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

Jeong Y, Lee KH, Park H, Choi J - Int J Nanomedicine (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bionano Technology, Graduate School, Hanyang University, Seoul, South Korea ; Department of Bionano Engineering, Hanyang University ERICA, Ansan, 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.

No MeSH data available.


Related in: MedlinePlus

Detection of human IgG protein in an immunochemistry assay on protein G or epoxy slides.Notes: (A) Typical structure of an antibody and its oriented positions that can affect binding affinity are illustrated. (B) Scheme of the different types of immobilization of antibodies on protein G or epoxy slides. Representative images of the fluorescence intensity of human IgG bound to the capture antibodies immobilized on either protein-G- or epoxy-terminated glass slides with decreasing concentration of the antigen (34–4.3 μg/mL).Abbreviations: IgG, immunoglobulin G; Fab, fragment antigen-binding; Fc, fragment crystallizable.
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f1-ijn-10-7197: Detection of human IgG protein in an immunochemistry assay on protein G or epoxy slides.Notes: (A) Typical structure of an antibody and its oriented positions that can affect binding affinity are illustrated. (B) Scheme of the different types of immobilization of antibodies on protein G or epoxy slides. Representative images of the fluorescence intensity of human IgG bound to the capture antibodies immobilized on either protein-G- or epoxy-terminated glass slides with decreasing concentration of the antigen (34–4.3 μg/mL).Abbreviations: IgG, immunoglobulin G; Fab, fragment antigen-binding; Fc, fragment crystallizable.

Mentions: Antibodies (Ig) are relatively large proteins (~150 kDa) consisting of two light and two heavy chains. The typical structure of an antibody consists of a roughly asymmetric Y-shape containing many molecules, one type of which are amino acids. While the amino acids in the Fc region share the same sequence, the ones in the fragment antigen-binding (Fab) region have different sequences. This, therefore, allows the specific binding of antibodies to various antigens.26 In Figure 1A, schemes of three possible orientations of antibody binding with a substrate are illustrated, excluding the possible steric hindrance. Single-site attachment or multiple-site attachment occurs when the affinity ligand of an antibody is attached to the substrates by one or more functional groups, respectively. The proper orientation for alignment of the antibodies can only be obtained in the case where the Fc regions bind to a single ligand molecule on the surface. Therefore, any improperly orientated positions could block the Fab regions. For an effective binding moiety, two binding sites at two Fab regions should be exposed to the target analytes. As shown in Figure 1B, both types of slides were treated with human IgG antibodies (with decreasing concentrations from 34 to 4.3 μg/mL) and then compared in terms of their limits of detection. The protein G slides showed higher fluorescence intensities than those of the epoxy-coated slides and also displayed an extended detection limit. This result indicates that protein-G-coated substrate could be used as a suitable surface for the preparation of antibodies and detection of analytes at low concentrations.


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

Jeong Y, Lee KH, Park H, Choi J - Int J Nanomedicine (2015)

Detection of human IgG protein in an immunochemistry assay on protein G or epoxy slides.Notes: (A) Typical structure of an antibody and its oriented positions that can affect binding affinity are illustrated. (B) Scheme of the different types of immobilization of antibodies on protein G or epoxy slides. Representative images of the fluorescence intensity of human IgG bound to the capture antibodies immobilized on either protein-G- or epoxy-terminated glass slides with decreasing concentration of the antigen (34–4.3 μg/mL).Abbreviations: IgG, immunoglobulin G; Fab, fragment antigen-binding; Fc, fragment crystallizable.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664541&req=5

f1-ijn-10-7197: Detection of human IgG protein in an immunochemistry assay on protein G or epoxy slides.Notes: (A) Typical structure of an antibody and its oriented positions that can affect binding affinity are illustrated. (B) Scheme of the different types of immobilization of antibodies on protein G or epoxy slides. Representative images of the fluorescence intensity of human IgG bound to the capture antibodies immobilized on either protein-G- or epoxy-terminated glass slides with decreasing concentration of the antigen (34–4.3 μg/mL).Abbreviations: IgG, immunoglobulin G; Fab, fragment antigen-binding; Fc, fragment crystallizable.
Mentions: Antibodies (Ig) are relatively large proteins (~150 kDa) consisting of two light and two heavy chains. The typical structure of an antibody consists of a roughly asymmetric Y-shape containing many molecules, one type of which are amino acids. While the amino acids in the Fc region share the same sequence, the ones in the fragment antigen-binding (Fab) region have different sequences. This, therefore, allows the specific binding of antibodies to various antigens.26 In Figure 1A, schemes of three possible orientations of antibody binding with a substrate are illustrated, excluding the possible steric hindrance. Single-site attachment or multiple-site attachment occurs when the affinity ligand of an antibody is attached to the substrates by one or more functional groups, respectively. The proper orientation for alignment of the antibodies can only be obtained in the case where the Fc regions bind to a single ligand molecule on the surface. Therefore, any improperly orientated positions could block the Fab regions. For an effective binding moiety, two binding sites at two Fab regions should be exposed to the target analytes. As shown in Figure 1B, both types of slides were treated with human IgG antibodies (with decreasing concentrations from 34 to 4.3 μg/mL) and then compared in terms of their limits of detection. The protein G slides showed higher fluorescence intensities than those of the epoxy-coated slides and also displayed an extended detection limit. This result indicates that protein-G-coated substrate could be used as a suitable surface for the preparation of antibodies and detection of analytes at low concentrations.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bionano Technology, Graduate School, Hanyang University, Seoul, South Korea ; Department of Bionano Engineering, Hanyang University ERICA, Ansan, 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.

No MeSH data available.


Related in: MedlinePlus