Construction and Functional Validation of a High-Diversity Naive Phage Antibody Library

Phage display technology has emerged as an indispensable and highly successful platform for the in vitro generation of monoclonal antibodies (mAbs) against a vast spectrum of targets, ranging from disease biomarkers and native biologically important proteins to small molecules. The critical determinant for constructing a high-quality library capable of yielding high-affinity antibodies is the diversity of the source antibody genes. Naïve antibody libraries, the most common type, are typically derived from the IgM repertoire of healthy human donors, using B-cells isolated from peripheral blood mononuclear cells (PBMCs). The preferred format for these displayed antibodies is the single-chain fragment variable (scFv) due to its smaller size and functional suitability for the phage display system. This chapter details the technical workflow for harnessing this power, which involves a robust two-step cloning method for constructing the high-diversity library, followed by the iterative process of biopanning for the efficient selection and isolation of target-specific, high-affinity clones.