Isolation of the intact molecule and ectodomain of C-erbB-2 oncoprotein from SK-BR-3 cells and development of immunoassays on microplate.

We isolated both the intact molecule (p185) and the ectodomain (p120) of c-erbB-2 oncoprotein from SK-BR-3 breast tumor cells. The p120 was extracted from the cells by 0.05 M phosphate buffer, pH 7.2, whereas the extraction of the p185 required the presence of a detergent, such as 1% Triton X-100 in 0.05 M Tris buffer. Protease inhibitors were also included in the extraction buffer during the isolation of p185 in order to prevent cleavage of p185 to p120 by an unknown protease apparently also present in the extract. In case there was any p120 in the p185 preparation, the p120 could be separated from p185 by chromatography on a Superose 12 column. Using the p120 and p185 as calibrators, we have established two microplate sandwich immunoassays: one measures both p185 and p120 (total assay) and the other is specific for the p185. Since capturing and detecting antibodies used in the total assay react against the extracellular domain of the c-erbB-2 oncoprotein, they can therefore be used to measure the p120 in serum and p185 in breast tumor tissue cytosol. On the other hand, the p185 specific assay uses the capturing antibody against the cytosolic domain of the oncoprotein and consequently can only measure p185 in breast tumor tissue cytosol.