Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 14, Num. 1, 1997, pp. 47-48

APPLICATIONS OF MONOCLONAL ANTIBODIES TO GANGLIOSIDES IN NEUROSCIENCE

Tadashi Tai

Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku,

Tokyo 113, Japan. Phone, 3-3823-2101;
FAX, 3-3823-2965. E. mail: tai@rinshoken.or.jp

Code Number:BA97011
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Abstract

We established an improved method for the generation of mouse monoclonal antibodies (MAbs) to gangliosides by immunizing gangliosides. These MAbs enabled us to examine the distribution of ganglioside in the brain. Immunohisto- and immunocyto-chemical studies suggested that there is a cell type-specific expression of gangliosides in the central nervous system.

Introduction

Gangliosides, sialic acid-containing glycosphingolipids, are normal membrane constituents and are highly expressed in the vertebrate central nervous system (1). Owing to their topological localization on the outer surface of neural plasma membranes and their unique chemical structure, gangliosides have been implicated in a variety of phenomena involving cell- cell recognition, neurite outgrowth, synaptogenesis, transmembrane signaling, and cell growth and differentiation (2-5). An understanding of the cellular localization of gangliosides in the brain could provide insight into the possible function of these molecules. In the past decade, cholera and tetanus toxins, and several polyclonal and monoclonal antibodies (MAbs) reacting with gangliosides have been used as probes for detecting gangliosides in neurons and glia (6, 7). It was, however, difficult to generate MAbs specific for individual gangliosides. We recently established an improved method for the generation of mouse MAbs to gangliosides by immunizing mice with purified gangliosides (8-15). These MAbs enabled us to examine the distribution of ganglioside in the central nervous system. These studies revealed the differential distribution patterns of gangliosides in the brain regions (16-19).

Materials and Methods

MAbs to gangliosides

The production and characterization of MAbs has been described previously (9-15). Briefly, all of the MAbs were generated by immunizing C3H/HeN mice with purified gangliosides adsorbed to Salmonella minnesota mutant R595. The binding specificity of these MAbs was determined by an enzyme- linked immunosorbent assay and an immunostaining on thin-layer chromatogram. Most of these MAbs show highly restricted binding specificity, reacting only with the immunizing ganglioside. None of other various authentic gangliosides or neutral glycolipids were recognized. Although most of the MAbs were of IgM, some MAbs belonged to IgG.

Immunohistochemistry

The expression of gangliosides in frozen sections of rat brain was determined by the indirect immunofluorescence technique with specific MAbs as previously described (16).

Immunocytochemistry

Cells were fixed with paraformaldehyde and stained in an immunofluorescence procedure as previously described (18).

Conclusions

Immunohistochemical studies of gangliosides in the rat brain

At first, we attempted to investigate the localization of major gangliosides in the adult rat brain by an immunofluorescence technique with mouse MAbs. Five MAbs that specifically recognize gangliosides GM1, GD1a, GD1b, GT1b and GQ1b, respectively, were used. We have found that there is a cell type-specific expression of the gangliosides in the rat central nervous system (16). As a next step, we studied the distribution of minor gangliosides in the adult rat brain by an immunofluorescence technique with mouse MAbs. Ten MAbs that specifically recognize GM3, GM2, GT1a, GD3, O-Ac- disialoganglioside, GD2, GM1b, GM4, IV^3NeuAca-nLc4Cer, and IV^6NeuAca- nLc4Cer, respectively, were used. Our study revealed that there is a cell type-specific expression of minor gangliosides as well as major gangliosides in the rat brain. (17) Subsequently, we studied the distribution of gangliosides during the development of postnatal rat cerebellum by an immunofluorescence technique with mouse MAbs. Eleven MAbs that specifically recognize each ganglioside changed dramatically during the development (19).

Immunocytochemical study of gangliosides in primary cultured neuronal cells

Then, we studied the expression of ganglioside antigens in primary cultures of rat cerebellum using an immunocytochemical technique with mouse MAbs specific for various gangliosides. Twelve MAbs that specifically recognize each ganglioside were used. Our study revealed that there is a cell type- specific expression of ganglioside antigens in the primary cultures (19). Some caution must be used in interpreting the expression of ganglioside antigens based on immunocytochemistry, since a lack of immunorecognition of ganglioside epitope on cells does not necessarily mean that a ganglioside is absent. There are indications that a number of factors are involved in influencing the reactivity of MAbs with specific cells: (i) the density of ganglioside on cells is involved in the reactivity of antibodies, (ii) other components of the cell surface may influence antibody reactivity; and (iii) the ceramide portion of gangliosides may be involved in the reactivity (20-22). Further study will be needed for elucidating the precise mechanisms of immunoreactivity, particularly in normal cells, since previous reports were based mainly on the studies of cancer cells. An immunoelectron microscopy study will be necessary to further evaluate the localization of the gangliosides in cells in the rat brain.

Acknowledgments

The author wishes to thank Drs. I. Kawashima, H. Ozawa, M. Kotani, and K. Ogura (Tokyo Metropolitan Institute of Medical Science) for their collaborations. He also thanks Drs. T. Terashima and Y. Nagata (Tokyo Metropolitan Institute of Neuroscience) for their valuable suggestions.

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Copyright 1997 Elfos Scientiae