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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 cytokines on protein-G-coated glass slides.Notes: (A) Recombinant human IL-2 spots on protein G slides were detected using a sandwich immunoassay. Various concentrations of IL-2 (50 ng/mL, 5 ng/mL, 500 pg/mL, 50 pg/mL, 5 pg/mL, and 0.5 pg/mL) were spotted onto both epoxy and protein G slides and detected using the corresponding fluorescently labeled antibody. (B) Comparison of the IL-2 fluorescence intensities on epoxy or protein G slides. (C) Recombinant human IFN-γ spots on protein G slides were detected by an immunoassay. (D) Comparison of the IFN-γ fluorescence intensities on epoxy or protein G slide.Abbreviations: IL-2, interleukin-2; IFN, interferon.
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f2-ijn-10-7197: Detection of human cytokines on protein-G-coated glass slides.Notes: (A) Recombinant human IL-2 spots on protein G slides were detected using a sandwich immunoassay. Various concentrations of IL-2 (50 ng/mL, 5 ng/mL, 500 pg/mL, 50 pg/mL, 5 pg/mL, and 0.5 pg/mL) were spotted onto both epoxy and protein G slides and detected using the corresponding fluorescently labeled antibody. (B) Comparison of the IL-2 fluorescence intensities on epoxy or protein G slides. (C) Recombinant human IFN-γ spots on protein G slides were detected by an immunoassay. (D) Comparison of the IFN-γ fluorescence intensities on epoxy or protein G slide.Abbreviations: IL-2, interleukin-2; IFN, interferon.

Mentions: Recombinant human IL-2 or IFN-γ was spotted on the epoxy and protein G slides coated with capture antibodies, and these cytokines were each detected using the respective antibodies labeled with fluorescent molecules (Figure 2). The results for the epoxy-slide spots are not shown in this figure due to negligible affinity and fluorescence signal observed for IL-2 and IFN-γ. A quantitative evaluation and comparison of the spotting results is shown in Figure 2C and D, respectively. On the protein-G-terminated slides, it was possible to detect the target antigens in the low-concentration sample and also to obtain higher fluorescent signals than those obtained on the epoxy-coated slides. More Fab and Fc regions were expected to bind onto the epoxy surfaces in a random fashion, and subsequently, decreased binding signal was observed. While it was expected that the protein-G-modified surfaces prepared with antibodies immobilized in an oriented manner would yield greater binding signal. Our results clearly show that the fluorescence intensities observed for the cytokines in the concentration range of ng/mL to pg/mL on the protein-G-modified slides were greater than those observed on the epoxy slides. These results indicate that using the protein G slides would provide the sensitivity needed to detect cytokine concentrations of less than 1 pg/mL, and that the antigen-binding sites (Fab regions) on the surface of protein G slides were more accessible to the analytes, whereas many antigen-binding sites might have been blocked on the epoxy-coated slides. Hence, site-oriented immobilization plays an important role in enhancing the detection signals for the protein-G-modified slides.


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 cytokines on protein-G-coated glass slides.Notes: (A) Recombinant human IL-2 spots on protein G slides were detected using a sandwich immunoassay. Various concentrations of IL-2 (50 ng/mL, 5 ng/mL, 500 pg/mL, 50 pg/mL, 5 pg/mL, and 0.5 pg/mL) were spotted onto both epoxy and protein G slides and detected using the corresponding fluorescently labeled antibody. (B) Comparison of the IL-2 fluorescence intensities on epoxy or protein G slides. (C) Recombinant human IFN-γ spots on protein G slides were detected by an immunoassay. (D) Comparison of the IFN-γ fluorescence intensities on epoxy or protein G slide.Abbreviations: IL-2, interleukin-2; IFN, interferon.
© Copyright Policy
Related In: Results  -  Collection

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

f2-ijn-10-7197: Detection of human cytokines on protein-G-coated glass slides.Notes: (A) Recombinant human IL-2 spots on protein G slides were detected using a sandwich immunoassay. Various concentrations of IL-2 (50 ng/mL, 5 ng/mL, 500 pg/mL, 50 pg/mL, 5 pg/mL, and 0.5 pg/mL) were spotted onto both epoxy and protein G slides and detected using the corresponding fluorescently labeled antibody. (B) Comparison of the IL-2 fluorescence intensities on epoxy or protein G slides. (C) Recombinant human IFN-γ spots on protein G slides were detected by an immunoassay. (D) Comparison of the IFN-γ fluorescence intensities on epoxy or protein G slide.Abbreviations: IL-2, interleukin-2; IFN, interferon.
Mentions: Recombinant human IL-2 or IFN-γ was spotted on the epoxy and protein G slides coated with capture antibodies, and these cytokines were each detected using the respective antibodies labeled with fluorescent molecules (Figure 2). The results for the epoxy-slide spots are not shown in this figure due to negligible affinity and fluorescence signal observed for IL-2 and IFN-γ. A quantitative evaluation and comparison of the spotting results is shown in Figure 2C and D, respectively. On the protein-G-terminated slides, it was possible to detect the target antigens in the low-concentration sample and also to obtain higher fluorescent signals than those obtained on the epoxy-coated slides. More Fab and Fc regions were expected to bind onto the epoxy surfaces in a random fashion, and subsequently, decreased binding signal was observed. While it was expected that the protein-G-modified surfaces prepared with antibodies immobilized in an oriented manner would yield greater binding signal. Our results clearly show that the fluorescence intensities observed for the cytokines in the concentration range of ng/mL to pg/mL on the protein-G-modified slides were greater than those observed on the epoxy slides. These results indicate that using the protein G slides would provide the sensitivity needed to detect cytokine concentrations of less than 1 pg/mL, and that the antigen-binding sites (Fab regions) on the surface of protein G slides were more accessible to the analytes, whereas many antigen-binding sites might have been blocked on the epoxy-coated slides. Hence, site-oriented immobilization plays an important role in enhancing the detection signals for the protein-G-modified slides.

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