Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.
Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the 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. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.
Department of Chemical and Biomolecular Engineering, ‡Department of Chemistry and Biochemistry, §Advanced Diagnostics and Therapeutics, ∥Mike and Josie Harper Cancer Research Institute, and ⊥Center for Rare and Neglected Diseases, University of Notre Dame , Notre Dame, Indiana 46556, United States.
Nur
N
Mustafaoglu
Department of Chemical and Biomolecular Engineering, ‡Department of Chemistry and Biochemistry, §Advanced Diagnostics and Therapeutics, ∥Mike and Josie Harper Cancer Research Institute, and ⊥Center for Rare and Neglected Diseases, University of Notre Dame , Notre Dame, Indiana 46556, United States.
Nathan J
NJ
Alves
Department of Chemical and Biomolecular Engineering, ‡Department of Chemistry and Biochemistry, §Advanced Diagnostics and Therapeutics, ∥Mike and Josie Harper Cancer Research Institute, and ⊥Center for Rare and Neglected Diseases, University of Notre Dame , Notre Dame, Indiana 46556, United States.
Basar
B
Bilgicer
D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology*; D000941:Antigens / Q000032:analysis* / Q000276:immunology*; D001665:Binding Sites; D019921:Biotinylation; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology*; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry / Q000276:immunology*
D055910:Antibodies, Immobilized; D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D009711:Nucleotides