, or cultured chick nervous cells (see below) as antigen; the antigen amount was optimized as 1.5 pg, 15 pg of protein, and 10,000 cells/well, respectively. The cultured chick neurons were prefixed with methanol when they served as antigen. In the early phase of hybridoma growth the antibodies produced were screened for the interaction with K-opioid ligand binding by a competitive ELISA. In this assay parallel samples were tested in the presence and absence of a high concentration ( M ) of opioid ligands (naloxone, EKC, DAGO, or ICI174,864) (Maderspach and Simon, 1987). The hybridomas selected by this way were cloned twice by the limiting dilution method. Clones were expanded, and ascitic fluid was obtained by intraperitoneal injection of 5 X lo6 hybridoma cells into BALB/c mice previously sensitized by Pristane (2,6,10,14tetramethyl pentadecane). The subclass of the mAbs produced was determined by an Amersham isotyping kit. For purification of the antibodies we used a protein A-Sepharose CL4B column according to the generally applied procedure (Campbell, 1984).

Materials The sources of the radioligands were as follows: 9[3H]ethylketocyclazocine(9-[3H]EKC;0.74 TBq/mmol) was from New England Nuclear, and [ Tyr-3,5-'H] [D-Ala2,NMePhe4,Gly-o15]-enkephalin([3H]-DAGQ 1.67 TBq/mmol) was from Amersham. [ Tyr-3,5-3H][~AlaZ,~-Leu5]-enkephalin (['HIDADLE 1.37 TBq/mmol) and [3H]naloxone(3.1 TBq/ mmol) were produced by G. T6th at the Isotope Laboratory, J Neurochem , Vol 56, No 6, 1991

Testing of mAb specificity At screening and testing of our mAb, the myeloma and an unrelated hybridoma supernatant and ascitic fluid served as controls. In some experiments commercially available mouse IgG was also used. In radioligand binding studies parallel measurements were performed with heat-inactivated (9OoC, 30 min) mAb (Bidlack and Denton, 1985).

MONOCLONAL ANTIBODY TO K-OPIOIDRECEPTOR Radioligand binding to frog brain receptors Ligand binding assays were performed in triplicate by incubating the particulate membrane fraction of frog brain (300-400 pg of protein) or the digitonin extract of these membranes (79-100 pg of protein) with tritiated opioid ligands ( 1 nM [3H]naloxone; 2, 4, or 5 nM ['HIEKC; 2 nM ['HIDAGO; or 4 nM [3H]DADLE)in the presence or absence of different concentrations of ascitic fluid, hybridoma supernatant, or purified mAb KA8. Samples were preincubated with the antibody for 20 rnin at room temperature in 50 mM Tris-HC1 (pH 7.4), 1 mMEDTA, 40 kallikrein IU ofTrasylo1, 20 mg/ml of bacitracin, and 1 mM phenylmethylsulfonyl fluoride buffer and then incubated with [3H]naloxone (60 min, 0°C) or ['HIEKC (40 min, 24°C). The p- and 6- sites were suppressed with I00 nM DAGO plus 100 nMDADLE. In some experiments 5 phf DADLE was added to the samples together with the radioligand. ['HIDAGO (40 min, 35°C) and [3H]DADLE(45 min, 24°C) specific binding to particulate membrane fractions was also measured. Nonspecific binding was determined in the presence of the appropriate unlabeled ligand or levorphanol at 10 phf. Binding was terminated by filtering the samples through Whatman GF/B or GF/C (for peptide Iigands) glass fiber filters. When solubilized material was used for the binding assay, the samples were filtered on polyethylenimine-treated Whatman GF/B filters (Bruns et al., 1983). The filters were then washed twice with 10 ml of ice-cold 50 mM Tris-HC1 (pH 7.4) buffer, and radioactivity was counted in a toluene-based scintillation fluid. Protein content was determined according to the technique of Bradford ( 1976).

Radioligand binding to cultured chick neurons The specific ['Hlnaloxone equilibrium binding to chick neuronal cell cultures was described earlier (Maderspach and Solomonia, 1988). The cultures were established from 7-dayold embryo forebrains according to the procedure of Sensenbrenner et al. (1978) and cultivated for 4 days. Intact cultures were preincubated with serial dilutions of KA8 or control ascitic fluid (500-16,000-fold dilution) or mouse immunoglobulin in 1 ml of serum-free growth medium (Eagle's minimal essential medium) for 20 rnin at 37°C and an atmosphere of 5% C02/95%air. [3H]Naloxone(1 nM) was then added, and cultures were incubated for another 4 rnin under the same conditions. Receptor binding was terminated by washing the cultures three times with 25 ml of physiological saline at room temperature. Bound radioactivity was transferred to Bray's scintillation fluid by treating the cultures twice each with 500 pl of methanol. Nonspecific binding was measured in the presence of l phfunlabeled naloxone. [3H]EKC equilibrium specific binding was measured similarly. The neuronal cultures were incubated with 1-5 nM radioligand in the presence or absence of 10 phf unlabeled EKC for 4 min in 1 ml of serum-free growth medium. The p- and 6sites were suppressed with 100 nM DADLE plus 100 nM DAGO. The effect of purified mAb KA8 on [3H]EKCbinding was measured at a radioligand concentration of 3 nM.The cells were preincubated with the antibody (0.04-2 pg of proteinlsample) for 20 min, followed by incubation with the radioligand for another 4 rnin under the same conditions as mentioned above.

Gel filtration on Sepharose 6B Frog brain membranes were solubilized with 1% digitonin, and the K-opioid receptor subtype was separated from the pand 6- sites on a Sepharose 6B column (Simon et al., 1984,

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1985). Two-milliliter fractions were collected, and their ['HIEKC specific binding was determined. The mAb binding capacity of the chromatographic fractions was measured by ELISA. Aliquots of fractions were immobilized on a microtiter plate (1.5 gg of protein/well) by overnight incubation at 4°C and then developed with 50 p1 of 100-fold diluted KA8 ascitic fluid as a first antibody, peroxidase-conjugated goat anti-mouse IgG as a second antibody, and o-phenylenediamine and H202as substrates.

Immunoprecipitation Experiments were carried out as described by Fraser (1984) for P-adrenergic receptors with minor modifications. Solubilized frog brain membrane proteins (-20 pg of protein/ sample), the partially purified K-opioid receptor fraction from a Sepharose 6B column (10 pg of protein/sample), or affinitypurified K-opioid receptor protein (according to the technique of Simon et al., 1990) (nearly 50 ng of protein/sample) was incubated with serial dilutions of KA8 or control ascitic fluid at 4°C for 18 h. Then, 100 pg of anti-mouse IgG or 50 pl of 20% protein A immobilized on Sepharose was added to the samples and incubated for a further 4 hat 4°C. The precipitate was pelleted by centrifugation at 12,000 rpm for 5 rnin in an Eppendorf microfuge. The amount of precipitated antigen was measured by two different techniques. In the ELISA, 50pl aliquots of the supernatants were immobilized on the microtiter plate, incubated with 50 p1 of 50-fold diluted KA8 ascitic fluid as a first antibody, and developed as above. For reference, an equal amount of nonprecipitated antigen, antigen incubated similarly but without antibody, and antibody incubated without antigen was applied. The values measured with the control ascites series were taken as a background. Specific binding of ['HIEKC (10 nM) to the supernatant and the precipitate-resuspended in phosphate-buffered saline-was also determined similarly as described above.

RESULTS

Our attempt to raise mAbs against the K-SUbtype of opioid receptor was successful, Several hybridoma cell clones producing high levels of an IgG-type antibody were identified. During the whole screeningprocedure the antibodies were selected for the interaction with opioid ligand binding. The modified competitive ELISA made this possible in the very early phase of the selection. We describe here in detail a monoclonal IgG1 (K) immunoglobulin produced by the cell clone KA8. The antigen specificity of the mAb KA8 was determined first by testing Sepharose 6B chromatographic fractions of frog brain solubilized membrane proteins by ELISA and by opioid ligand binding. The results of the parallel assay are presented in Fig. 1. The binding of the mAb KA8 and the specific binding of [3H]EKCto the fractions correlate very well, whereas the sensitivities of these measurements are essentially different. No antibody binding was measured in the fractions without ['HIEKC specific binding, and vice versa. The KA8 hybridoma supernatant and ascitic fluid inhibited the specific binding of 4 nM ['HIEKC to the frog brain particulate membrane fraction in a concentration-dependent manner. The binding of p- and 6J . Neurochem.. Vol. 56, No. 6. 1991

K. MADERSPACH ET AL.

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FRACTION NUMBER FIG. 1. Comparison of K-OpiOidligand bindingand mAb KA8 binding to gel-filtered fractions of frog brain solubilized membrane proteins. For measurement of [3H]EKCspecific binding (O),aliquots of fractions from a Sepharose 66 column were incubated with 5 nM [3H]EKC and 100 nM DAGO plus 100 nM DADLE for 40 min at 24°C. Nonspecific binding was measured with 1 pM levorphanol the same fractions or EKC. For performance of the ELSA (0). were immobilized on a microwell plate (1.5 pg of protein/well) and incubated with KA8 ascitic fluid diluted 1:lOO as the first antibody, as the second peroxidaseconjugated anti-mouse IgG (1:1,000) antibody, and o-phenylenediamine plus H202 as substrates. Absorbance was measured at 492 nm.

receptors present in the membranes was suppressed with 100 nM DAGO and 100 nM DADLE, respectively. As Fig. 2 demonstrates, the KA8 supernatant diluted 10-fold decreased the binding of 13H]EKCby 60%, whereas this value was -20% even at a 1,000fold dilution. The supernatant of an unrelated hybridoma clone decreased the binding of [3H]EKC to frog brain membranes by > 10%. [3H]EKC specific binding data in the presence of KA8 ascitic fluid and purified antibody are summarized in Table 1. A 10-fold dilution of the ascitic fluid decreased the binding by 72%. When p- and &receptor binding was suppressed as indicated above, this value was higher (86%)and peaked ( 100%)when 5 p M DADLE was present during the incubation. Pretreatment of the ascitic fluid at 90°C for 30 min eliminated the inhibitory effect. Specific binding of 2 nM[3H]DAG0 (a p-opioid specific ligand) or 4 nM [3H]DADLE(a 6preferring ligand) to frog brain membranes was not influenced by KA8 ascitic fluid. Specific binding of [3H]naloxone to cultured chick neurons was also inhibited by the mAb KA8 (Fig. 3). The equilibrium binding of the ligand was measured earlier (Maderspach and Solomonia, 1988). The KA8 ascitic fluid diluted 500-fold (4 pg of protein) decreased the specific binding of 1 nM [3H]naloxone by 80%. The inhibition was >50% even at a 16,000-folddilution (0.1 pg of protein), whereas complete blocking was achieved at a 100-fold dilution. Mouse IgG was much less effective at decreasing the specific [3H]naloxone binding. Equilibrium binding of [3H]EKC to chick neurons is demonstrated in Fig. 4. The characteristics of the binding are similar to those of [3H]naloxone.The paJ. Neurochem.. Vol. 56, No. 6 , 1991

rameters of the biphasic curve were estimated as a KD of 2 nM, a B,, of 6 fmol/106cells, and a Hill coefficient of >2, using the Hill equation as given earlier (Maderspach and Fajszi, 1982). The purified mAb KA8 decreased the specific binding of 3 nM [3H]EKC in a concentration-dependent manner. The inhibition was 80% at 2 pg of purified antibody/sample. Even as little as 0.2 pg of purified mAb KA8 was effectiveat causing >50% inhibition in the specific [3H]EKCbinding. The results were similar in the presence and absence of 100 nMDAGO plus 100 nMDADLE. The mAb KA8 proved to be capable of precipitating the K-OpiOid binding sites from solubilized frog brain membrane preparations. Figure 5A shows data when digitonin-solubilized frog brain membrane proteins were precipitated. In the experiment presented by Fig. 5B,the K-opioid receptor-enriched protein fraction, established by Sepharose 6B chromatography, was precipitated by serial dilutions of KA8 ascitic fluid. A maximum of 80% of the opioid binding sites was precipitated by the mAb in a concentration-dependent manner. These results were obtained with ELISA, by measuring the remaining binding capacity in the supernatant. Furthermore, radioligand binding to the supernatant and precipitate was also determined. Data are summarized in Table 2. The KA8 ascitic fluid diluted threefold precipitated 90% of the [3H]EKCspecific binding capacity from the K-OpiOid receptor-enriched, p- and 6-receptor-free protein fraction. The corresponding data are 36 and 396 cpm of [3H]EKC specific binding in the supernatant and the precipitate, respectively. When ascitic fluid was diluted by 94-fold, no specific binding was measurable in the precipitate. The mAb KA8 similarly precipitated the binding sites from the affinity-purified frog brain K-opioid re-

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DlLUTl ONS FIG. 2. Inhibition of I3H]EKC specific binding to frog brain rnembranes by rnAb KA8. Frog brain membranes(3-400 pg of protein/ supersample) were preincubated with KA8 (0)or myeloma (0) natant for 20 min, and then 4 nM [3H]EKCplus 100 nM DAGO and 100 nM DADLE were added and further incubated for 40 min at 24OC. Nonspecific binding was measured in the presence of 10 pM EKC. Results are averages of three determinations.

MONOCLONAL ANTIBODY TO K-OPIOIDRECEPTOR

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TABLE 1. Inhibition of specific radioligand Q3H]DADLE, [3H]DAG0, or [3H]EKC) binding to the frog brain particulate membranefraction by mAb KA8 Inhibition Radioligand

Antibody, dilution

Added unlabeled liaand

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I'HIDADLE [3H]DAG0 [3H]EKC ['HIEKC [3H]EKC ['HIEKC ['HIEKC ['HIEKC ['HIEKC

KA8 ascitic fluid 1:lO 1:lO 1:lO 1:lO 1:lOO 1:1,000 1:lO 1:lOO 1:1,000

100 nM DADLE + 100 nMDAGO 100 nM DADLE + 100 nMDAGO 100 nM DADLE + 100 nMDAGO 5 pM DADLE 5 pM DADLE 5 pM DADLE

['HIEKC 13H1EKC

KA8 purified antibody 1:lOO 1: 1,000

100 nMDADLE 100 nM DADLE

+ 100 nMDAGO + 100 nM DAGO

73 52

Frog brain membranes were incubated with KA8 ascitic fluid (20 mg of protein/ml) or purified KA8 antibody (0.36 mg of protein/ml) for 20 rnin at 24°C and then incubated with the radioligands as follows: 4 nM['HIEKC, 40 rnin at 24°C; 2 nM ['HIDAGO, 40 rnin at 35°C; and 4 nM ['HIDADLE, 45 rnin at 24°C. Nonspecific binding was measured in the presence of the appropriate unlabeled ligand at 10 pM. The 100%specific binding of ['HIEKC was 340 fmol/mg of protein, that in the presence of 100 nM DAGO and 100 nM DADLE was 23 1 fmol/mg of protein, and 5 pM DADLE decreased this value to 1 12 fmol/mg of protein. The basal specific binding of 13H]DAG0 was 59 fmol/mg of protein whereas that of ['HIDADLE was 127 fmollmg of protein. Percent inhibition values represent averages of at least three separate experiments.

ceptor preparation. The data obtained are 60 and 30% at three- and 30-fold dilutions, respectively, of the ascitic fluid, if determined by ELISA.

DISCUSSION We describe here an IgGl (K)mAb that is secreted by the hybridoma clone KA8 and recognizes primarily the K-SUbtype of the opioid receptor. The antigen used for immunization of BALB/c mice was derived from the frog (R. esculenta) brain membrane fraction after solubilization with 1% digitonin (Simon et al., 1984) and separation by Sepharose 6B chromatography (Simon et al., 1985). This procedure resulted in an opioid receptor preparation enriched in the K-Subtype and free from the p- and 6-subtypes. Application of such an antigen as well as of the competitive

ELISA during the early screening increased the probability of finding hybridoma clones producing antibodies with the required K-OpiOid receptor specificity. Earlier described antibodies (Bidlack and Denton, 1985; Roy et al., 1988) were raised against antigens containing mainly the p-opioid receptor subtype. Our data support the proposal that the mAb KA8 interacts specifically with the K- but not with the p- and 6-opioid binding sites in the frog brain. It is also suggested that the molecular epitope recognized by the mAb is at the ligand binding site. These conclusions are based primarily on the results of the competition experiments. The mAb KA8 (ascitic fluid, supernatant, or purified antibody) inhibited very potently the specific binding of the K-ligand [3H]EKC. This inhibition was concentration dependent and was further increased by suppression of the p- and &binding sites by the proper

6

FIG. 3. Inhibition of [3H]naloxone specific binding to chick cultured neurons by mAb KA8 and mouse IgG. A Equilibrium [3H]naloxonebinding (from Maderspachand Solomonia, 1988). B: Inhibition of specific binding measured at a [3H]naloxone concentration of 1 nM by mAb (0)or mouse Intact cultures were preincubated IgG (0). with ascitic fluid or mouse IgG (Sigma) for 20 min at 37°C in 1 ml of serum-free Eagle's minimal essential medium and then further incubated with the radioligand for 4 rnin. Nonspecific binding was measured in the presence of 1 pM unlabeled naloxone. Results are averages of three independent measurements.

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unlabeled ligands. However, neither myeloma nor unrelated hybridoma supernatant or ascitic fluid inhibited this radioligand binding. The mAb KA8 did not decrease the specific binding of the p-selective ligand [3H]DAG0 and 6-prefemng ligand [3H]DADLE. The exclusive K-specificity of the mAb KA8 was supported by the analysis of the Sepharose 6B chromatographic fractions of frog brain membrane proteins. This mAb recognized antigens only in those fractions where t3H]EKC specific binding was measurable, and the data were highly correlated. No antibody binding

FIG. 4. Inhibitionof f'H]EKC specific binding to chick cultured neurons by mAb KA8. A: Equilibrium [3H]EKC binding measured at 37°C incubated for 4 min. One hundred percent corresponds to 6 fmol of [3H]EKC/106 cells. B: Inhibitionof specific binding of 3 nM r3H]EKCby purified mAb KA8. Intact cultures were preincubated with the purified antibody for 20 min at 37°C in 1 ml of serum-free Eagle's minimal essential medium and then further incubated with [3H]EKC plus 100 nM DAGO and 100 nM DADLE for 4 min. Nonspecific binding was measuredin the presence of 1 pM unlabeled EKC. Results are averages of three independent measurements.

was found in those fractions where p- and &receptor subtypes can be detected (Simon et al., 1985). The competition experiments also revealed that complete blocking of the [3H]EKCspecific binding by the mAb KA8 can only be achieved if 5 p M DADLE is added to the incubation mixture. As Attali et al. (1982) defined, 5 pM DADLE is blocking, besides pand 6-, also the K2-subclassof opioid receptors. Presumably, the mAb KA8 would be preferentially recognizing the K,-subclass. However, we must take into account that the basal [3H]EKCbinding in the presence of 5 p M DADLE was essentially reduced (1 12 instead of 340 fmol/mg of protein) and that the K,-subtype represents only 20% of the K-opioid receptors in frog brain (Benyhe et al., 1989). Furthermore, the KA8 ascitic fluid also inhibited the specific [3H]EKCbinding (in the presence of 100 nM DAGO plus 100 nMDADLE) to rat brain membranes to 70-loo%, whereas 15% if guinea pig cerebellum the inhibition was membranes were assayed under the same conditions (data not shown). Zukin et al. (1988) found that the rat brain contains the K ~ rather than the K1-SubclaSSof opioid receptors but that in the guinea pig cerebellum the subclass predominates. The mAb KA8 (ascitic fluid or purified antibody) is able to precipitate the K-opioid binding sites from solubilized frog brain membrane fractions, the K-opioid receptor-enriched fraction, and an affinity-purified Kopioid receptor preparation. After the immunoprecipitation nearly 90% of the [3H]EKCspecific binding capacity can be measured in the precipitate. This result provides us with an excellent possibility for immunoaffinity purification of the receptor protein. The purified mAb KA8 recognizes a 45-kDa protein band on the Western blot of frog brain membrane proteins (data not shown). This molecular mass is similar to that of the minor band in our affinity-purified Kopioid receptor preparation, which was attributed to a degradation product of the major 65-kDa receptor protein (Simon et al., 1990). Further studies are currently in progress to understand why the major 65kDa component of our purified K-opioid receptor preparation was not stained on the Western blot.

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DILUTIONS FIG. 5. lmrnunoprecipitationof frog brain antigen by KA8 ascitic fluid. Serial dilutions of ascitic fluid were incubated for 18 h at 4OC with either (A) digitonin-solubilizedfrog brain membrane proteins (20 pg of proteinlsample) or (B) a K-OpiOid receptorenriched, pand 6-receptor-freefraction (10 pg of protein/sample), obtained by Sepharose6B chromatography.After the incubation with the ascitic fluid, 100 r g of anti-mouse IgG was added, and samples were further incubated for 4 h at 4°C. The precipitate was pelleted by centrifugation at 12,000 rpm for 5 rnin. The supernatant was immobilized on the microwell plate, and the remaining free antigen was quantifiedby ELISA. Precipitation by unrelated ascitic fluid is also shown (A).

J. Neurorhern., Vol 56.No. 6. 1991

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TABLE 2. Immunoprecipitation of solubilized frog brain antigen by the mAb KA8 Precipitation Antigen

Antibody, dilution

%-Receptorenriched fraction

KA8 purified antibody 1:lO 1: 100 1:1,000 KA8 ascitic fluid 1.3 1:92 Unrelated ascitic fluid 1:I

Affinity-purified K-receptor

KA8 ascitic fluid 1:l 1:lOO KA8 purified antibody 1:lO 1: 100

Assay

("/.)

ELISA

40 17 15 [3H]EKCbinding 90 2 ELISA

28 ELISA

70 4 ELISA

35 14

Protein concentrations were as follows: ascitic fluid, 20 mg/ml; purified KA8, 4 mg/ml. The K-receptorenriched fraction was obtained by Sepharose 6B chromatography (Simon et al., 1984, 1985); the affinitypurified K-receptor was obtained by dynorphin chromatography (Simon et al., 1987, 1990). For precipitation, antigen was incubated with antibody at 4°C for 18 h, and then anti-mouse IgG was added and incubated for 4 h at 4°C. The precipitate was pelleted by centrifugation at 12,000 rpm for 5 min. For the ELISA, an aliquot of supernatant was immobilized on a microwell plate and developed by KA8 ascitic fluid as the first antibody to quantify the remaining free antigen. For radioligand binding, an aliquot of supernatant and precipitate was incubated with 10 nM [3H]EKC for 45 min at 20°C. Nonspecific binding was measured in the presence of 1 pM levorphanol. Percent precipitation values represent averages of three determinations.

Our data suggest that the mAb KA8 cross-reacts also with the chick brain K-opioid receptors. It was recently reported that the avian-like the amphibian-brain contains the K-Opioid subtype in a high ratio (80%) (Mansour et al., 1988; Leander, 1988). The receptors were characterized by radioligand binding in chick brain tissue (Csillag et al., 1989) and in isolated chick neurons as well (Hendrickson and Lin, 1980; Maderspach and Solomonia, 1988).Earlier we described that the specific binding of the opioid ligand [3H]naloxone to both gllal and neuronal cells of chick brain in culture displays apparent positive cooperativity with a Hill coefficient of >2. This was interpreted as heterogeneity of the binding sites. Displacement experiments supported the high ratio of K-opioid receptors (Maderspach and Solomonia, 1988). In the present study we found that the mAb KA8 (ascitic fluid or purified antibody) potently inhibited the specific binding of both [3H]naloxoneand [3H]EKCto chick cultured neurons. Our observation that the antibody raised against the frog antigen recognizes chick and rat receptors suggests homologies in the K-OpiOid ligand binding site of these species. The mAb KA8 characterized in this article provides an excellent tool for further analysis of the K-opioid receptors: For example, it makes possible the immunocytochemical visualization by light and electron microscopy. Studies on systematic mapping of the Kopioid receptor in rat brain and the arrangement of the receptors on chick neurons during the in vitro differentiation are already in progress in our laboratory.

Acknowledgment: This work was supported by OKKFT, Ttl, Hungary. We thank Miroslav Nakoniecny and Assadullah Khan for their help in antigen preparation and Miss Zsuzsa Magyar for excellent assistance. K. NCmeth was the recipient of an ITC grant.

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