characterization of monoclonal antibodies to protoplast membranes of nicotiana tabacum identified by...

Planta (1987)171:453M65 P l a n t a �9 Springer-Verlag 1987

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

M.G. Hahn*, D.R. Lerner**, M.S. Fitter, P.M. Norman*** and C.J. Lamb Plant Biology Laboratory, Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92138-92/6, USA

Abstract. Murine monoclonal antibodies to protoplast membrane antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofiuorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodate- sensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of Mr 14 kilodal- tons (kDa) and a smear of bands of M~45-- 120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to

* To whom correspondence should be addressed. Present address." Complex Carbohydrate Research Center, University of Georgia, P.O. Box 5677, Athens, GA 306113, USA ** Present address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312, USA *** Present address. Friedrich-Miescher-Institut, Postfach 2543, CH-4002 Basel, Switzerland

Abbreviations." ELISA = Enzyme-linked immunosorbent assay; Ig=immunoglobulin; kDa=kilodalton; Mr=relative molecular mass; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel clectrophoresis

bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equili- brating at a density of approx. 1.12kg.1-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman etal. 1986, Planta 167, 452-459) available for the study of plant plasma-membrane structure and function.

Key words: Cell surface - Enzyme-linked immunosorbent assay (ELISA) - Glycoprotein - Mono- clonal antibody - Nicotiana (plasma membrane) - Plasma membrane - Protoplast surface.

Introduction

Hybridoma technology allows the generation of specific monoclonal antibodies starting from im- pure, heterogeneous immunogen preparations (K6hler and Milstein 1975; Milstein 1986) and has proved to be a valuable and powerful tool in the study of the structure, function, and biogenesis of the plasma membrane of animal cells (Hosking and Georgiou 1982; Williams 1980). We have recently characterized a number of monoclonal antibodies to epitopes present on the plasma-membrane of plant cells (Norman et al. 1986). Crude preparations of total cellular membrane from suspension- cultured Nicotiana glutinosa cells were used as immunogen to ensure that, in the absence of appropriate biochemical markers (see Quail 1979), plasma-membrane antigens were not inadvertently lost by a prior fractionation procedure. The monoclonal antibodies so obtained were directed against plasma-membrane epitopes widely distributed with respect to both cell-type and plant species.

In these initial studies, antibody secretion at

454 M.G. Hahn et al. : Monoclonal antibodies to protoplast membranes

various stages during the cloning of hybridoma cell lines was routinely monitored by radioimmunoassay of antibody binding to the total cellular membrane preparation used as immunogen. The radioimmunoassay was time-consuming, could handle only about 200 samples per day and because of the instability of radiolabeled second antibody, required the routine handling of large amounts of radioactivity in the regular preparation of iodin- ated antibody. Moreover, it was not possible by this assay to screen directly for antibodies that were directed against plasma-membrane antigens. Identification of the subset of monoclonal antibodies of interest required a second, laborious screen to monitor antibody binding to the surface of isolated protoplasts by immunofluorescence mi- croscopy.

We report here the development, optimization, and characterization of a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for binding of antibodies to immobilized plant membrane preparations or immobilized protoplasts. The assay is safer and more convenient than the radioimmunoassay described previously and allows handling of up to 800 samples per day. Fur- thermore, when protoplasts are used as the immobilized antigen, the ELISA appears to be selective for antibodies that bind to epitopes present on the external face of the plasma membrane. This ELISA has facilitated the generation of additional monoclonal antibodies to epitopes present on the plasma membrane. We describe here two new classes of antibody generated by immunization with intact leaf mesophyll protoplasts.

Material and methods

Chemicals and supplies. Cellulysin and macerase were purchased from Calbiochem-Behring (San Diego, Cal., USA). Goat anti- mouse immunoglobulin (Ig)-/~-galactosidase conjugate and o- nitrophenyl-fl-D-galactopyranoside were from Zymed (San Francisco, Cal.). Heat-inactivated horse serum and fluorescein- labeled goat anti-mouse immunoglobulins were obtained from HyClone (Logan, U., USA). Glutaraldehyde, poly-L-lysine, 4- chloro-l-naphthol, polyoxyethylene sorbitan monolaurate (Tween 20), lactalbumin, ovalbumin, and bovine serum albumin were from Sigma Chemical Co. (St. Louis, Mo., USA), and goat anti-mouse-IgG-horseradish peroxidase conjugate, protein-assay dye reagent concentrate and bovine ~-globulin were from BioRad (Richmond, Cal.). Polystyrene fiat-bottom micro-titer plates (Immulon 1 and 2) and flat-bottom polyvin- ylchloride plates (Falcon No. 3912) were from Dynatech (Alex- andria, Va., USA) and Becton-Dickinson (Oxnard, Cal.), re- spectively. A monoclonal antibody to a surface antigen of the phytopathogenic bacterium Pseudomonas syringae pv. glycinea was provided by V.P.M. Wingate (Salk Institute). A number of monoclonal antibodies generated against membranes from Nicotiana glutinosa have been described previously (Norman et al. 1986).

Plant material. Seeds of Nicotiana tabacum L. cv. Xanthi, N. glutinosa L., N. plumbaginifolia L. (from F. Hoffmann, Univer- sity of California, Irvine, USA) and Lycopersicon eseulen- tum (L.) Mill. cv. UC82 (from L. Fitzmaurice, SIBIA, La Jolla, Cal.) were surface-sterilized with 0.5% sodium hypochlorite for 20 min and were germinated on Murashige and Skoog medium (Murashige and Skoog 1962) containing 3% (w/v) sucrose and 0.8% (w/v) agar (MS). A single plant of each species was arbi- trarily selected and divided into segments of 1-2 cm length, with a nodal bud and leaf on each section. These nodal sections were planted aseptically in 100 ml of MS in Magenta GA7 vessels (Magenta Corp., Chicago, Ill., USA). Plants were grown at 28~ under cool-white fluorescent lamps (20 W; General Electric Co., Cleveland, Oh., USA) (14 h photoperiod) and new explants propagated every six to eight weeks.

Phaseolus vulgaris L. cv. Canadian Wonder seeds (from Im- perial Chemical Industries, Runkorn, UK) were surface-sterilized with 95% ethanol (30 s) and 0.26% sodium hypochlorite (30 rain). The seeds were then soaked overnight in sterile, deionized H20 and placed on MS (5 ml/well) in multiwell plates (Falcon No. 3046). Seeds that germinated and remained uncon- taminated were transferred to 100 ml of MS in Magenta GA7 vessels and grown as above until the primary leaves had expanded, at which time the leaves were used for isolation of protopiasts (see below). Phaseolus vulgaris plantlets were not propagated.

Isolation ofprotoplasts. Conditioning, Digestion and Rinse me- dia were those described by Haberlach et al. (1985). Leaves (1 3 g FW) of aseptically grown plants were excised and placed in 150 ml of Conditioning medium overnight in the dark at 4 ~ C. Protoplasts were prepared from the conditioned leaves (Haberlach et al. 1985) or from cell-suspension cultures (Nor- man et al. 1986) as described, and yield determined using a hemocytometer. For the ELISA, protoplasts were either used immediately or after overnight storage in Rinse medium at 4 ~ C, and were diluted to about 105 .ml- 1 in 20 mM 2-amino-2-(hyd- roxymethyl)-l,3-propanediol (Tris)-HC1, pH 7.5, containing 0.3 M NaC1 (ELISA Buffer).

Membrane preparation. A crude total membrane fraction was prepared from suspension cultured cells of N. glutinosa and N. tabacum, and freshly isolated N. tabacum leaf protoplasts as described in Norman et al. (1986), except that for N. tabacum suspension cells the homogenization buffer contained 0.1 M sodium metabisulfite. Protein concentrations of the membrane preparations were estimated by the "BioRad protein assay" based on the method of Bradford (1976) using bovine y-globulin as the standard.

Production of monoelonal antibodies. Balb/c female mice (bred by the Salk Institute Animal Facility) were immunized by injection with either freshly isolated N. tabacum leaf protoplasts or crude total cellular membranes from N. tabacum leaf protoplasts by one of two immunization schedules. Schedule A consisted of four intraperitoneal injections of 6" 105 to 8.5-105 protoplasts in 0.5 ml of Rinse medium at weekly intervals, followed 10 d later by a single intravenous injection of 6.6 gg crude total protoplast membrane protein in 0.2 ml of phosphate-buffered saline. Schedule B consisted of a single intraperitoneal injection of 6.4-105 protoplasts in 0.5 ml of Rinse medium followed a month later by a single intravenous injection of 6.6 gg crude total protoplast membrane protein in 0.2 ml phosphate-buffered saline. All mice were sacrificed four days after final injection. The freshly isolated splenic lymphocytes were fused with SI94/5.XXO.BUI myeloma cells as described by Trowbridge (1978). Following fusion, the cell suspensions were plated into

M.G. Hahn et al. : Monoclonal antibodies to protoplast membranes 455

48-well plates (Costar No. 3548) and grown as described in Norman et al. (1986). Supernatants harvested from the expanded hybridoma colonies were screened for antibodies to the immunogen by ELISA (see below). Hybridoma colonies that secreted antibodies of interest were cloned by limiting dilution at 2 and l0 cells/well in the absence of feeder cells. Secret- ing clonal cell lines were expanded, cryopreserved, and their culture supernatants collected for further analysis.

Enzyme-linked immunosorbent assay. All operations were performed at room temperature. Microtiter plate(s) (96 wells; Ira- toulon 2) were loaded with about 104 protoplasts or 7.5 gg crude total cellular membrane protein (in 50/A of ELISA Buffer) per well and the antigen allowed to settle for 10 rain. Glutaral- dehyde (1% (v/v) in ELISA Buffer, 50 IA/well) was added, and the plate(s) centrifuged for 3 rain at 100-g~v for protoplasts or 700'ga~ for membranes, and incubated for 15 min. The supernatant was removed by aspiration with a Nunc-Immuno- wash 12 (Nunc, Roskilde, Denmark) to leave about 25 gl in the bottom of each well. The wells were washed once with ELISA Buffer (200 1/well). Following aspiration of the supernatant, non-specific antibody-binding sites were blocked by incubation with 1% (v/v) horse serum in ELISA Buffer (200 gl/well) for 30 rain. Blocking solution was removed by aspiration, and immobilized antigen was then incubated with hybridoma supernatants (50 gl/well) for 30 rain. The plates were washed once with ELISA Buffer containing 0.1% (v/v) horse serum. Following aspiration of the supernatant, goat anti-mouse Ig-fl-galactosidase-conjugate (diluted 1:200 in 10 mM Tris-HC1 buffer, pH 8.0, containing 0.3 M NaC1, 0.1% (v/v) horse serum, 1 mM MgC12, 0.02% (w/v) sodium azide) was added (50 ~d/well) and the plate(s) incubated for 2 h. The plate(s) were then washed three times with ELISA Buffer containing 0.1% (v/v) horse serum (200 gl/well). Substrate (1 mg.ml 1 o-nitrophenyl-fl-D- galactopyranoside in 10 mM 2-mercaptoethanol and 10 mM NaC1) was added to each plate (50 gl/well) and the plate(s) incubated until color developed (usually 30 to 60 rain). The reaction was stopped with 0.5 M Na2CO3 (100 ~d/well) and the absorbance measured at 405 nm using an ELISA plate reader (BioRad, Richmond, Cal.). Unless otherwise indicated, the absorbance obtained with hybridoma supernatant VW40.2B2, generated against a surface epitope of Pseudomonas syringae pv. glycinea, was subtracted as a measure of background, non- specific antibody binding. A hybridoma clone was selected as positive if it gave an absorbance of 0.1 or higher above background.

Immunofluorescence assay. Direct visualization of antibody binding to protoplast cell surfaces was accomplished by an indirect immunofluorescence procedure described previously (Nor- man etal . 1986; Fitter et al. 1987) for small numbers of samples (< 20). Large numbers of samples were screened by a procedure adapted from the ELISA protocol described above, but in which the glutaraldehyde fixation and associated washes as well as substrate and stop incubations of the ELISA procedure were eliminated. Fluorescein-labeled goat anti-mouse antibody was used as the second antibody at a ] :20 dilution in ELISA Buffer. The ELISA protocol was otherwise unchanged. Proto- plasts were examined directly in the microtiter plate wells as described previously (Norman et al. 1986).

lsotyping of hybridoma clones. The immunoglobulin classes to which the various monoclonal antibodies belonged were determined using the HyClone Mouse Monoclonal Sub-isotyping kit (HyClone).

Detection of antibodies to periodate-sensitive membrane epitopes. Monoclonal antibodies recognizing periodate-sensitive epitopes

in membrane preparations were determined essentially by the method of Woodward et al. (1985). Briefly, crude total membrane preparations from N. glutinosa were fixed to the bottom of micro-titer plate wells (20 gg/well) as described for the ELISA above. The plates were then washed twice with 50 mM sodium-acetate buffer, pH 4.5 (SAB) (200 gl/well). Selected wells (usually every other set of three columns in a micro-titer plate) were then treated with 10 mM periodate in SAB (200 gl/ well) for 90 rain at room temperature in the dark. The remain- ing wells (controls) were treated with SAB. The plate(s) were washed three times with SAB (200 gl/well), and then treated with 1% (w/v) glycinc in ELISA Buffer for 30 min to block any newly formed aldehyde groups. The plate(s) were washed twice with ELISA Buffer and then carried through the re- mainder of the ELISA procedure beginning at the blocking step as described above. Each hybridoma supernatant was applied to a set of periodate-treated and untreated wells.

Western blotting. Extracts of cellular proteins from actively growing cultures of N. glutinosa (7-d-old culture) were prepared by grinding cells using a mortar and pestle in i ml .g-1 FW of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Sample Buffer (80 mM Tris-HC1 buffer, pH 6.8, containing 10% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate, and 0.005% (w/v) bromo- phenol blue) for 3-5 rain at room temperature. The cell homog- enate was heated in a boiling water bath for 10 rain, and the undissolved material pelleted at 48000.gay for 20 rain at 25 ~ C. The supernatant was decanted and stored frozen at - 2 0 ~ until needed. The cellular extract (0.5 ml) was applied to a dis- continuous SDS polyacrylamide slab gel (5% acrylamide stacking gel, 10% acrylamide running gel), 0.7 mm thick, in a 12-cm- wide sample well. The stacking gel also contained a small sepa- rate well for molecular-weight standards. Electrophoresis was carried out in the presence of 0.1% SDS as described (Laemmli /970).

Etectroblotting of the proteins from the gel to nitrocellulose (0.45 ~tm pore size, Schleicher and Schuetl, Keene, N.H., USA) was carried out in Transfer Buffer (80 mM Tris, 13 mM glycine, 20% (v/v) methanol, pH 9.2) for 3 h at 60 V constant voltage using a Hoefer Transphor cell (Hoefer, San Francisco, Cal.) as described (Towbin et al. 1979). After blotting, the strip of nitrocellulose containing the molecular-weight markers was removed and stained with 0.1% (w/v) amido black in 20% (v/v) methanol, 7.5% (v/v) acetic acid in water for 15 rain at room temperature, followed by destaining in distilled water for 30 rain to reveal the protein bands. The rest of the nitrocellulose sheet was incubated in about 50 ml of 5% (w/v) bovine serum albumin in 20 mM Tris-HC1, pH 7.5, containing 150 mM NaC1 (TBS) for 30 rain at room temperature with continuous agitation to block non-specific antibody-binding sites. The sheet was cut into vertical strips (approx. 3-5 mm width) and the strips placed !n individual wells of a multi-well incubation tray (LKB, Broma, Sweden). Hybridoma supernatant (5 ml/well of a 1 : 10 dilution in TBS containing 1% (w/v) bovine serum albumin) was added to each well, and the trays were covered and incubated overnight at room temperature with continuous agitation. Each strip was washed three times for 5 rain with 5 ml of 0.05% (v/v) Tween 20 in TBS, and then incubated with goat anti-mouse IgG-horseradish peroxidase conjugate (5 ml/strip of a 1:3000 dilution in TBS containing 1% (w/v) bovine serum albumin) for 2 h at room temperature with continuous agitation. The strips were washed three times with 0.05% (v/v) Tween 20 in TBS as above, followed by five 1-min washes with TBS. Immunoreactive bands were visualized by incubating the strips in TBS containing 0.5 mg.m1-1 4-chloro-l-naphthol, 0.015% (v/v) H202 and 17% (v/v) methanol until color devel-


oped (not longer than 30 rain), after which the strips were washed several times in deionized HzO and stored in the dark.

Antigen distribution in sucrose density gradients, Total cellular membranes prepared as above from approx. 50 g FW of suspension-cultured N. glutinosa cells were centrifuged for 18 h at 100000'g,v in a linear 15-45% (w/w) sucrose gradient containing 2.5 mM Tris-2-(N-morpholino)ethanesulfonic acid (Mes), 0.5 mM ethylenediaminotetraacetic acid (EDTA) and 1 mM dithiothreitol at pH 7.4. A portion (0.2 ml) of each gradient fraction (approx. 1.4 ml) was diluted fivefold in TBS and aliquots (20 pl) of the diluted fractions were absorbed to nitrocellulose using a slot-blot manifold (Schleicher and Scbuell). The nitrocellulose sheet was removed from the manifold and incubated in 50 ml of 5% bovine serum albumin in TBS for 30 rain at room temperature with continuous agitation. The sheet was cut into strips, each containing all the slots from a gradient. Each strip was incubated with hybridoma superna- rant (25 ml of a 1 : 50 dilution in TBS containing 1% (w/v) bovine serum albumin) overnight at room temperature with continuous agitation. Immunodetection of antibody binding to immobilized membranes was performed as described above. Anti- body binding was quantified by reflectance densitometry using a Scanning Densitometer (Hoefer).

Results

Optimization of the ELISA

The goal of the optimization was to obtain high absorbance signals with test hybridoma supernatants containing antibodies to plant plasma-membrane epitopes relative to background absorbance with samples containing control supernatants from hybridoma VW 40.2B2. Previous radioimmunoas- says had shown that VW 40.2B2, which contains antibodies to epitopes on the surface of the phytopathogenic bacterium Pseudomonas syringae pv. glycinea, does not bind to plant membrane preparations. Optimization of the ELISA protocol was carried out using leaf protoplasts as the immobilized antigen and, where appropriate, subsequently rechecked with crude total cellular membrane preparations.

Multi-well plate type. Three different multi-well plate types (Immulon 1 and 2, and Falcon No. 3912) were tested for suitability in the ELISA. Antigen could be immobilized to all three plate types. Slightly higher signals as measured by absorbance with test-positive supernatants relative to control VW 40.2B2 supernatant were obtained with Immulon 2 plates and hence these were used routinely.

Immobilization ofprotoplasts. Optimization of the immobilization of antigens to the bottom of the micro-titer plate wells proved to be a key step in the development of the assay. Several methods

were tried alone or in combination, including: (a) centrifugation of the protoplasts to the bottom of the microtiter plate wells; (b) coating the wells with poly-L-lysine (1 mg 'ml-1) ; and (c) glutaraldehyde (0.5%) fixation. Of these, glutaraldehyde fixation followed by centrifugation of the protoplasts in the micro-titer plates proved most effective. While a final glutaraldehyde concentration greater than 0.25% (v/v) was required to obtain satisfactory adhesion of protoplasts, the duration of fixation did not affect the efficacy of the treatment. Coating the wells with poly-L-lysine did not notably im- prove the immobilization of the protoplasts.

Care is required in subsequent handling of the plates since the immobilized protoplasts are quite easily dislodged, for example by flicking or blotting. For removal of wash solutions, careful aspiration of supernatant to leave about 25 gl of liquid in the bottom of each well avoids disturbance of the immobilized protoplasts. For addition of liquid to the wells, an eight-channel micropipetter with tips cut to give openings of at least I mm was found to be satisfactory. Detergents in the wash buffers reduced protoplast adhesion and were omitted from all solutions used in the assay. The optimal wash protocol was defined by the mini- mum number of washes required at each stage to reduce to lowest attainable levels the absorbance obtained with control hybridoma supernatant VW 40.2B2.

Maintenance of protoplast integrity. In an effort to prevent bursting of protoplasts during the assay, which would result in accessibility of cell-internal epitopes, NaC1 was included in all solutions at a concentration corresponding to the osmolarity of the medium in which the protoplasts were isolated. NaC1 was used instead of the usual protoplast os- motica (e.g. mannitol, sorbitol, sucrose) to avoid potential problems with non-specific antibody binding encountered previously (Norman et al. 1986). When NaC1 was included, most protoplasts adhering to the plates at the end of the assay had not burst (Fig. 1).

Blocking of non-specific antibody-binding sites. Non-specific antibody binding to protoplasts has been reported (Larkin 1977; Norman et al. 1986). Several reagents (ovalbumin, lactalbumin, bovine serum albumin, horse serum) were tested at various concentrations for their efficacy in blocking non- specific antibody binding to the plates and to adhered antigen. All reagents except lactalbumin blocked non-specific antibody binding to the plas- tic and to antigen at concentrations of 1% or


Fig. 1 A, B. Adhered protoplasts from leaves of Nicotiana tabacum: A during glutaraldehyde fixation; B during the third wash after incubation with goat anti- mouse-Ig-/?-galactosidase conjugate, x 95 ; bar = 200 gm

greater. Lactalbumin behaved anomalously in that concentrations of 1% or greater resulted in a high background signal in the absence', of either antigen or first antibody. Horse serum was chosen as the blocking reagent for convenience and cost-effec- tiveness. No time dependence was observed for the blocking reaction.

Amount of antigen. The absorbance obtained in the assay was dependent on the amount of antigen applied. Thus, signal strength increased with in- creasing number of protoplasts up to about 10 4

protoplasts/well (Fig. 2A). Inclusion of greater numbers of protoplasts did not result in a corresponding increase in signal, probably because there is no net increase in the number of protoplasts that remain adhered. A similar concentration dependence was observed with crude total cellular membrane preparations as the immobilized antigen (Fig. 2 B).

Antibody incubations. Hybridoma supernatants could be used directly in the assay without prior treatment. The protocol was equally effective for

458 M.G. Hahn etal. : Monoclonal antibodies to protoplast membranes

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Number of Protoplasts (x lO4/well) Amount of Membrane (/~g protein/well) Fig. 2A, B. Dependence of ELISA signal on protoplast number or amount of membrane protein. Varying numbers of N. tabacum leaf protoplasts (A) or amounts of N. glutinosa membrane protein (B) were applied to the micro-titer plate wells and carried through the ELISA. Hybridoma supernatants utilized were PN 16.1B3 (zx--A) and PN 16.4B4 ( v - - v ) containing antibodies directed to epitopes present on the external face of plasma membranes; PN 17.3A6 ( ~ - - n ) containing antibody directed to a cell-internal epitope; and VW 40.2B2 ( o - - o ) containing antibody directed to a surface epitope of the phytopathogenic bacterium Pseudomonas syringae pv. glycinea. Each point is the average of three replicates

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Incubation Time (min) Fig. 3A, B. Dependence of ELISA signal on the length of incubation with second antibody-enzyme conjugate. The immobilized antigen was either N. tabacum leaf protoplasts (A) or crude total membrane preparation from N. glutinosa (B). The absorbance obtained with control hybridoma supernatant VW 40.2B2 was subtracted from each point. Details are the same as in Fig. 2

hybridoma supernatants containing either IgG or IgM subclasses of antibodies. Varying the first antibody incubation time between 15 and 120 rain did not affect the signal strength. In contrast, the incubation parameters for the second antibody-enzyme conjugate had a significant effect on the final absorbance. The optimum dilution of the antibody-enzyme conjugate must be determined for each batch of the conjugate. The signal strength was very dependent on the length of the incubation with second antibody (Fig. 3). An incubation time of 2 h was generally used as a compromise between satisfactory signal strength and rapidity of assay. A longer incubation may be appropriate when an-

tibodies to rare epitopes are sought, although the background measured by controls with VW 40.2B2 becomes correspondingly greater. Both the goat anti-mouse-Ig-alkaline phosphatase and the goat anti-mouse-Ig-fi-galactosidase conju- gates were used successfully in the ELISA, the latter being used routinely.

Reproducibility of the ELISA. A set of 16 identical replicates with the same protoplast preparation and hybridoma supernatant (PN 16.4B4) processed in parallel in adjacent wells of the same plate gave a mean absorbance above control supernatants of 0.112 with a standard deviation of 0.015


Fig. 4A, B. Indirect immunofluorescence assay in micro-titer plate wells. Micrographs of N. glutinosa protoplasts in the wells at the end of the assay under (1) bright-field illumination and (2) epifluorescence illumination. Monoclonal antibodies used were A VW 40.2B2 ( x 68) and B PN 16.4B4 ( x 136). Bars= 100 gm

(13%) about the mean. The comparable absorbance and standard deviation wit!h membrane preparations as immobilized antigen was 0.282_+0.017(6%). Greater variation was observed between identical samples in different plates processed in parallel. As with other colorimetric assay procedures, absorbance values obtained in different experiments were not directly comparable and therefore we routinely included monoclonal antibody PN 16.4B4 in each plmLe, both as a positive control and to provide a comparative refer- ence. For routine plus-minus screening of hybridoma supernatants (such as during cloning by limiting dilution), a single well per sample was sufficient. Comparative and/or more quantitative assays were performed in triplicate and all samples were tested in one assay.

Applicability of the ELISA procedure. The ELISA protocol was tested using: protoplasts from leaves of Nicotiana tabacum, N. plumbaginifolia, N. gIu- tinosa, Phaseolus vuIgaris, Lycopersicon esculen-

turn, and suspension-cultured ceils of N. glutinosa and N. tabacum; crude total membrane preparations from suspension cultured cells of N. glutinosa and N. tabacum; and suspension cultured ceils of N. glutinosa. Protoplasts, irrespective of their ori- gin, could be used as the immobilized antigen. The absorbance obtained with the control hybridoma supernatant (VW 40.2B2) was low (<0.2) compared to signals with supernatants containing antibodies to plasma-membrane antigens, which typi- cally were in the absorbance range 0.3-1.1.

Similarly, crude membrane fractions prepared from either protoplasts or suspension-cultured cells could serve as the immobilized antigen, and generally gave a stronger signal than intact protoplasts (Table 1). This may, in part, reflect the relative amount of antigen that can be immobilized when presented in the form of isolated membranes compared to intact protoplasts. While suspension- cultured cells could also be immobilized, unaccep- tably high absorbance was obtained with a control antibody.

460

Adaptation of the ELISA protocol to indirect immunofluorescent visualization of antibody binding to protoplast and cell surfaces

The success with the ELISA protocol led us to adapt the procedure for visualization of antibody binding to protoplast and cell surfaces by indirect immunofluorescence. The major modification was elimination of the glutaraldehyde fixation step since such fixation results in high levels of auto- fluorescence (Norman et al. 1986). This led to sub- stantial losses of protoplasts and cells from the micro-titer plate wells during the procedure. In addition, a greater number of protoplasts burst. However, sufficient numbers of protoplasts remained in the wells to inspect microscopically for immunofluorescent labeling of the protoplast plasma-membrane (Fig. 4). More recently, we have found that diluting hybridoma supernatants with ELISA buffer alleviates the breakage problem somewhat (data not shown). This immunofluorescent procedure is able to handle substantially more samples per day (several hundred vs. 20) than does the immunofluorescence assay described previously (Norman et al. 1986; Fitter et al. 1987).

Selection and characterization of monoclonal antibodies

Selectivity of the protoplast ELISA for external plasma-membrane epitopes. The ELISA was used to test a number of monoclonal antibodies that had been characterized previously (Norman et al. 1986), to determine whether the protoplast ELISA could function as a selective screen for antibodies to the plant plasma membrane. All of these monoclonal antibodies gave a strong signal in the ELISA with crude total cellular membranes as the immobilized antigen (Table 1). When the same monoclonal antibodies were tested versus protoplasts, the ELISA became more selective. In particular, monoclonal antibody PN 17.3A6, which binds to cell-internal epitopes, and PN 17.3B4, which binds to cell-wall epitopes (Norman et al. 1986), gave very low signals in the ELISA with protoplasts as immobilized antigen.

This selectivity was further confirmed in a subsequent screen of newly generated hybridomas. About 350hybridoma cultures were recovered from two fusions using splenocytes from mice immunized with N. tabacum leaf protoplasts. When crude total cellular membranes were used as the immobilized antigen, 24 clones tested positive in the ELISA (Table 2). However, only six of these cell lines tested positive in the protoplast-ELISA

M.G. Hahn et al. : Monoclonal antibodies to protoplast membranes

Table 1. ELISA of binding of previously characterized monoclonal antibodies using protoplasts or crude total cellular membranes as immobilized antigen

Clone Target b A405" Ratio a

Protoplasts c Membrane ~

PN 16.1B3 PM 0.928 1.417 0.65 PN 17.3A3 PM 0.474 0.772 0.61 PN 17.3D4 PM 0.464 0.897 0.52 PN 16.4B4 PM 0.393 0.850 0.46 PN 16.2C6 PM 0.448 1.064 0.42 PN 16.3C1 PM 0.509 1.472 0.35 PN 17.4B5 PM 0.123 0.381 0.32 PN 16.4A6 PM 0.127 0.640 0.20

PN 17.3A6 I 0.129 0.902 0.14

PN 17.3B4 CW 0.119 0.813 0.15

All hybridoma supernatants were assayed against both antigens in one experiment. Each datum is the average of three replicates. The absorbance obtained with control hybridoma supernatant VW 40.2B2 has been subtracted b Determined by indirect immunofluorescence (Norman et al. 1986). P M = epitope on the external face of the plant plasma- membrane; I = plant celMnternal epitope; C W = plant cell-wall epitope c Protoplasts were prepared from Nicotiana tabacum leaves. Crude total cellular membranes were prepared from N. glutinosa suspension-cultured cells a Absorbance with protoplasts/absorbance with membranes

(Table 2), and, of these, four were later shown to bind to the external face of the protoplast plasma membrane (see below). Thus, the protoplast- ELISA preferentially selected clones secreting antibodies that bind to the outer surface of intact protoplasts. Furthermore, the protoplast-ELISA identified all outer-surface-reactive monoclonal antibodies subsequently picked by an indirect immunofluorescence screen (see below).

Identification of monoclonal antibodies to plasma- membrane epitopes by indirect immunofluorescence. In addition to screening the newly generated hybridoma cell lines by ELISA, all were screened using the micro-titer plate immunofluorescence assay. This screen identified four clones (MH 3.2B4, MH 4.2A4, MH 4.3E5, MH 4.4E4), also identified in the protoplast ELISA, as secreting antibodies that yielded the ring fluorescence characteristic of labeling of the protoplast surface (Fig. 5 B). Three additional clones (MH 3.2D1, MH 3.3C2, Mid 3.5E7), which tested positive in the membrane ELISA but not in the protoplast ELISA (Table 2), were shown to secrete antibodies that appear to bind to the surface of broken protoplasts but do not bind to intact protoplasts (Fig. 5 C). The re-


Table 2. ELISA of initial hybridoma supernatants from fusions using splenocytes from mice immunized with Nicotiana tabacum leaf protoplasts

Clone b A~os a Immuno- fluorescence a

Protoplasts c Membranes c

MH 3.1F5 0.080 0.186 MH 3.2B4" 0.274 0.467 + (PM, CW) MH 3.2C2 0.0 0.132 MH 3.2D1 * 0.050 0.186 + (BPM) MH 3.3C2 0.0 0.171 + (BPM) MH 3.3D2 0.024 0.174 - MH 3.3D3 0.047 0.209 MH 3.3E1 0.069 0.104 - MH 3.4F5" 0.045 0.342 - MH 3.5E7" 0.0 0.215 + (BPM) MH 3.5F4 0.024 0.294 - MH 3.8C4 0.010 0,113 -

MH 4.1C1 0.306 0.051 - MH 4.1D1 0.244 0.048 - MH 4,1E6" 0.004 0.247 MH 4.1F7 0.030 0.143 - MH 4.2A4" 0.183 0.505 + (PM, CW) MH 4.2C4 0.0 0.104 - MH 4.2C5 0.017 0.136 MH 4.2E2 0.014 0.127 - MH 4.3E5" 0.229 0.498 + (PM, CW) MH 4.3F7 0.013 0.084 MH 4.4C7 0.062 0.669 - MH 4.4E4" 0.315 0.893 + (PM, CW) MH 4.5A4 0.041 0.112 - MH 4.7D1 0.055 0.097 -

PN 16.4B4" 0.282 0.556 + (PM)

Each datum is the average of three replicates. The absorbance obtained with control hybridoma supernatant VW 40.2B2 has been subtracted b Clones marked with an asterisk have been cloned by limiting dilution

Protoplasts were prepared from N. tabacum leaves. Crude total cellular membranes were prepared from N. tabacum suspension-cultured cells a Indirect immunofluorescence assay against N. tabacum leaf protoplasts or N. glutinosa suspension-cultured cells (see Fig. 5). PM=labeling of plasma-membrane of intact protoplasts; CW=labeling of surface of suspension-cultured cells; BPM= labeling of plasma membrane of broken protoplasts

mainder of the monoclonal antibodies did not show any binding to protoplast plasma membranes (Table 2), and it was concluded that these antibodies recognize cell-internal membrane epitopes.

Interestingly, an immunofluorescence screen with intact cells from suspension cultures showed that the four antibodies exhibiting strong binding to protoplasts also bound to the surface of intact cells (Fig. 5 E, F). This is an indication that these newly identified monoclonal antibodies must recognize epitopes different from those characterized previously which only bind to protoplast surfaces

(Table 1; Norman et al. 1986). The monoclonal antibodies did not always show uniform binding to all ceils in a given culture. Some cell clumps, particularly larger ones (Fig. 5 E), showed uniform binding over the entire cell surface. Other cell clumps, most notably smaller ones, showed local- ized antibody binding to portions of the cell surface or no antibody binding (Fig. 5 F).

Species and tissue specificity of antibody reactivity. The four monoclonal antibodies that bound strongly to epitopes on the external surface of N. tabacum leaf protoplasts (MH 3.2B4, MH 4.2A4, MH 4.3E5, MH 4.4E4) were tested in the protoplast ELISA for cross-reactivity with protoplasts from N. glutinosa, N. plumbaginifolia, Phaseolus vulgaris, and Lycopersicon escuIentum leaves, and N. glutinosa and N. tabacum cell suspension cultures. The antibodies tested positive in the protoplast ELISA irrespective of the source of the protoplasts; they showed no species specificity and did not discriminate between protoplasts from leaves and suspension-cultured cells of Nicotiana spp.

Effect of periodate treatment on the subsequent binding of monoclonal antibodies to cellular membranes. Pre-treatment of total cellular membrane preparations with 10 mM periodate abolished the binding of the four antibodies reactive with epitopes present on the external face of the plasma membrane (Table 3). Such mild periodate treatment at acid pH has been shown to cleave vicinal hydroxyl groups in carbohydrates while leaving the polypeptide intact (Bobbitt 1956). The data indicate that the antigen(s) recognized by monoclonal antibodies MH 3.2B4, MH 4.2A4, MH 4.3E5, and MH 4.4E4 are glycoconjugates of some kind, the epitope(s) being located in the carbohydrate moiety.

The binding of the "broken protoplast" antibodies MH 3.2D1 and MH 3.5E7 was also reduced substantially (approx. 75%) by the periodate pre- treatment. The binding of several other antibodies to "cell-internal" epitopes was affected to a lesser degree (Table 3), indicating that these epitopes do not contain carbohydrate as important parts of the antibody binding site(s).

Molecular species carrying reactive epitopes. The supernatants from hybridomas selected by the membrane ELISA were screened for binding to Western blots of proteins extracted from N. glutinosa suspension-cultured cells. The four antibodies (MH 3.2B4, MH 4.2A4, MH 4.3E5, MH 4.4E4) that showed strong binding to protoplasts strongly


Fig. 5A-F. Indirect immunofluorescence visualization of the binding of monoclonal antibodies to the surfaces of N, tabacum leaf protoplasts (A-C) and suspension-cultured N. glutinosa cells (D--F). Micrographs were taken under (I) bright-field illumination, and (2) epifluorescence illumination. Monoclonal antibodies were A, D VW 40.2B2, B MH 4.3E5, C MH 3.2D1, E M H 3.2B4, F MH 4.4E4. Solid arrow in C denotes an intact protoplast, open arrow denotes a broken protoplast. A-C x 136; D x 34; E, F x 68; ba r s=100 gm

Table 3. Effect of pre-treatment of membranes with periodate on the subsequent binding of monoclonal antibodies

Clone Ig class A405a Reduction (%)

- P e r i o d a t e + Periodate

MH 3.2B4 G3 1.139 0.134 88 MH 4.2A4 M 0.873 0.067 92 MH 4.3E5 M 0.731 0.089 88 MH 4.4E4 M 0.837 0.041 95

MH 3.2DI G3 0.858 0.249 71 MH 3.5E7 G2b 0.766 0.194 75

MH 3.4F5 M 0.153 0.176 0 MH 3.5F4 - 0.158 0.070 56 MH 4.4C7 M 0.518 0.420 19

a All hybridoma supernatants were assayed against treated (10 mM periodate) and untreated crude total cellular membranes from Nicotiana glutinosa in one experiment. Each datum is the average of three replicates. The absorbance obtained with control hybridoma supernatant VW 40.2B2 has been subtracted

Antigen distribution in sucrose density gradients. The distributions in linear sucrose density gradients of the membrane antigens recognized by the antibodies that, by immunofluorescence, appear to bind to plant plasma membranes are shown in Fig. 7. For comparison, the antigen distribution seen with a previously characterized plasma-membrane-reactive monoclonal antibody, PN 16.4B4 (Norman et al. 1986), is also shown. The monoclonal antibodies that bind to the exterior ofprotoplasts and cells appeared to uniformly label all membranes within the gradient. In contrast, while the "broken protoplast" antibodies also bound to most membrane fractions, there was a clear peak in reactivity at a buoyant density of 1.12 kg.1-1 Thus, the "intact" and "broken" protoplast antibodies showed two different reactivity distribution patterns, which moreover were different from the patterns observed with previously characterized antibodies (Norman et al. 1986).

labeled a species of Mr 14 kDa and polydisperse species of Mr between 45 and 120 kDa (Fig. 6). The antibodies which bind to broken protoplast (MH 3.2D1, MH 3.5E7) labeled very weakly material undergoing electrophoresis in the stacking gel.

Discussion

In this paper we report the successful generation of monoclonal antibodies to plant plasma membranes using Nicotiana tabacum leaf protoplasts as the initial immunogen. The selection of hybridoma

M.G. Hahn et aI. : Monoclonal antibodies to protoplast membranes 463

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clones secreting the antibodies of interest was greatly facilitated by the development of protoplast and membrane ELISAs. Protocols used for equivalent animal cellular ELISAs (e.g. Cobbold and Walkmann 1981; Effros etal. 1985; Morris et al. 1982; Sutter et al. 1980) could not be used with the plant system, primarily because of the in- herent fragility of the protoplasts which led to bursting and loss of the protoplasts from the microtiter plates. A recently described "whole cell" assay for antibody binding to protoplasts proved not to be useful for screening large numbers of samples (Villanueva et al. 1986). The ELISA developed here overcomes these difficulties, and has

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Fig. 7A-C. Distribution of membrane antigens at equilibrium in a linear sucrose density gradient. Total cellular membranes were prepared from suspension-cultured 27. glutinosa cells. An- tigen distribution in the gradient was monitored by reactivity with monoclonal antibodies A PN 16.4B4, B MH 4.3E5, and C MH 3.5E7

been optimized with respect to immobilization of antigen, blocking of non-specific antibody binding, and incubation times.

An ELISA which utilizes plant protoplasts as the immobilized antigen has been briefly outlined in an abstract (Galbraith and Maddox 1983). This assay employed secondary and tertiary antibodies to detect monoclonal antibody binding to protoplast epitopes, took 2 d to complete, and apparent- ly did not exhibit any selectivity toward external plasma-membrane epitopes. In contrast, the present protocol requires only a secondary antibody to detect monoclonal antibody binding and can be completed within 5-6 h. When care is taken to keep the protoplasts intact throughout the assay, the ELISA protocol is selective for external epitopes on the plant plasma membrane as indicated by the weak signals with an antibody to a cell-


internal epitope (PN 17.3A6) and antibodies that bind to broken protoplasts (MH 3.2D 1, MH 3.5E7) (Tables 1, 2).

Preparation of protoplasts using digestive enzyme mixtures is likely to alter aspects of the surface architecture of the plasma membrane. For example, potential antigenic determinants present in vivo may be cleaved and new antigenic determinants uncovered during the digestion of the cell walls by the crude enzyme mixtures. The latter may explain why antibodies MH 4.1C1 and MH 4.1D1 gave strong signals in the protoplast ELISA, but only a weak response in the membrane ELISA (Ta- ble 2).

Some discrepancies between the protoplast ELISA and the data obtained from the indirect immunofluorescence assays were also noted (Ta- bles 1, 2). Thus, PN 16.4A6 and PN 17.4B5, which by immunofluorescence bind to the protoplast plasma membrane, do not test positive in the protoplast ELISA. Conversely, MH 4.1C1 and MH 4.1D1 test positive in the protoplast ELISA, but show no binding to protoplasts by indirect immunofluorescence. We cannot explain these discrepancies at present. Our experience clearly points out the need to use multiple and, preferably, or- thogonal screens (e.g. membrane and protoplast ELISA, immunofluorescence, Western blots) in scanning a library of hybridomas so as not to miss potentially interesting clones. Nonetheless, the ELISA provides a very useful, rapid assay to monitor secretion of antibodies to plant membrane epitopes during cloning of hybridoma cell lines as well as a rapid screen to initiate identification of those monoclonal antibodies to epitopes on the external face of the plasma membrane.

The four plasma-membrane-positive monoclonal antibodies identified in this study (MH 3.2B4, MH 4.2A4, MH 4.3E5, MH 4.4E4) differ in important aspects from antibodies to the plant plasma membrane that were characterized previously. The latter labeled protoplasts but not intact cells by indirect immunofluorescence (Nor- man et al. 1986), showed two peaks of reactivity in sucrose density gradients (Fig. 7A; Norman et al. 1986), and labeled a restricted series of bands on protein blots of electrophoresed solubilized membrane preparations (Mr 60 to 120 kDa). In contrast, the newly identified antibodies labeled both protoplasts and cells by indirect immunofluorescence (Fig. 5). Moreover, the corresponding membrane epitopes were broadly distributed when centrifuged to equilibrium in a sucrose gradient (Fig. 7 B), and were found on a polydisperse array of molecular species (Fig. 6). Taken together with

the periodate sensitivity of these epitopes (Table 3), the data indicate that these four antibodies recognize a glyco-epitope that is found not only on the external face of the plasma membrane, but also on a variety of internal cellular membranes as well as the cell wall. The broad, possibly ubiquitous, distribution of these epitopes on plant cellular membranes may reflect a central role for these epitopes in the functional insertion of membrane glycoproteins,

The monoclonal antibodies MH 3.2D1 and MH 3.5E7 constitute a novel class of antibodies since by indirect immunofluorescence, they appear to bind to the plasma membrane only of broken protoplasts (Fig. 5 C). However, the reactivity pat- tern of these antibodies to plant membranes equilibrated in a sucrose density gradient is markedly different from that seen for plasma-membrane- reactive antibodies (Fig. 7C), and hence the immunofluorescence data may reflect binding of these antibodies to epitopes that reside on cell-internal membranes which adhere to the internal face of the plasma membrane of broken protoplasts. The epitopes recognized by these antibodies are periodate sensitive (Table 3), but were not characterized further.

It is noteworthy that immunization with intact protoplasts did not result in a greater proportion or variety of hybridoma cell lines secreting antibodies to plasma-membrane epitopes compared to previous studies in which total cellular membrane preparations were used as immunogen. Immuniza- tion with intact protoplasts might be expected to enrich the immune response to epitopes present not only on the external face of the plasma membrane but also on internal cellular membranes since these epitopes would be exposed to immune surveillance both immediately following introduction of intact protoplasts and after subsequent protoplast lysis in the animal.

The antibodies described in this and a previous study that are reactive with the external face of the plasma membrane are directed toward glyco- epitopes. This may reflect extensive glycosylation of the external face of the plant plasma membrane such that these carbohydrate components effec- tively mask underlying polypeptide domains of cell-surface glycoproteins. Anderson et al. (1984) reported the apparent immunodominance of glyco- epitopes on arabinogalactan glycoproteins in style extracts. Competition-binding experiments using simple sugar and methylglycosides (data not shown) indicate that the four cell-surface-reactive antibodies identified in the Present study do not recognize the same epitopes as those antibodies


recovered by Anderson et al. (3979), though they may still be located on the same family of glycoproteins. Thus, it will be necessary to remove or suppress these immunodominant epitopes to en- hance the recovery of hybridomas secreting antibodies to epitopes located in polypeptide domains of plant plasma-membrane (glyco)proteins.

We thank Ross Allen for advice on ELISA techniques, Vincent P.M. Wingate for helpful discussions and supplies of hybridoma supernatant VW 40.2B2, and Carol L. Gubbins Hahn for preparation of figures. This work was supported by grants to CJL from the Seaver Institute and the U.S. Department of Agriculture, Competitive Grants F'rogram (83-CRCR-I- 1251).

References

Anderson, M.A., Sandrin, M.S., Clarke, A.E. (1984) A high proportion of hybridomas raised to a plant extract secrete antibody to arabinose or galactose. Plant Physiol. 75, 1013 1016

Bobbitt, J.M. (1956) Periodate oxidation of carbohydrates. Adv. Carbohydrate Chem. 11, 1~41

Bradford, M. (1976) A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Anal. Biochem. 72, 248-254

Cobbold, S.P., Waldmann, H. (1981) A rapid enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens. J. Immunol. Methods 44, 125 133

Effros, R.B., Zeller, E., Dillard, L., Walford, R.L. (1985) Detec- tion of antibodies to cell surface antigens by a simplified cellular ELISA (CELISA). Tissue Antigens 25, 204-211

Fitter, M.S., Norman, P.M., Hahn, M.G., Wingate, V.P.M., Lamb, C.J. (1987) Identification of somatic hybrids in plant protoplast fusions with monoclonal antibodies to plasma- membrane antigens. Planta 170, 49-5.4

Galbraith, D.W., Maddox, J.M. (1983) Production of monoclonal antibodies directed against deve, lopmentally regulated protoplast antigens. Experientia Suppl. 45, 234-235

Haberlach, G.T., Cohen, B.A., Reichert, N.A., Baer, M.A., Towill, L.E., Helgeson, J.P. (1985) llsolation, culture and regeneration of protoplasts from several Solanum species. Plant Sci. 39, 67 74

Hosking, C.S., Georgiou, G.M. (1982) Application of monoclonal antibodies to the study of human lymphocyte surface antigens. In: Monoclonal hybridoma antibodies: Tech-

niques and applications, pp 177-192, Hurrell, J.G.R., ed. CRC Press, Boca Raton, Fla., USA

K6hler, G., Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685

Larkin, P.J. (1977) Plant protoplast agglutination and membrane-bound fl-lectins. J. Cell Sci. 26, 31-46

Milstein, C. (1986) From antibody structure to immunological diversification of immune response. Science 231, 1261-1268

Morris, R.E., Thomas, P.T., Hong, R. (1982) Cellular enzyme- linked immunospecific assay (CELISA). I. A new micro- method that detects antibodies to cell-surface antigens. Hum. Immun. 5, 1-19

Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497

Norman, P.M., Wingate, V.P.M., Fitter, M.S., Lamb, C.J. (1986) Monoclonal antibodies to plant plasma-membrane antigens. Planta 167, 452-459

Quail, P.H. (1979) Plant cell fractionation. Annu. Rev. Plant Physiol. 30, 425~484

Sutter, L., Bruggen, J., Sorg, C. (1980) Use of an enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens. J. Immunol. Methods 39, 407411

Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350M354

Trowbridge, I.C. (1978) Interspecies spleen-myeloma hybrid producing monoclonal antibodies against mouse lymphocyte surface glycoprotein, T200. J. Exp. Med. 148, 313-323

Villanueva, M.A., Metcalf, T.N., III, Wang, J.L. (1986) Mono- clonal antibodies directed against protoplasts of soybean cells. Generation of hybridomas and characterization of a monoclonal antibody reactive with the cell surface. Planta 168, 503-511

Williams, A.F. (1980) Cell-surface antigens of lymphocytes: markers and molecules. Biochem. Soc. Symp. 45, 27-50

Woodward, M.P., Young, W.W., Jr., Bloodgood, R.A. (1985) Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J. Immunol. Meth- ods 78, 143-153

Received 24 April 1986; accepted 16 March 1987

characterization of monoclonal antibodies to protoplast membranes of nicotiana tabacum identified by...

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