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The generation of monoclonal antibodies to neural cell type-specific antigenic determinants

University of British Columbia

Somatic cell hybridization techniques were used to produce a panel of anti-neural cell monoclonal antibody-secreting murine hybridomas. Whole living cultures of bovine oligodendrocytes were used to inject (intra-splenically and without adjuvants) Balb/C mice with a total of 2.0 to 2.5 x 10⁶ oligodendrocytes. Polyethylene glycol was subsequently used to fuse these sensitized splenic lymphocytes to the murine NS-1 myeloma cell line. After fusion, supernatants were screened immunocytochemically using murine neural cell cultures. Initial screening identified a total of 17 clones, each of which primarily labelled a surface antigen specific to galactocerebroside-positive oligodendrocytes; as demonstrated by double-immunostaining characterization. The monoclonal antibodies were determined to be of either the IgM class or IgG[sub 2b] subclass. Moreover, these antibodies recognized protein bands possessing an apparent molecular weight of 29,000 and/or 59,000 Da on Western blots of homogenized bovine oligodendrocytes. This may, therefore, represent immunolabelling of a previously unrecognized surface constituent of oligodendrocytes. Culture supernatants from a second separate fusion experiment involving an intrasplenic injection with 20 μg of SDS-PAGE subfractionated human newborn brain particulate fraction were tested using first, an ELISA, and subsequently, indirect immunofluorescence microscopy. Screening demonstrated 2 clones which produced antibodies immunoreactive to an astrocyte subclass (glial fibrillary acidic protein-positive cells) found only in cultures of newborn murine brain. These antibodies were determined to be of an IgM class and were shown to specifically reactivity with electroblots of a protein constituent of whole newborn brain particulate fraction having an apparent molecular weight of 50,000 Da.

Text

2010-09-10

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http://hdl.handle.net/2429/28399

Pathology

University of British Columbia

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