[Preparation of monoclonal antibodies to phosphotyrosine and their use for identification of phosphotyrosine-containing proteins]

Abstract

Protein kinases phosphorylating proteins at tyrosine residues play an essential role in the cell growth regulation and neoplastic transformation. However, the functions of the majority of tyrosine protein kinases are still obscure, thus creating hindrances in the identification and isolation of phosphotyrosine-containing proteins. The use of the phosphotyrosine structural analog, aminobenzyl phosphonate, as a hapten group enabled the preparation of monoclonal antibodies capable of reacting to phosphotyrosine. The phosphotyrosine specificity of six clones of monoclonal antibodies was tested by a competitive solid phase immunoenzymatic assay. Using fluorescence quenching, the values of constants of binding for antibodies of four clones to phosphotyrosine (2.5-4.0 x 10(6) M-1) were determined. Using two independent methods, it was shown that clone B4 antibodies reveal the highest specificity towards phosphotyrosine. An immunoadsorbent based on clone B4 antibodies was obtained; this immunoadsorbent possessed an ability to selectively interact with an EFR receptor phosphorylated at tyrosine residue. Using eluate acid hydrolysis from the immunoadsorbent, it was demonstrated that clone B4 antibodies interact only with the phosphotyrosine-containing proteins. The experimental results are suggestive of clone B4 monoclonal antibody specificity to phosphotyrosine and of the feasibility of their application for the isolation and identification of tyrosine protein kinases and their substrates.