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

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.

An investigation into non-covalent functionalization of a single-walled carbon nanotube and a graphene sheet with protein G:A combined experimental and molecular dynamics study

“…Studies have shown that the content of the loaded antigen in well-oriented antibodies is at least 3 times higher than the randomly-oriented antibodies 7,8 . Besides, it has been demonstrated that glass slides coated with the PrG could be used as a suitable substrate to orient the antibodies resulting in the detection of low concentration of antigens 9 . Furthermore, it was shown that using the PrG-coupled beads significantly improved the multiplex immunoassay approach and simultaneously quantified analytes in a limited volume 10 .…”