a. Overview. Monoclonal antibodies (mAbs) are antibodies of a single specificity derived from an immortalized B cell [251]. Human monoclonal antibodies can be made by applying traditional hybridoma technology to mice transgenic for the human immunoglobulin loci [252], by phage display [253], in which antibody fragments, such as single chain Fvs (scFv) or Fabs, are displayed on the surface of phage [254,255], or by yeast display [256- 258]. Naive phage antibody libraries, from which antibodies can be selected against most targets, are derived from natural unimmunized [255,259-262] or synthetic [263-266] rearranged (usually human) V genes, and are the commonest in vitro source used to derive antibody fragments. The use of phage display to select antibodies has two great advantages: first the ability to select antibodies with specific performance or recognition characteristics, and secondly, the fact that the gene for the antibody is cloned simultaneously with selection. This allows antibody fragment genes to be subjected to downstream engineering, including affinity maturation to affinities not attainable in natural immune systems [256,267-270], and reformatting to include effector functions for downstream applications [271-275], such as enzymatic activity (e.g. alkaline phosphatase) [272,275], immobilization tags for orientated coupling to surfaces [276,277], tandem affinity purification tags [278] for immunoprecipitation, dimerization domains [279], fluorescent labeling vectors [24,271,274] and recloning into the full length immunoglobulin format [38,280,281]. ScFvs have great advantages over conventional mAbs for STMC research, including their small size, useful for studies on the composition of signaling complexes at the membrane (Aims 1 and 2);their exquisite specificity, as needed to develop Abs that recognize specific phosphotyrosine sites on signaling proteins (Aim 1);and their monovalency, which is critical for the analysis of membrane dynamics (Aim 2). Technology for the generation of scFvs is limited to only a few labs woridwide. Thus the STMC Affinity Reagents Core, located in the Bradbury lab at LANL, is an enormous asset to the center.

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