(335c) Oriented Fab Fragment Immobilization By Site-Specific Biotinylation at the Nucleotide Binding Site for Enhanced Ebola Detection

Oriented immobilization of antibodies and antibody fragments has become increasingly important because of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. Fab fragment, with its small size and simple production methods compared to intact antibody, are good candidates for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. The nucleotide-binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that binds specifically to this site. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS^Biotin) in which a Fab fragment is site-specifically biotinylated via UV energy exposure (1 J/cm²) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS^Biotin method yielding 1031 fold and 2 fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS^Biotin method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.