title: 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.
authors.affiliation: Department of Bionano Technology, Graduate School, Hanyang University, Seoul, South Korea ; Department of Bionano Engineering, Hanyang University ERICA, Ansan, South Korea.
authors.forename: Yoon
authors.initials: Y
authors.lastname: Jeong
authors.affiliation: Department of Bionano Technology, Graduate School, Hanyang University, Seoul, South Korea ; Department of Bionano Engineering, Hanyang University ERICA, Ansan, South Korea.
authors.forename: Kwan Hong
authors.initials: KH
authors.lastname: Lee
authors.affiliation: School of Integrative Engineering, Chung-Ang University, Seoul, South Korea.
authors.forename: Hansoo
authors.initials: H
authors.lastname: Park
authors.affiliation: Department of Bionano Technology, Graduate School, Hanyang University, Seoul, South Korea ; Department of Bionano Engineering, Hanyang University ERICA, Ansan, South Korea.
authors.forename: Jonghoon
authors.initials: J
authors.lastname: Choi
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004044:Dietary Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005898:Glass / Q000737:chemistry*; D006801:Humans; D006825:Hybridomas; D007118:Immunoassay / Q000379:methods; D007074:Immunoglobulin G / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism*; D007376:Interleukin-2 / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004044:Dietary Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D007376:Interleukin-2; C012551:single cell proteins; D007371:Interferon-gamma
keywords: antibody’s orientation; microwell array; protein-G-terminated surface; secreted cytokine; single cell analysis