Proc. Nati. Acad. Sci. USA Vol. 77, No. 8, pp. 4741-4745, August 1980 Cell Biology Microtubule-associated proteins: A monoclonal antibody to MAP2 binds to differentiated neurons (microtubule assembly/tubulin/high molecular weight component/lymphocyte hybridoma/neuronal development) JONATHAN G. IZANT AND J. RICHARD MCINTOSH Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 Communicated by William B. Wood, April 28, 1980 ABSTRACT Hybridomas that secret IgG reacting specifi- the lower molecular weight HMW protein, MAP2 (13), is found cally with the brain microtubule-associated protein MAP2 have only in differentiated neuronal cells in a wide variety of been prepared with spleen cells from BALB/c mice hyperim- munized with high molecular weight neurotubule-associated mammals. Our monoclonal antibody to MAP2 does not stain proteins. Immunofluorescence microscopy using dual fluoro- the mitotic apparatus or cytoplasmic microtubules in non- chrome labeling of tubulin and MAP2 antigens revealed iden- neuronal tissue or cell lines. Examination of neuroblastoma cell tical patterns of interphase fiber networks in cells from explants lines which differentiate in culture suggests that the appearance of newborn mouse brain. The anti-MAP2 antibody did not stain of MAP2 may be associated with neuronal differentiation both primary mouse kidney cells or CHO, 3T3, HeLa, or PtKI cell lines. Immunoprecipitation and antibody gel staining tech- in vitro and in vivo. niques failed to demonstrate any crossreacting antigen in these cells. MAP2 antigen was not seen in association with the mitotic MATERIALS AND METHODS spindle in any of the cells examined. Radioimmunoassay Antigen Preparation. Microtubule protein (MTP) was pre- showed species crossreactivity of the anti-MAP2 antibody wi mammalian but not avian neural cell extracts. Glial cells and pared from the brain of freshly slaughtered hogs by two cycles some neuroblastoma cell lines did not appear to contain MAP2. of assembly/disassembly (3) and stored as pellets at -700C. However, in the B104 rat neuroblastoma cell line the MAP2 MAP2 was prepared by the method of Kim et al. (13). HMW antigen appeared to be associated with the cytoskeleton con- antigen was prepared by separation of MAPS from MTP on a comitant with differentiation induced by dibutyryl cyclic AMP. phosphocellulose column (3) and electrophoresis of the MAPs In disagreement with most previously published reports, our on NaDodSO4/polyacrylamide slab gels (2.5 mm thick and 100 data suggest that MAP2 is found only in differentiated neuronal mm 6% cells and raises the possibility that MAP2 is involved in neuronal wide, acrylamide) (14). The bands containing HMW differentiation or neuron-specific processes. were excised and the protein was extracted by electroelution and acetone precipitation. Protein assays were done by the Two classes of microtubule-associated proteins (MAPs) copurify method of Bradford (15). with brain tubulin through cycles of assembly/disassembly: tau Hybridoma Fusion. Female BALB/c mice were injected factor, 55,000-62,000 daltons (1, 2); and the high molecular intraperitoneally with 100 .g of HMW in Freund's complete weight protein (HMW), 250,000-350,000 daltons (3, 4). These adjuvant (Difco) and boosted intravenously 21 and 24 days later MAPs increase rates of both nucleation and elongation during with 100 ,gg of HMW in Earle's phosphate-buffered saline tubulin polymerization in vitro (2, 4), and it has been suggested without calcium (Pi/NaCI). On day 27, two mice were sacri- that they play a role in the control of the in vivo utilization of ficed and their spleens were excised; 1.65 X 108 spleen cells microtubules during interphase and cell division. Consistent were fused to 1.65 X 107 logarithmic phase SP2/0 mouse with this hypothesis, antisera prepared against MAPs stain in- myeloma cells in 30% (wt/vol) polyethylene glycol 1000 by terphase microtubule networks and mitotic spindles in a wide using modifications of the methods of Gefter et al. (16) and variety of cells (5-9). Recently, however, carefully character- Claflin (17). Fourteen days after fusion, samples of the cell ized antisera to high molecular weight assembly MAPs from supernatants were screened for the production of specific brain and cultured cells have been shown to have limited antibody, and the cells from positive wells were cloned twice crossreactivity between different cell types (ref. 10; unpublished in RPMI-1640 containing 10% heat-inactivated fetal calf serum, results) and species (11). 8% heat-inactivated horse serum (GIBCO), and 0.5% agarose The reasons for the discrepancy between the different anti- (Seaplaque, Marine Colloids). Antibody-producing hybrids sera are not easily understood. Serum contains many different were grown in RPMI-1640 supplemented with 5% NCTC 109 immunoglobulins, and it is possible that one subset of immu- medium and 10% heat-inactivated fetal calf serum (R/N.10) noglobulin is giving rise to immunofluorescent staining patterns in roller bottles or as ascites tumors in Pristane-primed BALB/c whereas another is responsible for Ouchterlony immunopre- mice. Antibody produced in tissue culture was concentrated cipitation lines. This complexity is not remedied by affinity 50-fold before storage. Rabbit serum raised against tubulin was chromatography purification of the antiserum because, during the generous gift of J. Olmsted. elution, partial denaturation of antibody may occur, possibly Solid-Phase Assay. Four-day cell supernatants were assayed modifying the specificity of the immunoglobulins. in 60-well Terasaki plates (Falcon 3034). A 0.2- to 2-,ag sample To alleviate some of the problems inherent in the use of an- of antigen was dried in each well or, alternatively, the plates imal sera, we have been studying the in vvo function of HMW were pretreated with 1.0% 1-ethyl-3(dimethylaminopropyl)- by using monoclonal antibody probes produced against MAPs carbodiimide (Sigma) and thoroughly rinsed with Pi/NaCl according to the lymphocyte hybridoma method originated by before the addition of antigen. The plates were washed twice Kohler and Milstein (12). In this report we present evidence that with Pi/NaCl and once for 10 min with Pi/NaCl containing ovalbumin (1 mg/ml). The pretreated plates were washed for The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: MAP, microtubule-associated protein; HMW, high vertisement" in accordance with 18 U. S. C. §1734 solely to indicate molecular weight protein; MTP, microtubule protein; Pi/NaC1, Earle's this fact. phosphate buffered saline without calcium. Downloaded by guest on September 27, 2021 4741 4742 Cell Biology: Izant and McIntosh Proc. Natl. Acad. Sci. USA 77 (1980) 10 min with 0.1 M Tris (pH 7.4) containing ovalbumin (1 glycerol in Pi/NaCl. For dual fluorochrome labeling, the order mg/ml). After two more rinses with Pi/NaCl, 15 A1 of super- of incubation was rabbit anti-tubulin, fluorescein-conjugated natant to be tested was added to each well and the plates were goat anti-rabbit IgG (Miles), monoclonal anti-MAP2, and incubated at 370C for 1 hr. After two rinses with Pi/NaCl, one rhodamine B-conjugated rabbit anti-mouse IgG (Miles). lO-min wash with Pi/NaCi plus ovalbumin, and two more rinses with Pi/NaCl, all at 0-40C, approximately 50,000 cpm of RESULTS 125I-labeled Staphylococcus protein A (Pharmacia) labeled to Fourteen days after the fusion of the myeloma and spleen cells, a specific activity of 20 mCi/mg (1 Ci = 3.7 X 1010 becquerels) one to eight colonies of hybrid cells were growing per well. by the chloramine-T method (18) was added to each well. After Solid-phase immunoassays revealed nine wells containing IgG a 15-min incubation at room temperature the plates were which bound to electroeluted HMW antigen, phosphocellu- washed as before with Pi/NaCl at 0-40C, washed with cold lose-purified MAPs, or MTP. Cells from two wells were suc- distilled water, dried, and autoradiographed with X-Omat RP cessfully cloned. Two of the clones, D-1D1.16.6 and D- film (Kodak) and a Cronex intensifying screen (DuPont) at 1D1.43.2, were used in subsequent experiments. -700C for 6-18 hr. The specificity of the antibody was first examined by anti- Antibody Specificity. The specificity of the monoclonal body staining of NaDodSO4 gel slices. Fig. la shows the Coo- antibody preparations was determined by staining of NaDod- massie blue staining pattern of a gel slice containing MTP and SO4 gels and radioimmunoassay. Antigens were detected in homogenates of four cultured cell lines. Fig. 1 b and c shows slices of 7.5% NaDodSO4/polyacrylamide gels fixed in 15% the autoradiographs of identical gel slices incubated with a trichloroacetic acid and methanol/acetic acid by the method control monoclonal antibody (B-3A3.2, an IgG-secreting hybrid of Burridge (19). lodinated Staphylococcus protein A was used from a fusion using unimmunized mice) and with D-1D1.16.6 to probe for bound IgG. Tritiated monoclonal antibody was antibody, respectively. The nonspecific binding of the 125I- obtained by growing the IgG-secreting hybridoma cells in labeled Staphylkccs protein A was low, and the D-1D1.16.6 medium containing [3H]leucine (New England Nuclear) at 0.5 antibody bound to a single band, corresponding to the lower ,uCi/ml. For radioimmune determination of the presence of molecular weight HMW band, MAP2. This band reportedly antigen, tissue and cell samples were homogenized or sonicated runs as a doublet (4) but we could resolve only one evenly in 4 vol of 50 mM piperazine-NN'-bis(2-ethanesulfonic acid), staining band, suggesting there may be some antigenic rela- pH 6.95/1.0 mM MgCl2/1.0 mM ethylene glycol bis(,B-ami- tionship between the two MAP2 bands, but MAPI and MAP2 noethyl ether) N,N,N',N'-tetraacetic acid (EGTA)/0.1% have at least some antigenically distinct domains.