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Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling.

Shang J, Zrazhevskiy P, Postupna N, Keene CD, Montine TJ, Gao X - Sci Rep (2015)

Bottom Line: Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters.In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion.Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, β-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the "Alzheimer's disease" cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics.

No MeSH data available.


Related in: MedlinePlus

Same-sample quantitative analysis of Aβ and PHF-tau abundance in brain tissue sections.FFPE brain tissue sections from “Alzheimer′s disease (AD)” (n = 10) and healthy “Control” (n = 9) groups were simultaneously labeled for Aβ and PHF-tau pathologic proteins. AP reporters corresponding to each target were sequentially released via DNA displacement and measured with a plate reader. Chemiluminescence signal was normalized by the gray matter area on each tissue section. (a) Comparison of Aβ expression in “AD” and “Control” groups. (b) Comparison of PHF-tau expression in “AD” and “Control” groups. Average expression level is indicated by a solid line on each scatter plot. (c) Pairwise comparison of Aβ and PHF-tau expression in “AD” (circles) and “Control” (diamonds) groups.
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f5: Same-sample quantitative analysis of Aβ and PHF-tau abundance in brain tissue sections.FFPE brain tissue sections from “Alzheimer′s disease (AD)” (n = 10) and healthy “Control” (n = 9) groups were simultaneously labeled for Aβ and PHF-tau pathologic proteins. AP reporters corresponding to each target were sequentially released via DNA displacement and measured with a plate reader. Chemiluminescence signal was normalized by the gray matter area on each tissue section. (a) Comparison of Aβ expression in “AD” and “Control” groups. (b) Comparison of PHF-tau expression in “AD” and “Control” groups. Average expression level is indicated by a solid line on each scatter plot. (c) Pairwise comparison of Aβ and PHF-tau expression in “AD” (circles) and “Control” (diamonds) groups.

Mentions: Assay validation was performed by simultaneous same-sample labeling and quantification of Aβ and PHF-tau in FFPE brain tissue sections from 19 subjects—10 from the AD cohort and 9 from the control group (Fig. 5). As expected, all “Control” specimens were negative for both pathologic proteins, whereas the AD group yielded significantly elevated levels of Aβ and PHF-tau (two-tailed t-test P < 0.001) consistent with the molecular manifestation of Alzheimer′s disease. Greater variation in protein abundance was also observed among different AD cases, which, given similar disease stage, might indicate involvement of different mechanisms in disease pathogenesis. More importantly, the multiplexed in-cell immunoassay produced quantitative data for both pathologic proteins from the same specimen, enabling direct comparison and correlation of protein expression levels (Fig. 5c).


Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling.

Shang J, Zrazhevskiy P, Postupna N, Keene CD, Montine TJ, Gao X - Sci Rep (2015)

Same-sample quantitative analysis of Aβ and PHF-tau abundance in brain tissue sections.FFPE brain tissue sections from “Alzheimer′s disease (AD)” (n = 10) and healthy “Control” (n = 9) groups were simultaneously labeled for Aβ and PHF-tau pathologic proteins. AP reporters corresponding to each target were sequentially released via DNA displacement and measured with a plate reader. Chemiluminescence signal was normalized by the gray matter area on each tissue section. (a) Comparison of Aβ expression in “AD” and “Control” groups. (b) Comparison of PHF-tau expression in “AD” and “Control” groups. Average expression level is indicated by a solid line on each scatter plot. (c) Pairwise comparison of Aβ and PHF-tau expression in “AD” (circles) and “Control” (diamonds) groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556981&req=5

f5: Same-sample quantitative analysis of Aβ and PHF-tau abundance in brain tissue sections.FFPE brain tissue sections from “Alzheimer′s disease (AD)” (n = 10) and healthy “Control” (n = 9) groups were simultaneously labeled for Aβ and PHF-tau pathologic proteins. AP reporters corresponding to each target were sequentially released via DNA displacement and measured with a plate reader. Chemiluminescence signal was normalized by the gray matter area on each tissue section. (a) Comparison of Aβ expression in “AD” and “Control” groups. (b) Comparison of PHF-tau expression in “AD” and “Control” groups. Average expression level is indicated by a solid line on each scatter plot. (c) Pairwise comparison of Aβ and PHF-tau expression in “AD” (circles) and “Control” (diamonds) groups.
Mentions: Assay validation was performed by simultaneous same-sample labeling and quantification of Aβ and PHF-tau in FFPE brain tissue sections from 19 subjects—10 from the AD cohort and 9 from the control group (Fig. 5). As expected, all “Control” specimens were negative for both pathologic proteins, whereas the AD group yielded significantly elevated levels of Aβ and PHF-tau (two-tailed t-test P < 0.001) consistent with the molecular manifestation of Alzheimer′s disease. Greater variation in protein abundance was also observed among different AD cases, which, given similar disease stage, might indicate involvement of different mechanisms in disease pathogenesis. More importantly, the multiplexed in-cell immunoassay produced quantitative data for both pathologic proteins from the same specimen, enabling direct comparison and correlation of protein expression levels (Fig. 5c).

Bottom Line: Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters.In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion.Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, β-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the "Alzheimer's disease" cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics.

No MeSH data available.


Related in: MedlinePlus