A general approach to the generation of monoclonal antibodies against members of the tetraspanin superfamily using recombinant GST fusion proteins

Abstract

Tetraspanins belong to a rapidly growing family of proteins characterized by the presence of four conserved transmembrane segments and are involved in such diverse functions as cellular activation, adhesion, migration and differentiation. In an effort to develop reagents against newly discovered tetraspanins, we have devised a simple method for the screening of monoclonal antibodies (mAbs) using recombinant GST fusion proteins. GST fusion proteins containing the second extracellular domain of different tetraspanins (CD9, CD63, CD53, CD81, A15 or CO-029) were produced separately. Mice were immunized with cells having a high expression of the chosen tetraspanin and the constructs were used to screen hybridomas in a solid phase ELISA. Several clones binding the fusion protein were identified for each construct tested: four anti-CD9 hybridoma clones, four anti-CD63, two anti-CD53, two anti-CD81, three anti-A15 and one anti-CO-029. All the newly developed mAbs recognized the native proteins by flow cytometry, immunofluorescence staining of cells and immunoprecipitation and bound to the denatured proteins on immunoblotting. Use of GST fusion protein constructs in a simple ELISA can facilitate screening for mAbs to members of the tetraspanin family, especially in cases where information is limited to the nucleotide sequence. The mAbs obtained by this strategy should prove to be valuable tools for functional studies of newly discovered tetraspanins.