Discipline: Biological Sciences
Subcategory: Biomedical Engineering
Room: Exhibit Hall A
Oriana Esteves-Ruiz - Indiana University Purdue University Indianapolis
Co-Author(s): Ziqian Zeng, IU School of Medicine, Indianapolis; Dr. Nathan J. Alves, PhD., IU School of Medicine, Indianapolis.
The effects of temperature on antibody conjugation efficiency utilizing a UV photocrosslinking method is described here. We hypothesized that temperatures ranging from 0-70C will affect the number of resulting conjugations per antibody. This site-specific conjugation method takes advantage of the nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, for covalent functionalization of antibodies with indole-3-butyric acid (IBA)-FITC while preserving antibody activity. IBA is a small molecule that has high affinity to the NBS allowing for FITC modified IBA to bind to the NBS during incubation. The UV-NBS method is a novel antibody labeling strategy with many benefits over alternative antibody conjugation methods. Seeing that the nucleotide binding site is in all antibody isotypes, it allows this method to be use in all antibodies. This technique also creates a way to add new functional groups to antibodies easier, site-specifically, and in a chemically efficient manner. Non-site-specific methods can reduce antibody activity by conjugations occurring at the complementary determining region or the Fc domain preventing binding of secondary antibodies. Having a method that functionalizes antibodies not only in a very simple way, but also site-specifically while still preserving the antibody activity is critical for the success of any application where antibodies are utilized. To demonstrate the impact temperature has on conjugation efficiency, triplicate antibody conjugate experiments consisting of 15µM antibody (Rituximab or Tocilizumab) and 300 µM IBA-FITC were incubated for 10 minutes, and photocrosslinked by exposure to 1 J/cm2 for each temperature (0,15, 25,37,50, 70C). Absorbance at 280 and 494nm were used to quantify the average number of conjugations per antibody using Beer-Lambert Law to calculate the concentration of the antibody and FITC. Conjugation efficiency was lowest at 0°C at 0.8 conjugation/antibody (Rituximab), while the range of temperatures between 15-50C yielded a consistent number of conjugations (1.5-2.0 conjugations/antibody (Rituximab)) with a significant deviation in conjugation efficiency at higher temperatures. At 70°C thermal denaturing of the antibody was observed causing non-specific crosslinking, increasing the average number of conjugations while reducing the antibody recovery yield. In summary, the results presented in this study show that antibody-ligand incubation temperature has an effect on the number of conjugations per antibody using the UV-NBS photocrosslinking technique. Future studies will focus on the stability of the conjugations and maintaining such for a set amount of time. References: Biomaterials. Nathan J Alves, Matthew M. Champion, Jared F. Stefanick, Michael W. Handlogten. 2013, Selective photocrosslinking of functional ligands to antibodies, pp. 5700-5710.
Funder Acknowledgement(s): This study was supported, in part, by a grant from NSF awarded to the IN Louis Stokes Alliance for Minority Participation.
Faculty Advisor: Nathan J. Alves, email@example.com
Role: Tested all temperatures by photocrosslinking and measuring conjugations following the procedure on the abstract.