 Hello, I'm Bashar Bilgeiser from the Department of Chemical and Biomolecular Engineering at the University of Notre Dame. Hello, I'm Nur Mustafa Olu, grad student in bioengineering program at the University of Notre Dame. Welcome to our bio-technology and bioengineering video highlights. We would like to introduce our recent article and titled site-specific pet fragment biochallination at the consort nucleotide binding site for enhanced Ebola detection. Antibodies typically have extraordinary specificity and affinity for their antigens, which makes them very useful in a vast area of applications including detection, diagnosis, catalysis and therapy. Each antibody typically has two antigen binding fragments, also called FAB fragments, where antigen binding and the recognition takes place. The FAB fragments can be separated from the rest of the antibody without losing their antigen binding ability. Due to their smaller size and comparably easier production methods, they promise to be better candidates for use in miniaturized diagnostic devices as well as therapeutic applications. Unfortunately, commonly used modification techniques are not well suited for FAB fragment modification as they are often more delicate than intact antibodies. FAB fragments are of particular interest for sensor surface functionalization. However, because of their truncated size, they provide a smaller surface that can be used for attaching to surfaces without damaging the antigen binding site structure. Consecutively, typical conjugation methods end up impeding FAB's antigen binding activity. Here, we describe a UV photofrosting infactionalization method that we have developed in our labs. Using this method, we cite specifically biotin-related FAB fragments with an IBA biotin linker at the conserved nucleotide binding site without unfavorably impacting its antigen binding activity. In this study, we used the KZ52 antibody that recognizes Ebola GP proteins. We functionalize the KZ52 FAB fragments with an IBA biotin linker we synthesized. They were then immobilized on the lysoplate surfaces to be used in the detection of Ebola GP proteins. Nucleotide binding site located between the heavy and light chains of the antibody within the variable region of the FAB arms is a highly conserved region present in near lowland bodies. While this binding site is not widely known, it has been shown that indole-tributric acid has a moderate binding affinity to the nucleotide binding site. The site-specific binding of IBA to the antibody nucleotide binding site can be used for conjugating various peptide linkers and functionalities that contain a terminal IBA molecule to antibodies. Covalent conjugation of IBA to the antibody at the nucleotide binding site can be accomplished without reduction in antigenbionic activity by utilizing UV energy in a photocrossing reaction known as the UV and BS conjugation technique. The structure stability of FAB fragments upon UV exposure was determined with a direct ELISA assay. Antigenbionic activity of biotinulated FAB fragments was determined with an indirect ELISA assay. According to these results, there is no observable reduction in antigenbionic activity of the FAB and detectable damage to the FAB structure up to UV energies of one joule per centimeter square. The biotinulated FAB was analyzed using ELISA in two separate experiments to examine biotinulation efficiency and evaluate antigenbionic activity. The results of direct ELISA established that the photocross-linking efficiency of IBA biotin to the FAB fragment reached a maximum FAB biotinulation at one joule per centimeter square UV exposure, and the pleta was observed above this energy, suggesting that conjugation site had been saturated. The indirect ELISA result validated that almost 100% photocross-linking efficiency was reached at an UV energy of one joule per centimeter square and there was no observable reduction in antigenbionic activity up to this level of UV energy. The FAB fragment was biotinulated using increasing levels of UV energy and drawn on STS page show. The STS page show demonstrates that the light chain band of the intake KZ53 antibody matches with the light chain band from the FAB fragment at 25 kD. To show the specific location of the biotinulation on the FAB fragment, a worst-in-blood assay was performed by transferring protein to another solar's membrane and detected the biotinulated FAB fragment with HRP strapped gravity. The results of the blotted film indicate that the biotinulation of FAB fragments at the nucleotide binding site is occurring specifically at the light chain. We compared the UBNBS method with two other commonly used immobilization methods, NHS biotin and physical absorption. KZ52 FAB fragment was immobilized to 96 well-placed using all three methods. Antigen detection sensitivity was determined using increasing concentrations of ZepoGP DTM as the antigen and then an anti-ZepoGP detection antibody was added and quantified by an HRP conjugated anti-FZ antibody as a reporter. This study demonstrates successful immobilization of biotinulated Ebola-detectant FAB fragments via the UBNBS method yielding a 1031-fold bad-rentigen detection sensitivity compared to direct physical absorption and two-fold bad-rentigen detection sensitivity compared to NHS biotin functionalization methods. Consequently, the utilization of UBNBS method for the immobilization of FAB fragments in order to design antigen-recognition systems instead of incansable, provides further advantage to miniaturized and nanoscale devices. Thank you for watching this video. We hope to enjoy reading our article.