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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

The detection of IgG proteins secreted from hybridomas in microwells.Notes: (A) Schematic diagram of the microwell array used to capture cytokines secreted from viable hybridomas deposited onto an array (~20×50 mm2) consisting of 72×24 blocks that contain a 7×7 array of microwells. (B) Representative bright-field image of live hybridomas loaded into microwells at an average density of ~1–2 cells/well. The array was sealed with glass slides bearing a mixture of capture antibodies. After the incubation period, the glass slide was recovered and the bound proteins were detected using secondary antibodies. Both the protein-G-terminated glass slides (C) and the epoxy-based slides (D) were coated with anti-human antibodies (IgG) for the respective cytokines. The immunofluorescence images of a region of a microarray show captured IgG from hybridomas on each substrate.Abbreviations: IgG, immunoglobulin G; PDMS, polydimethylsiloxane.
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f3-ijn-10-7197: The detection of IgG proteins secreted from hybridomas in microwells.Notes: (A) Schematic diagram of the microwell array used to capture cytokines secreted from viable hybridomas deposited onto an array (~20×50 mm2) consisting of 72×24 blocks that contain a 7×7 array of microwells. (B) Representative bright-field image of live hybridomas loaded into microwells at an average density of ~1–2 cells/well. The array was sealed with glass slides bearing a mixture of capture antibodies. After the incubation period, the glass slide was recovered and the bound proteins were detected using secondary antibodies. Both the protein-G-terminated glass slides (C) and the epoxy-based slides (D) were coated with anti-human antibodies (IgG) for the respective cytokines. The immunofluorescence images of a region of a microarray show captured IgG from hybridomas on each substrate.Abbreviations: IgG, immunoglobulin G; PDMS, polydimethylsiloxane.

Mentions: In order to confirm the increased sensitivity of the protein assay, we evaluated the protein-G-modified glass slide for the capturing of single-cell-secreted proteins using an array of microfabricated wells. Because the cells in microwells can be retained after printing to utilize their cytokine release, the system would be particularly useful for the monitoring of nonsacrificed, viable cell responses and also for the repeated capturing of cytokines from the same cells.25 In Figure 3A, a cross-sectional schematic illustration of the microwell system is shown for the printing of cell-secreted proteins. After the suspension of cells was placed onto the array of microwells, the cells were settled down into the wells by gravity at an average density of 1–2 cells/well (Figure 3B). Next, the microwells were covered with two different types of glass slides that were precoated with specific IgG capture antibodies. After incubation (~1 hour), fluorescently labeled reagents were added to the glass slides for analyte detection. After the detachment of the glass slides, hybridomas were retained in the microwells for a continuous incubation period. We first hypothesized that the protein-G-terminated glass substrate could adsorb capture antibodies in the proper orientation. The comparative fluorescent microscope images (Figure 3C and D) for the IgG detection clearly showed that the protein-G-terminated glass slides had higher signal intensities than those on the epoxy-based slides. A greater detection range would be beneficial for capturing the small amounts of single cell-secreted proteins, and proper immobilization significantly improves the antibody–antigen binding ratio in a sandwich immunoassay. Previous studies11,25 used epoxy-based slides to capture the cytokines or immunoglobulins secreted from isolated individual cells, while our results show that, first, the application of protein-G-terminated substrates for the immobilization of antibodies in an immunoassay increases its sensitivity for the detection of cytokines from hybridomas. This is due to the correct orientation of antibodies that occurs on a surface coated with such substrates.


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)

The detection of IgG proteins secreted from hybridomas in microwells.Notes: (A) Schematic diagram of the microwell array used to capture cytokines secreted from viable hybridomas deposited onto an array (~20×50 mm2) consisting of 72×24 blocks that contain a 7×7 array of microwells. (B) Representative bright-field image of live hybridomas loaded into microwells at an average density of ~1–2 cells/well. The array was sealed with glass slides bearing a mixture of capture antibodies. After the incubation period, the glass slide was recovered and the bound proteins were detected using secondary antibodies. Both the protein-G-terminated glass slides (C) and the epoxy-based slides (D) were coated with anti-human antibodies (IgG) for the respective cytokines. The immunofluorescence images of a region of a microarray show captured IgG from hybridomas on each substrate.Abbreviations: IgG, immunoglobulin G; PDMS, polydimethylsiloxane.
© Copyright Policy
Related In: Results  -  Collection

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

f3-ijn-10-7197: The detection of IgG proteins secreted from hybridomas in microwells.Notes: (A) Schematic diagram of the microwell array used to capture cytokines secreted from viable hybridomas deposited onto an array (~20×50 mm2) consisting of 72×24 blocks that contain a 7×7 array of microwells. (B) Representative bright-field image of live hybridomas loaded into microwells at an average density of ~1–2 cells/well. The array was sealed with glass slides bearing a mixture of capture antibodies. After the incubation period, the glass slide was recovered and the bound proteins were detected using secondary antibodies. Both the protein-G-terminated glass slides (C) and the epoxy-based slides (D) were coated with anti-human antibodies (IgG) for the respective cytokines. The immunofluorescence images of a region of a microarray show captured IgG from hybridomas on each substrate.Abbreviations: IgG, immunoglobulin G; PDMS, polydimethylsiloxane.
Mentions: In order to confirm the increased sensitivity of the protein assay, we evaluated the protein-G-modified glass slide for the capturing of single-cell-secreted proteins using an array of microfabricated wells. Because the cells in microwells can be retained after printing to utilize their cytokine release, the system would be particularly useful for the monitoring of nonsacrificed, viable cell responses and also for the repeated capturing of cytokines from the same cells.25 In Figure 3A, a cross-sectional schematic illustration of the microwell system is shown for the printing of cell-secreted proteins. After the suspension of cells was placed onto the array of microwells, the cells were settled down into the wells by gravity at an average density of 1–2 cells/well (Figure 3B). Next, the microwells were covered with two different types of glass slides that were precoated with specific IgG capture antibodies. After incubation (~1 hour), fluorescently labeled reagents were added to the glass slides for analyte detection. After the detachment of the glass slides, hybridomas were retained in the microwells for a continuous incubation period. We first hypothesized that the protein-G-terminated glass substrate could adsorb capture antibodies in the proper orientation. The comparative fluorescent microscope images (Figure 3C and D) for the IgG detection clearly showed that the protein-G-terminated glass slides had higher signal intensities than those on the epoxy-based slides. A greater detection range would be beneficial for capturing the small amounts of single cell-secreted proteins, and proper immobilization significantly improves the antibody–antigen binding ratio in a sandwich immunoassay. Previous studies11,25 used epoxy-based slides to capture the cytokines or immunoglobulins secreted from isolated individual cells, while our results show that, first, the application of protein-G-terminated substrates for the immobilization of antibodies in an immunoassay increases its sensitivity for the detection of cytokines from hybridomas. This is due to the correct orientation of antibodies that occurs on a surface coated with such substrates.

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