| The proper orientation of primary ('coating') antibodies on solid substrates is an area of increasing interest, particularly in immunoassays, microarray and biosensor technologies. The detection of test articles requires optimal attachment of primary antibodies in which the Fc moiety of the molecule is adsorbed to the substrate, leaving the binding sites on the FAb moiety freely accessible.; The choice of substrate material (generally a polymer, most commonly, polystyrene) can play a crucial role in the successful alignment of the primary antibody in these assays. Most of the focus lies in increasing the overall non specific protein-binding capacity of the substrate, generally by increasing the hydrophobicity of the substrate surface. While this may increase overall binding, it does not automatically ensure retention of biological activity of the antibody. Several strategies for directed adsorption of antibodies to solid substrate have been investigated. Typically these strategies involve chemical modification of the Fc moiety in concert with altered substrate surface chemistry.; Rather than chemical conjugates in combination with specifically treated substrates, protein linkers can be used to achieve directed adsorption. Several bacterial proteins are known to bind to the Fc region of the antibody, one, Protein G, is used extensively in antibody purification. A recombinant form of Protein G, Protein G' encompasses only the Fc binding domains of the native protein, thus making it an ideal protein linker for directed antibody adsorption. In the process of examining directed adsorption of antibodies by Protein G' bound to a series of different polymeric substrates it was observed that Protein G' bound to poly(methyl methacrylate) resulted in a high concentration of properly aligned primary antibodies. Through a series of experiments it was determined that a substrate consisting of a Protein G'-poly(methyl methacrylate) complex consistently yielded a higher percentage of correctly-oriented primary antibody than that of a polystyrene control, suggesting that poly(methyl methacrylate) might interact with Protein G' in such a way as to increase the capacity of Protein G' to capture and align the primary antibody. |
