Highly specific characterization of tyrosine phosphoproteins in tumor cells based on monoclonal antibodies defined by conjugated phosphotyramine.

Phosphotyrosine proteins of four different tumor cell lines were characterized by monoclonal antibodies exhibiting high affinity binding to phosphotyrosine. For the preparation of the antibody-producing mouse hybridoma cell lines we used a novel kind of immunizing antigen with phosphotyramine conjugated directly to carboxylic groups of carrier proteins. Screening for high affinity binding antibodies was based on their selective reactivity in immunoprecipitation, affinity chromatography and immunofluorescence. By means of affinity chromatography we established a one-step purification of phosphotyrosine proteins yielding substantial quantities of highly pure 170kDa EGF receptor from A431 tumor cells, 210kDa bcr-abl gene product from K562 tumor cells and 120 kDa transforming protein of the Abelson murine leukemia virus from TK tumor cells. Cross-reactivity with phosphoproteins containing no phosphotyrosine was not observed.