Journal of Neuroscience Research 27:642-650 (1990)

Monoclonal Antibodies to the Cell Surface and a Soluble Form of the Human Nerve Growth Factor Receptor M. Clagett-Dame, C. Chung, M.V. Chao, and P.S. DiStefano School of Pharmacy. University of Wisconsin-Madison, Madison (M.C.-D.); Neuroscience Research Division, Pharmaceutical Discovery, Abbott Laboratories, Abbott Park, Illinois (C.C.. P.S.D.); Department of Cell Biology and Anatomy and Division of HematologyiOncology , Cornell University Medical College, New York. New York (M.V.C.)

Monoclonal antibodies (designated IIIGS, VIIDl, VIIICS, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface (Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan AA, Buck CR, Sehgal A [1986]: Science 232:51&521). Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross-linked to [125-I]-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgG,,, subclass. All monoclonal antibodies bound to both NGFR and NGF-Rt. Two monoclonal antibodies (VIIDl, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SYSY human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIGS) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added [125-I]-labeled NGF. Key words: truncated receptor, 2-site RISA, E9b cells 0 1990 Wiley-Liss, Inc.

INTRODUCTION Nerve growth factor receptors (NGF-R) are present on the cell surface of sympathetic neurons and neural crest-derived sensory neurons, as well as in populations of neurons in the CNS (Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980; Johnson and Taniuchi, 1987: Misko et al., 1987; Springer, 1988). NGF-Rs have also been identified in a variety of tumor cell lines (Fabricant ct al., 1977; Grecne and Tischler, 1982; Sonnenfeld and Ishii, 1985; Rettig et al., 1987) and non-neuronal cells such as peripheral nerve Schwann cells (Carbonctto and Stach, 1982; Zimmerman and Sutter, 1983; Rohrer, 1985; Taniuchi et al., 1986; DiStefano and Johnson, 1988a). The receptor cxists in both a high affinity (Kd = 10-100 pM) and a low affinity (Kd = 1-10 nM) form (Sutter et al., 1979; Thoenen and Barde, 1980; Johnson and Taniuchi, 1987; Misko et al., 1987). The human NGF-R (low affinity form) has been cloned and sequenced (Chao et al., 1986; Johnson et al., 1986). The 3.8 kb mRNA codes for a 427 amino acid protein with a molecular weight of 49,689 dalton (Da). The mature NGF-R protein contains both N- and 0-linked carbohydrate (Grob et al., 1985). When the human NGF-R is cross-linked to [ 125-I]-NGF using 1-ethyl-3-(3-dimethylaminopropy1)carbodiimide and is resolved on sodium dodccyl sulfate (SDS)-polyacrylamide gels, a labeled band appears in the molecular weight range of 80,000100,000 Da (Puma et al., 1983; Ross et al., 1984; Grob et al., 1985; Chao et al., 1986; Marano et al., 1987). A less abundant 180-200,000 Da species has also been reported and may represent an incompletely reduced or an aggregated form of the receptor. In addition to the cell Received July 11, 1990;revised August 3 1, 1990; accepted September 1, 1990.

Address reprint requests to Peter S . DiStefano. Neuroscicnce Research Division, Abbott Laboratories, Dept. 47W, APLO, .4bhott Park, 1L 60064.

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surface NGF-R, a soluble protein has been identified that and eluted with 4 M MgCI, in acetate buffer (0.2 M , pH binds to NGF and is immunoprecipitated by an antibody 6.5). This resulted in an approximate 100-fold purificawhich recognizes the cell surface NGF-R (DiStefano and tion of NGF-Rt from E9b conditioned medium. The colJohnson, 1988bj. Affinity labeling with [ 125-Il-NGF umn eluate was extensively dialyzed against PBS and and resolution on reducing SDS-polyacrylamide gels re- concentrated prior to use in immunization. A 2-monthveals a protein with a molecular weight of 6 3 4 6 , 0 0 0 old fcmalc BALBicByJ mouse (The Jackson Laboratory, Da. The soluble protein has been observed in thc condi- Bar Harbor, ME) received a primary intraperitoneal intioned medium of variouc NGF-R bearing cells, partic- jection of partially purified NGF-Rt (-100 pg total proularly Schwann cells. The protein has also bcen identi- tein) in MPL TDM adjuvant (RIB1 IminunoChem Refied in amniotic fluid, urine and plasma, and the levcls of search, Inc., Hamilton, MT) followed by a booster this protein are regulated both developmentally and in injection containing the same amount of protein at 3 response to sciatic ncrve leion. Based on molecular weeks. The mouse was anesthetized and bled by retrosize, NGF-binding specificity, and immunologic reactiv- orbital puncture prior to the primary immunization and at ity with NGF-R antibody, it has been proposed that the 10 days following the boost. An IV injection (-140 pg soluble protein comprises the extracellular domain of the total protein) was administered 7 weeks after the 3 week cell surface NGF-R. This conclusion is supported by the boost. Three days later, spleen cells were fused with similarity of the amino terminal portion of the soluble mouse myeloma cells (NS 1) according to established protein isolated from human urine to the deduced amino procedures (Kohler and Milstein, 1975, 1976; Oi and acid bequence from the human NGF-R cDNA (Zupan et Herzenberg, 1980). Following selection with hypoxanal., 1989). The protein has been referred to as the trun- thine, aminopterin, and thymidine, the hybridomas were cated NGF-R or NGF-Rt. The cellular mechanisms in- maintained in Dulbecco's modified Eagle's medium volved in generating this protein as well as the physio- (DMEM) (GIBCO, Grand Island, NY) supplemented logical significance of this receptor form remain unclear. with 15% fetal calf serum (FCS) (Hyclone, Logan, VT), The present study was initiated to develop mono- glutamine (2 mM), sodium pyruvate ( 1 mM). nonessenclonal antibodies to the NGF-Rt in order to facilitate tial amino acids (GIBCO) (10 mM), 2-mercaptoethanol investigations on its origin and biological significance. A ( S O pM), and n-(2-hydroxyethylj-l-piperazine-N'-2-ethmouse fibroblast L cell transformant cell line (E9b) that anesulfonic acid (Hepes), pH 7.3 (10 mM). expresses large numbers of human NGF-R on its cell surface was used as a preparative source of the truncated Affinity Labeling of NGF-R With [125-I]-NGF NGF was purified from male mouse submaxillary receptor. A partially purified preparation of NGF-Kt was used to generate monoclonal antibodies. We report here glands according to the procedure of Bocchini and Anfour newly developed anti bodies that recognize both the geletti (1969) and was labeled with [l25-I] according to cell surface and the soluble, NGF-Rt. Antibodies that thc method of Marchalonis (1969). Affinity labeling of bind to distinct epitopes on both the truncated and cell cell surface and NCF-Rt species was performed as desurface receptor have been used to develop a sensitive, scribed previously (Tdniuchi ct al., 1986; DiStefano and 2-site radiometric immunosorbent assay (RISA) in which Johnson, 1988b). receptor can be assayed rapidly from large numbers of RISA biological samples in a ligand-independent fashion. Hybridoma supernatants were screened for the presence of antibodies using the RlSA as described preMATERIALS AND METHODS viously (Pierce et a]., 1986). Briefly, Immulon 2 ReImmunization/Cell Fusion movawell strips (Dynatech Labs, Alexandria, VA) were Partially purified NGF-Rt was used as an immuno- coated with goat anti-mouse IgG ( S O pl, SO p/ml) in PBS gen. E9b cells were grown as described previously (Chao (pH 8.0) overnight at 4°C or at room temperature for I et al., 1986). Conditioned medium was decanted from hr. After removal of the unbound goat anti-mouse IgG, the cells, brought to 0.02% with sodium azide, and the wells were blocked with 1 .S% bovine serum albumin stored at 4°C. An immunoaffinity chromatography resin (BSA) (1.50 plj in PBS for 30 min at room temperature. was prepared by coupling affinity purified monoclonal The wells were washed three times with cold PBS folantibody ME20.4 (Ross et al., 1984) to cyanogen bro- lowed by the addition of hybridoma supernatants (50 p.1) mide activated Sepharose-4B (Pharmacia, Uppsala, and incubation for 2 hr at room temperature. The wells Sweden) (4 mg antibodyiml resin) (Axen et al., 1967). were washed three times with cold PBS, placed on ice, E9b conditioned medium (580 ml) was passed over the and 50 p.1 of NGF-R cross-linked to 112S-I-NGF ME20.4-Sepharose column, the column was washed (25,000 cpm) was added to each well and incubated for with 20 mM phosphate buffered saline (PBS, pH 7.4), 45 min at 4°C. The wells were washed four times with

+

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cold PBS containing 0.05% tween 20 and the wells were analyzed for the presence of radioactivity.

Immunoprecipitation of NGF-R and NGF-Rt Receptor-containing samples affinity labeled with [125-I]-NGF (50-100 p1) were incubated with hybridoma supernatant (50-100 pl), mouse serum (50 yl of a 1:100 dilution in PBS), or purified monoclonal antibodies (5 ~ g for ) 2-4 hr at 4°C. A suspension of goat antimouse IgG Sepharose (Organon Teknika Corp., Durham, NC) in PBS was added (1070, volume/volume) and the mixture was incubated for 1 hr. Sepharose in tubes containing NGF-R was washed twice with 500 pl of PBS containing 0.05% tween 20, whereas Sepharose in tubes containing NGF-Rt was washed with PBS containing 0.5% BSA, 0.5 M sucrose, and 0.1% tween 20. The washed resin was either counted for radioactivity or mixed with reducing SDS-sample buffer. Sample buffer was incubated with resin for 1 hr at room temperature and was removed by centrifugation. Samples were boilcd for 90 sec and applied to 10% discontinuous SDS-polyacrylamide gels. Electrophoresis was performed using the buffer system of O’Farrell (1975). Dried gels were cxposed to Kodak (Rochester, NY) XAR film using an intensifying screen at -70°C. Analysis of NGF-K or NGF-Rt after affinity cross-linking to [ 125-11-NGFI immunoprecipitation, and gel electrophoresis is referred to as the cross-link-immunoprecipitation (CLIP) assay.

by one rinse with PBS containing 0.5% non-fat dry milk, and incubated for 2 hr with affinity purified monoclonal antibody (25 pgiml) in the same buffer. Membranes were rinsed three times each for 5 min with PBS containing 0.5% non-fat dry milk and 0.05% tween 20. Membranes were then incubated for 2 hr in the presence of [ 125I]-labeled goat-anti-mouse IgG (Kirkegaard & Perry Labs, Gaithersburg. MD). Radiolabeled antibody (3 X lo6 cpmiml) was diluted in PBS containing 0.5% non-fat dry milk. The membranes were washed five times with PBS containing 0.5% non-fat dry milk and 0.05% tween 20 and once with PBS alone. Dried membranes were exposed to Kodak XAR film using an intensifying screen at -70°C until films were developed.

Antibody Competition Studies Cells (E9b or Lrk-) were solubilized as described above. NGF-Rt in E9b cell conditioned medium was used directly or was purified by immunoaffinity chromatography on a resin constructed using an antibody (ME20.4) to the human NGF-R. The column was washed sequentially with PBS, 20 mM sodium phosphate buffer (pH 7.4) containing 0.55 M NaCl, PBS, and 50 mM 3-(cyclohexy1amino)- 1-propanesulfonic acid (CAPS) buffer (pH 9.8). NGF-Rt was eluted from the column using CAPS buffer (pH 11.5). Column fractions were brought to pH 7.4 by the addition of 1 M Hepes buffer, pH 7.0. Immulon 2 Removawells were coated with 50 p1 monoclonal antibodies (50 pgiml) in PBS overnight at Radiolabeling of Antibodies Affinity purified antibodies (50 pg) were radiola- 4°C. Additional binding sites were blocked with the adbeled with [ 125-I]-Bolton-Hunter reagent (New England dition of 150 pl of 1.5% BSA in PBS for 30 min at room Nuclear, Boston, MA) (2,200 Ciimmole, 1 mCi) accord- temperature. The wells were washed three times with ing to the procedure of Bolton and Hunter (1973). [125- cold PBS after which 50 yl of the receptor-containing I]-labeled antibody was separated from unreacted [ 125- preparation was added and incubated for 90 min at room I]-Bolton-Hunter reagent by chromatography on an temperature. The wells were washed three times with Econopac 10-DG column (BioRad Labs, Richmond, cold PBS followed by the addition of 50 p1 of [125CA) equilibrated in PBS Containing 0.2% gelatin and I]-labeled monoclonal antibody (2.5-5 x lo5 cpmiwell) and incubated for 45 min at 4°C. The wells werc then 0.01% sodium azide. washed four times with PBS containing 0.05% tween 20 Immunoblotting and counted for radioactivity. NGF-R was solubilized from E9b cells using 20 mM A 2-site RISA for NGF-R and NGF-Rt was develsodium phosphate buffer (pH 7.4) containing 2% n-octyl oped using antibodies that bound to distinct epitopes on glucoside, 0.65 M NaCl, I mM phenylmethylsulfonyl the receptor protein. These assays were conducted in fluoride (PMSF), and 1 mM iodoacetamide. The solubi- similar fashion to the antibody competition studies. XIFl lized preparation was mixed with either SDS-sample was chosen as the anchoring antibody and was used to buffer containing 5% beta-mercaptoethanol followed by bind NGF-R from solubilized cell preparations or NGFboiling for 90 sec or with non-reducing SDS-sample Rt to the solid phase. [135-I]-IIIG5 (5 x 10” cpmiwell) buffer and used directly without boiling. Samples were was used in the final incubation step to detect the presresolved on 10% discontinuous SDS-polyacrylamide ence of receptor. gels. Reduced [ 14-C]-methy kited protein molecular weight markers (Amersham, Arlington Heights, IL) were General Procedures run in a parallel lane. Protein was transferred electroProtein concentration was determined by the phoretically to nitrocellulose membranes (Towbin et al., method of Bradford (1976) using crystalline BSA as a 1979). Membranes were incubated for 1 hr at room tem- standard. Laser densitometry was performed using an perature in PHS containing 5% non-fat dry milk, followed LKB UltroScan XL laser densitometer. Antibodies were

NGF-Receptor Antibodies

affinity purified from hybridoma supernatants using a protein A-Sepharose monoclonal antibody purification system (BioRad Labs). Antibodies were isotyped using a ScreenType kit from Boehringer Mannheini (Indianapolis, IN).

RESULTS Antibody Production NGF-Rt from the conditioned medium of E9b cells was used as a source of receptor for immunization. To demonstrate that NGF-Rt from E9b cells is similar to that reported previously from other sources, receptor in E9b conditioned medium was affinity labeled with [ 125-11NGF, immunoprecipitated with antibody ME20.4, and resolved on SDS-polyacry Iamide gels. This resulted in the appearance of a labeled protein with the expected apparent molecular weight of 66,000 Da (Fig. 1j . Subtracting a monomer of NGF (13,000 Da) yielded a molecular weight for NGF-Rt of 53,000 Da. NGF-Rt was immunopurified from E9b conditioned medium and used to immunize a BALBicByJ mouse. Serum from a mouse receiving a primary immunization and one booster injection of partially purified NGF-Rt was positive for antibody activity to intact NGF-R by RlSA and immunoprecipitation assay (data not shown). Spleen cells from this mouse were fuscd with mouse myeloma cells (NS- 1). Hybridomas secreting antibody to the NGF-R were first identified using the RISA assay. Wells with a signal two times above background radioactivity (3411,056 wells) were rescreened by immunoprecipitation, and 1 8 of 34 wells remained positive for the presence of antibody to the NGF-R using this assay. Five lines continued to screen positive for antibody to NGF-R after expansion of the cell lines and these were cloned by limiting dilution. After cloning and expansion, four hybridoma lines remained positive for antibody to NGF-R. Characterization of the monoclonal antibodies using class- and subclass-specific anti-mouse immunoglobulin antisera in an ELISA showed that all are IgG,,, antibodies. Analysis of Antibodies Using the CLIP Assay Antibodies were tested for their ability to immunoprecipitate [ 125-I]-NGF affinity labeled receptor. Samples were resolved on SDS-polyacrylamide gels to confirm the molecular identity of the immunoprecipitated species. Immunoprecipitation of affinity labeled receptor from E9b cells by all four antibodies (IIIGS, VlID1, VIIIC8, XIFl) resulted in the appearance of a 90,000 Mr species on gel autoradiograms (Fig. 2). Subtracting the molcular weight of a monomer of NGF yielded a molecular weight for NGF-R of 77,000 Da. The same protein was immunoprecipitated using a monoclonal antibody (ME20.4) to human melanoma cell NGF-R (Ross et al., 1984). No affinity labeled receptor was immunoprecipitated when either a control murine IgG,., antibody

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Fig. 1. Identification of NGF-Rt in E9b conditioned medium. Samplcs of E9b conditioned medium (100 pl) were incubated with 4 nM [125-lj-NGF (lane 2) or radiolabeled NGF in the presence of 150-fold excess of unlabeled NGF (lane 3). Affinity labeling and immunoprecipitation of samples with antibody ME20.4 were performed as described in Materials and Methods using the CLIP assay. The samples were resolved on discontinuous, reducing 10% SDS-polyacrylamide gels and subjected to autoradiography. Lane 1: Standards are shown and include the following [ 14-C]-methylated proteins: myosin 1200,000), phosphorylase B (92,500), BSA (69,000), ovalbumin (46,000), and carbonic anhydrase (30,000).

(MOPC21j or a monoclonal antibody specific for the rat NGF-R (Ab 192) was used (Chandler et al., 1984). All antibodies produced in this study (IJTGS, VlIDl, VIIlC8. XIF1) as well as the antibody ME20.4 immunoprecipitated the 90,000 Da NGF-R-[ 125-I]-NGF complex from the clonal human neuroblastoma cell line, SH-SY5Y (Ross et al., 1983) (Fig. 3). As with E9b cells, no labeled material was immunoprecipitated from SH-SY5Y cells by either MOPC21 or Ab 192. All of the antibodies produced in this study were also capable of immunoprecipitating a protein with an apparent molecular weight of 66,000 from a preparation containing affinity labeled human NGF-Rt (Fig. 4). As with the intact NGF-R, NGF-Rt-[ 125-I]-NGF complex

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14.3 Fig. 3. Immunoprecipitation of NGF-R from SH-SYSY cells. SH-SYSY cell surface NGF-R was affinity labeled with [ 125I]-NGF followed by solubilimtion of cells with octyl glucoside as described in Materials and Methods. Samples were incubated with monoclonal antibodies and analyzed using the CLIP assay. Standards are as described in Figure 2 .

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Fig. 2. Immunoprecipitation of low affinity NGF-R from E9b cells. Monoclonal antibodies ( 5 p g ) were incubated with NGFR affinity crosslinked to 1125-IJ-NGF(100 p1) and solubilized from E9b cells (3 X 10')). The CLIP assay was performed as described. Standards include the following [ 14-C]-niethylated protcins: myosin (200,000). phosphorylase B (92,500), BSA (69.000), ovalbumin (46,000), carbonic anhydrase (30,000), and lysozyme (14,300).

was not immunoprecipitated by a control IgG,., antibody (MOPC21) or by a monoclonal antibody specific for the rat NGF-R (Ab 192).

NCF-R protein species (Fig. 5A). No immunoblotting occurred with the control antibody, MOPC2 1 . When receptor samples were exposed to a reducing agent (betamercaptoethanol) and boiled prior to electrophoresis there was no antibody binding (Fig. 5B). Additional experiments showed that all of the antibodies produced in this study immunoblotted non-reduced NGF-Rt, however, the intensity of the autoradiographic signal obtained was less for antibodies IIIG5 and VIIIC8 (data not shown).

Epitope Mapping and Development of the 2-Site RISA Species Cross-Reactivity Solid phase competition studies were performed to All of the antibodies bound to NGF-Rt from mon- determine whether monoclonal antibodies generated in key urinc. However, antibodies did not bind to NGF-R this study bound to distinct receptor epitopes. All of the from chick embryonic dorsal root ganglia, rat supcrior antibodies were tested on the solid phase as well as servcervical ganglia, or PC12 cells (data not shown). ing as the radiolabeled (top) antibody. In this assay, the retention of radiolabel in the well indicates that the top Tmmunoblot of the Cell Surface NGF-R and bottom antibodies recogniLe distinct epitopes on the In order to further characterize the specificity of receptor protein. The only exception to this would occur antibody binding, immunoblotting studies were per- if a single antibody bound to repeated epitopes on the formed using NGF-R solubilized from E9b cells. When receptor. The results of the competition studies clearly receptor samples were prepared in the absence of reduc- showed that antibody IIIG5 bound to an epitope on the ing agent and were not boiled prior to resolution on gcls, NGF-Rt that was distinct from the epitope(s) rccognixd two of the antibodies (VIID1, X l F I ) bound to a ma-jor by monoclonal antibodies VTID1, VITIC8, IXFl , and the

NGF-Receptor Antibvdies

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Fig. 4. Immunoprecipitation of NGF-Rt. Monoclonal antibodies ( 5 pg) were incubated with [125-I]-NGF cross-linked to NGF-Rt (2.2 pg protein) that had been purified approximately 650-fold from E9b conditioned medium by chromatography on ME20.4 sepharose. Samples were analyzed using the CLIP assay. Standards are as described in Figure 2 .

previously developed monoclonal antibody ME20.4 (Fig. 6A-D). When each antibody was probed against itself, the retention of radioactivity in the wells was no greater than when a control antibody, MOPC21, was used. These results indicate that the antibodies generated in this study do not recognize repeated epitopes on the receptor. Two monoclonal antibodies recognizing distinct sites on the receptor were selected for use in the development of a 2-site RISA. Based on results of the previous competition studies, antibodies IIIG5 and XIF 1 were chosen. When either NGF-R (Fig. 7A) or NGF-Rt (Fig. 7B) was assayed, the combination of radiolabeled IIIG5 with XIFl as the anchoring antibody resulted in the maximum retention of specific counts per well. Ltk- cells or Ltk- cell conditioned medium were assayed in parallel with E9b cells and medium to assess non-specific binding. These studies showed that non-specific binding in the assay was higher when solubilized cell preparations were used compared to conditioned medium. The use of MOPC21 as the anchoring antibody also providcd a good assessment of non-specific binding. In order to test the linearity of the 2-site RISA, serial dilutions of NGF-Rt that had bcen concentrated by ammonium sulfate precipitation were tested. There was a linear relationship between the amount of NGF-Rt added

Fig. 5 . Immunoblot of E9b cell NGF-R. E9b cells were solubilized with 2% n-octyl glucoside. A: The solubilized prcparation was loaded directly on a non-reducing 10% SDS-polyacrylamidc gel followed by electrophoresis and transfer to a nitrocellulose membrane. The membrane was cut into strips and incubated with affinity purified monoclonal antibodies. Antibody bound to receptor on the nitrocellulose was detected using [ I25-I]-labeled goat-anti-mouse 1gG antibody. B: Samples were mixed with 5% beta-mercaptoethanol and boiled prior to resolution on gels and ininiunoblotting.

and the amount of [125-I]-IIIG5 retained per well up to at least 30,000 cpm per well (Fig. 8A). Data obtained from the 2-site RISA were compared to those obtained using the CLIP assay. Figure 8 shows a close correlation (r = 0.998) between the relative values obtained using the 2-site RlSA and the CLIP assay.

DISCUSSION Monoclonal antibodies have been produced against a soluble, NGF-Rt elaborated by E9b cells. NGF-Rt from E9b cells is similar to NGF-Rt found in the conditioned medium of culturcd Schwann cells (DiStefano and Johnson, 1988b) and melanoma cells (Zupan et al., 1989) with respect to molecular weight and displacement of [ 12S-II-NGF binding by unlabeled NGF. Although the four antibodies produced in this study immunoprecipitate affinity labeled receptor species to varying degrees, it is evident that they all recognize the whole (cell surface) and truncated forms of the NGF-R. Additionally, all antibodies bind to NGF-R localized on the cell surface of SH-SYSY human neuroblastoma cells. This cell line has been reported to express exclusively the high affinity form of the NGF-R (Sonnenfeld and Ishii, 1985), al-

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Fig. 7. 2-site RISA using [125-I]-labeled IIIGS antibody to detect NGF-R and NGF-Rt bound to immobilized antibodies. A: E9b cells (hatched bar) were solubilized and used as a source of NGF-R in these assays. Ltk- solubilized cell preparations (open bar) were examined in parallel wells to assess non-specific binding. B: E9b conditioned medium (solid bar) was used in the assay as a source of NGF-Rt. The conditioned medium from Lrk- cells was examined in parallel wells to assess non-specific binding. Samples were analyzed in triplicate and data are expressed as mean C SD. An average for the background binding when either Ltk- solubilized cells or conditioned medium was used in the assay is shown by the dotted line.

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Fig. 6. Identification of antibodies that bind to distinct receptor epitopes. Solid-phase competition studies were performed using the four [ 12S-I]-labeled monoclonal antibodies generated in this study to probe NGF-Rt bound to affinity purified antibodies. NGF-Rt used in these studies was purified approximately 240-fold from E9b conditioned medium. The dotted line represents background for each [ 12S-I]-labeled antibody probed against itself. Samples were analyzed in triplicate and data are expressed as mean SD.

*

though recent evidence suggests that SH-SY5Y cells may express both low and high affinity NGF-R (Chen et al., 1990). High and low affinity receptors may represent the same ligand binding protein with high affinity binding conferred on the core (low affinity) NGF-R protein by association with a modulatory protein (Hosang and Shooter, 1985; Green and Greene, 1986; Hempstead et al., 1989). It remains to be determined whether the antibodies developed in the present study bind to both high and low affinity NGF-R. The monoclonal antibodies produced in this study were screened on the basis of their ability to bind human NGF-R affinity labeled with [125-I]-NGF in both a solid phase (RISA) and an immunoprecipitation assay. The antibodies all cross-react with receptor from monkey but not rat o r chick. T w o other laboratories have reported the production of monoclonal antibodies to the human NGF-

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dislinct epitopes on the NGF-R has allowed the development of a highly sensitive assay which can be used to measure NCF-Rt in large numbers of samples. The assay can also be used to measure whole NGF-R extracted from a variety of sources. Measurement of receptor using the 2-site RTSA offers an added advantage over previously described methods because receptor can be assayed in the abscnce of ligand (NGF). Measurement of NGF-Rt using the 2-site RISA and the CLIP assay yields similar results. The 2-site RISA has been used to examine the regulation of NGF-Rt levels during the course of development in humans (DiStefano et al., in press). In this study as little as I to 2 ng (20-40 fmol) of NGF-Rt could be detected in 25 pl of human urine using the 2-site RISA. Antibodies that bind to different receptor epitopes provide important tools which can be used to confirm the specificity of antibody binding to receptor in tissue sections using immunohistochemical techniques (Loy et al., 1990; see next article). Jn conjunction with the overexprcssion of recombinant receptor, these new antibodies are currently being used to develop immunoaffinity purification strategies to facilitate studies of NGF-R structure.

CLIP (% mox)

Fig. 8. Validation of the 2-site RISA. A: Linearity of binding of [ 125-II-IIIG5to increasing amounts of NGF-Rt immobilized on Immulon 2 Removawells by antibody XIFl . B: Correlation of relative values obtained for NGF-Rt in serial dilutions of EYb conditioned medium concentrated by ammonium sulfate precipitation using the 2-site RISA and the CLIP assay. Data in each assay are expressed as a percent of the maximum value obtained for NGF-Rt.

ACKNOWLEDGMENTS We thank Dr. June Biedler, Memorial Sluan-Kettcring Cancer Center, New York, NY, for providing us with human neuroblastoma cells (SH-SY5Y). We also thank Dr. Jeffrey F. McKelvy for valuable suggestions. This work was supported in part by Abbott Laboratories.

REFERENCES R, none of which cross-react with receptor from chick or rat (Ross et al., 1984; Marano et al., 1987). A comparison of NGF-R cDNAs from human, rat, and chicken shows that the encoded receptor amino acid sequences are highly conserved between species (Johnson et al., 1986; Radeke et al., 1987; Ernfors et al., 1988; Escandon and Chao, 1989; Large et al., 1989). This high degree of homology may explain why the generation of antibodies that crossreact with a variety of species has been difficult. The immunoblotting experiments demonstrate that the monoclonal antibodies generated in this study do not bind to reduced forms of the NGF-R after transfer to nitrocellulose. This suggests that the antibodies recognize NGF-R species in a conformation-dependent fashion. Additional experiments in our laboratory (unpublished data) have revealed that the antibodies bind to receptor expressed in a bacterial system, implying that they recognize the core protein rather than sugar moieties present on the NGF-R (Grob et al., 1985). Identification of monoclonal antibodies that bind to

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