Generation and characterization of the murine monoclonal antibody M-KID 2 to VLA-3 integrin

Through the analysis of the antigenic phenotype of a recently established human renal carcinoma cell line (KJ29), we have demonstrated that alpha 3 subunit of the integrin family is selectively expressed by the plastic adherent cell subpopulation. Because of the scanty availability of monoclonal antibodies to this adhesion molecule, we have used KJ29 cell line as immunogen to raise novel murine monoclonal antibodies. We isolated an hybridoma secreting the mAb M-KID 2 of the IGg1k isotype that immunoprecipitates from intrinsically [35S]-Methionine labeled KJ29 cells, an heterodimer of 130/130 and 110/150 Kd, in reducing and nonreducing conditions respectively. This reactivity was completely abolished by immunodepletion of the cell extract with a polyclonal anti alpha 3 chain antiserum. Treatment of M-KID 2 immunoprecipitates with various solutions of pH ranging from 2 to 10.5, to dissociate alpha 3 from beta 1 chains, showed a retention of both alpha 3 beta 1 chains thus indicating that the epitope identified by mAb M-Kid 2 is likely to be constituted by the alpha 3 beta 1 heterodimer. Furthermore immunohistochemical studies on selected frozen and paraffin embedded tissues with mAb M-Kid 2 have provided staining pattern indicating the recognition of Vla-3. These findings demonstrate that mAb M-KID 2 can represent a valuable reagent for the study of Vla-3 integrin in normal and pathologic conditions.