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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 secreted cytokines (IL-2 and IFN-γ) from stimulated PBMCs in a microwell assay using two types of substrates.Notes: Microwells were placed in contact with each glass slide coated with IL-2 or IFN-γ capture antibodies, respectively. After 1 hour, the glass slide was removed and incubated with secondary antibodies tagged with fluorescent molecules. (A) Combined fluorescence images of microwells, and (B) their printed glass slides on the same region capturing cell-secreted IL-2 or IFN-γ. Two different cytokines released by PBMCs on each substrate were detected by dye-conjugated secondary antibodies. (C) Comparison of the numbers of positive events detected per 10,000 cells for two cytokines on the epoxy-based slide (white boxes) or on the protein-G-terminated slide (gray boxes). Statistical significance was determined by Fisher’s exact test (**P<0.01). The data represent composite measurements from three separate microarrays generated in parallel using divided aliquots from the same pool of stimulated PBMCs. (D) Box and whisker plot comparing relative fluorescence units detected on the epoxy-based slide (white boxes) and on the protein-G-terminated slide (gray boxes) for two different proteins secreted from PBMCs. Statistical significances were determined by the Student’s t-test (*P<0.05, **P<0.01).Abbreviations: IL-2, interleukin-2; IFN-γ, interferon-γ; PBMCs, peripheral blood mononuclear cells.
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f4-ijn-10-7197: Detection of secreted cytokines (IL-2 and IFN-γ) from stimulated PBMCs in a microwell assay using two types of substrates.Notes: Microwells were placed in contact with each glass slide coated with IL-2 or IFN-γ capture antibodies, respectively. After 1 hour, the glass slide was removed and incubated with secondary antibodies tagged with fluorescent molecules. (A) Combined fluorescence images of microwells, and (B) their printed glass slides on the same region capturing cell-secreted IL-2 or IFN-γ. Two different cytokines released by PBMCs on each substrate were detected by dye-conjugated secondary antibodies. (C) Comparison of the numbers of positive events detected per 10,000 cells for two cytokines on the epoxy-based slide (white boxes) or on the protein-G-terminated slide (gray boxes). Statistical significance was determined by Fisher’s exact test (**P<0.01). The data represent composite measurements from three separate microarrays generated in parallel using divided aliquots from the same pool of stimulated PBMCs. (D) Box and whisker plot comparing relative fluorescence units detected on the epoxy-based slide (white boxes) and on the protein-G-terminated slide (gray boxes) for two different proteins secreted from PBMCs. Statistical significances were determined by the Student’s t-test (*P<0.05, **P<0.01).Abbreviations: IL-2, interleukin-2; IFN-γ, interferon-γ; PBMCs, peripheral blood mononuclear cells.

Mentions: In addition, we studied the feasibility of obtaining high sensitivity in capturing primary cell-secreted cytokines using protein-G-terminated glass slides in a microwell assay. The protein-G-coated glass slides were functionalized with antibodies for human IL-2 and IFN-γ, and were then sandwiched with a microwell assay isolating human PBMCs. The approach we describe in Figure 4 reveals that it was possible to obtain results for the repetitive detection of different cytokines released from the same subset of PBMCs in the microwells. The combined fluorescence images (Figure 4A) show that the live PBMCs visualized in the microwells had different surface cluster-of-differentiation markers for immune cells. For the evaluation of the detection sensitivity, protein-G-terminated glass slides were compared with the epoxy-coated slides in terms of the number of positive spots and their fluorescence intensities, representing the capture of IL-2 and IFN-γ (Figure 4B). Figure 4C correlates the number of positive spots for each cytokine with the data collected from each microwell by image analysis. The number of secretion events detected in the protein G slides was two- to threefold higher than that in the epoxy slides. As shown in Figure 4D, the relative fluorescence intensity was also statistically significantly higher in the protein G slides than that in the epoxy slides for both IL-2 and IFN-γ detection.


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 secreted cytokines (IL-2 and IFN-γ) from stimulated PBMCs in a microwell assay using two types of substrates.Notes: Microwells were placed in contact with each glass slide coated with IL-2 or IFN-γ capture antibodies, respectively. After 1 hour, the glass slide was removed and incubated with secondary antibodies tagged with fluorescent molecules. (A) Combined fluorescence images of microwells, and (B) their printed glass slides on the same region capturing cell-secreted IL-2 or IFN-γ. Two different cytokines released by PBMCs on each substrate were detected by dye-conjugated secondary antibodies. (C) Comparison of the numbers of positive events detected per 10,000 cells for two cytokines on the epoxy-based slide (white boxes) or on the protein-G-terminated slide (gray boxes). Statistical significance was determined by Fisher’s exact test (**P<0.01). The data represent composite measurements from three separate microarrays generated in parallel using divided aliquots from the same pool of stimulated PBMCs. (D) Box and whisker plot comparing relative fluorescence units detected on the epoxy-based slide (white boxes) and on the protein-G-terminated slide (gray boxes) for two different proteins secreted from PBMCs. Statistical significances were determined by the Student’s t-test (*P<0.05, **P<0.01).Abbreviations: IL-2, interleukin-2; IFN-γ, interferon-γ; PBMCs, peripheral blood mononuclear cells.
© Copyright Policy
Related In: Results  -  Collection

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

f4-ijn-10-7197: Detection of secreted cytokines (IL-2 and IFN-γ) from stimulated PBMCs in a microwell assay using two types of substrates.Notes: Microwells were placed in contact with each glass slide coated with IL-2 or IFN-γ capture antibodies, respectively. After 1 hour, the glass slide was removed and incubated with secondary antibodies tagged with fluorescent molecules. (A) Combined fluorescence images of microwells, and (B) their printed glass slides on the same region capturing cell-secreted IL-2 or IFN-γ. Two different cytokines released by PBMCs on each substrate were detected by dye-conjugated secondary antibodies. (C) Comparison of the numbers of positive events detected per 10,000 cells for two cytokines on the epoxy-based slide (white boxes) or on the protein-G-terminated slide (gray boxes). Statistical significance was determined by Fisher’s exact test (**P<0.01). The data represent composite measurements from three separate microarrays generated in parallel using divided aliquots from the same pool of stimulated PBMCs. (D) Box and whisker plot comparing relative fluorescence units detected on the epoxy-based slide (white boxes) and on the protein-G-terminated slide (gray boxes) for two different proteins secreted from PBMCs. Statistical significances were determined by the Student’s t-test (*P<0.05, **P<0.01).Abbreviations: IL-2, interleukin-2; IFN-γ, interferon-γ; PBMCs, peripheral blood mononuclear cells.
Mentions: In addition, we studied the feasibility of obtaining high sensitivity in capturing primary cell-secreted cytokines using protein-G-terminated glass slides in a microwell assay. The protein-G-coated glass slides were functionalized with antibodies for human IL-2 and IFN-γ, and were then sandwiched with a microwell assay isolating human PBMCs. The approach we describe in Figure 4 reveals that it was possible to obtain results for the repetitive detection of different cytokines released from the same subset of PBMCs in the microwells. The combined fluorescence images (Figure 4A) show that the live PBMCs visualized in the microwells had different surface cluster-of-differentiation markers for immune cells. For the evaluation of the detection sensitivity, protein-G-terminated glass slides were compared with the epoxy-coated slides in terms of the number of positive spots and their fluorescence intensities, representing the capture of IL-2 and IFN-γ (Figure 4B). Figure 4C correlates the number of positive spots for each cytokine with the data collected from each microwell by image analysis. The number of secretion events detected in the protein G slides was two- to threefold higher than that in the epoxy slides. As shown in Figure 4D, the relative fluorescence intensity was also statistically significantly higher in the protein G slides than that in the epoxy slides for both IL-2 and IFN-γ detection.

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