UV Nucleotide Binding Site Photocrosslinking of Antibodies at Various Light Intensities

Undergraduate #117
Discipline: Biological Sciences
Subcategory: Microbiology/Immunology/Virology

Celia Ochoa - Indiana University-Purdue University Indianapolis
Co-Author(s): Nathan J. Alves, Indiana University School of Medicine, Indianapolis, IN

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The nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, is utilized in UV photocrosslinking methods for site-specific functionalization of monoclonal antibodies. UV exposure (254nm) to a small molecule, indole-3-butyric acid (IBA), that has high affinity to the NBS can be used to photocrosslink ligands to antibodies. Here, we propose a method to modify antibodies by photocrosslinking with various intensity UV light sources: UV crosslinker XLE-1000 (40-watt), handheld EF-160C (6-watt), and MiniMax UV-5NF (5-watt). The different UV sources possess different power levels and by modulating both time of UV exposure and distance from source site-specific crosslinking at the NBS, for affinity tags (IBA-Biotin) and fluorescent molecules (IBA-FITC) was optimized. Application of the UV-NBS photocrosslinking technique is possible by first incubating the antibodies with IBA-Biotin or IBA tagged ligand followed by 0.5-1.5J/cm2 of UV exposure. Conjugation efficiency was determined via Western Blot analysis of IBA-Biotin and absorbance/fluorescent measurements for the presence and quantity of conjugated IBA-FITC. The UV-NBS technique is a reproducible method of photocrosslinking antibodies. Optimization of UV energy exposure resulted in an increase of conjugations per antibody with maximized photocrosslinking efficiency, while antibody antigen binding activity and Fc recognition were preserved. This study demonstrates that the UV-NBS site-specific antibody modification technique can be accomplished using UV light sources with differing light intensities expanding its implementation potential through making the technology more accessible. Ultimately, the UV-NBS method is an efficient, practical, and accessible method of functionalizing antibodies in diagnostic, pharmaceutical, and therapeutic settings.

OCHOA_Abstract_ERN_2018.docx

Funder Acknowledgement(s): This study was supported, in part, by grants from the Diversity Summer Undergraduate Research Opportunity Program and Commitment to Engineering Excellence Research Fund, to Celia Ochoa, Biomedical Engineering, Indiana University-Purdue University Indianapolis, 2017.

Faculty Advisor: Nathan J. Alves, nalves@iu.edu

Role: I collected the exploratory data of the XLE-1000 UV crosslinker to determine the relationships between UV intensity, UV energy, time, and distance of the samples to light source. In addition, my contributions to this research project include photocrosslinking the antibody and ligand samples under UV exposure (254nm), running SDS-PAGE gels, western blot analysis (of anti-EMAP II), and performing absorbance assays of the anti-FITC photocrosslinked antibody.