Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

Abstract

Yoon Jeong,1,2 Kwan Hong Lee,1,2 Hansoo Park,3 Jonghoon Choi1,2 1Department of Bionano Technology, Graduate School, Hanyang University, Seoul, 2Department of Bionano Engineering, Hanyang University ERICA, Ansan, 3School of Integrative Engineering, Chung-Ang University, Seoul, South Korea Abstract: We present an evaluation of protein-G-terminated glass slides that may contain a suitable substrate for aligning the orientation of antibodies to obtain better binding moiety to the target antigen. The results of the protein-G-terminated slides were compared with those obtained with epoxy-based slides to evaluate signal enhancement for human immunoglobulin G (IgG) targets, and an increase in the average fluorescence intensity was observed for the lowest measurable amount of IgG target in the assay using protein-G-terminated slides. Applying this strategy for signal amplification to single-cell assays improves the limits of detection for human IgG protein and cytokines (interleukin-2 and interferon-γ) captured from hybridomas. Our data indicate that protein-G-terminated slides have a higher binding capacity for antigens and have better spot-to-spot consistency than that of traditional epoxy-based slides. These properties would be beneficial in the detection of fine amounts of single-cell-secreted proteins, which may provide key insights into cell–cell communication and immune responses. Keywords: microwell array, antibody’s orientation, single cell analysis, secreted cytokine, protein-G-terminated surface