title: Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A.
abstract: Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (K(d)) in the range between 0.5 and 3 microm. One variant (Z(IgA1)) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA(1) and IgA(2)) as well as secretory IgA were recognized with similar efficiencies. No detectable cross-reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the Z(IgA1) affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head-to-tail Z(IgA1) affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.
authors.affiliation: Department of Biotechnology, Royal Institute of Technology (SCFAB), Stockholm, Sweden.
authors.forename: Jenny
authors.initials: J
authors.lastname: Rönnmark
authors.forename: Hans
authors.initials: H
authors.lastname: Grönlund
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Per-Ake
authors.initials: PA
authors.lastname: Nygren
mesh_terms: D000911:Antibodies, Monoclonal; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism*; D008024:Ligands; D015202:Protein Engineering; D013205:Staphylococcal Protein A / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D008024:Ligands; D013205:Staphylococcal Protein A