CHARACTERIZATION OF MONOCLONAL-ANTIBODIES TO THE ESTROGEN-REGULATED MR52,000 GLYCOPROTEIN AND THEIR USE IN MCF7 CELLS

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  • 1 January 1985
    • journal article
    • research article
    • Vol. 45  (2) , 709-716
Abstract
The MW 52,000 glycoprotein is regulated by estrogen and released by breast cancer cells in culture. This rare protein was partially purified from 25 l of medium conditioned by MCF7 cells and injected into Biozzi''s selected mice. The spleen lymphocytes of 1 immunized mouse was fused with the murine myeloma P3-X63-Ag8-653. Sixteen hybridomas producing monoclonal antibodies to the MW 52,000 protein were isolated, and 7 were cloned and purified. The 7 monoclonal antibodies were all of the IgG/1 isotype, and their Kd ranged from 0.35 to 2.3 nM. The antibodies specifically recognized the secreted MW 52,000 protein as evidenced by double immunoprecipitation and by immunoblotting after electrophoretic separation and transfer. Double-determinant immunoradiometric assay indicated that the 7 purified monoclonal antibodies recognized 3 distinct regions of MW 52,000 protein, and it was used to assay the MW 52,000 protein in biological fluids. These antibodies did not react with the external plasma membrane of MCF7 cells, as shown by immunofluorescence analysis. By contrast, the cytoplasm of MCF7 cells (but not T47D and RBA cells) was stained by the peroxidase-immunoperoxidase complex after plasma membrane premeation, indicating that the protein is secreted by exocytosis rather than shed from the plasma membrane.