Monoclonal antibodies reactive with components in serum-free conditioned medium from a human breast cancer cell line...

Approximately 10,000 primary hybridomas were generated after immunization with either serum-free conditioned medium (SFCM) or extracts from the human breast cancer cell line MCF-7. A total of 11 different monoclonal antibodies (MAbs; 8 generated against SFCM and 3 generated against cell extracts) were selected on the basis of high specificity in cell-binding ELISA for human breast cancer cell lines. The 8 different MAbs obtained by immunization with SFCM all reacted with secreted components in SFCM from MCF-7 cells and 4 of these MAbs reacted with glycolipids extracted from MCF-7 cells. 1 of these MAbs (S2) also recognized a secreted glycoprotein of approximately 77 kilodaltons (kDa). The remaining 4 MAbs did not show specificity solely for carbohydrate determinants. 1 of these MAbs (S7) recognized a secreted protein of approximately 41 kDa. The 3 MAbs raised against cell extracts from breast cancer cells reacted with cytoplasmic antigens in immunofluorescence but also reacted with a secreted component in SFCM from MCF-7 cells. Immunoblotting experiments with proteins from cell extracts and with proteins in SFCM showed that these antibodies all reacted with a protein of a molecular weight of approximately 40 kDa. Our results suggest that this component is cytokeratin 19 or proteolytically processed cytokeratin 19.