Poster V6 - Optimizing Fc-Specific Antibody-Oligonucleotide Conjugation to Improve Sensitivity of Immunoassay Probes

Introduction: Many neurological diseases have specific hallmark protein-protein interactions that can be used for diagnosis. Immunoassays, such as proximity ligation assay (PLA), are often used to detect these protein-protein interactions of interest, in the femtomolar to nanomolar range. The PLA assay requires antibodies to be labeled with oligonucleotides that help to amplify the protein-protein interaction signals, however some labeling methods used such as non-site-specific antibody labeling methods, when used with a high payload-to-antibody conjugation ratio can drastically reduce the affinity of antibody to the epitope which decreases the sensitivity of these diagnostic assays.

Materials and

Methods: We believe that β-1,4-galactosidase deglycosylation and β-1,4 galactosyltransferase glycosidation can be used to swap the terminal galactose residues of IgG antibodies with GalNAz, or galactose molecules with azide modifications resulting in a high antibody conjugation ratio with preserved/non-altered antibody affinity, and a more sensitive diagnostic proximity ligation assay. The goal of this project is to determine which optimal payload-to-antibody ratio for the azide modified reaction would result in high conjugate yields while preserving antibody affinity by using native antibodies with azide attached via SiteClick Antibody Azido Modification Kit. 8:1 and 16:1 payload-to-antibody ratios were tested in-house. NanoDrop was used to measure the concentrations of the functionalized antibodies, oligonucleotides, and final products, which found a small number of functionalized antibodies and a large number of oligonucleotides. SDS-PAGE was used to compare antibody presence before and after conjugation, which showed some conjugate yield. Finally, Western blot was used to ascertain the affinity of the antibody before and after conjugation, where affinity was retained.

Results, Conclusions, and Discussions: We found that our payload to antibody ratio should be increased to improve the conjugation yield. Additionally, we found that that antibody affinity was retained throughout the conjugation process. Future experimentation will use antibody-oligonucleotide conjugate probes produced with optimized payload-to-antibody ratios, with the eventual goal to measure protein-protein interactions in neurons.

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