Tools of the trade: developing antibody-based detection capabilities for recombinant proteins.
ABSTRACT Protein-specific antibodies serve as critical tools for detection, quantification, and characterization of recombinant proteins. Perhaps the most important and widely used antibody-based procedures for recombinant protein applications are Western immunoblotting and enzyme-linked immunosorbent assays (ELISAs). These analyses require well-characterized, sensitive, and high-affinity antibodies that specifically and selectively recognize the recombinant target protein in the native or denatured form. Although the number of commercially available antibodies is quite substantial and rapidly growing, the appropriate antibody tools for many applications currently do not exist. In this chapter, strategies to develop and characterize both polyclonal and monoclonal antibodies directed against a specific protein of interest are discussed. Experimental strategies and methods are presented for producing and selecting the best antibodies and optimizing protocols for Western analyses, ELISAs, and other applications. Once antibody and procedure optimization is completed to ensure specificity, sensitivity, accuracy, and reliability, these immune-based approaches can now serve as powerful and enabling tools in the characterization, detection and diagnostics, structure/function analysis, and quality assessment of recombinant proteins.