Limits...
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

Schematic illustration of the multiplexed in-cell immunoassay.The specimen is first incubated with a library of self-assembled 1′Ab/PrA-ssDNA probes and then with a cocktail of complementary ssDNA′-functionalized reporters (e.g., an enzyme alkaline phosphatase, AP). Target labeling occurs through (i) antibody-antigen binding and (ii) ssDNA-ssDNA′ hybridization. As a result, all targets of interest are simultaneously labeled by the same reporter, but each target is linked to a reporter via a displaceable DNA bridge with a unique sequence (e.g., X1-X2, Y1-Y2, or Z1-Z2). This enables the release of subsets of target-bound reporters into solution via sequence-specific DNA bond displacement with a longer complementary ssDNA probe (X3, Y3, or Z3, respectively), yielding unbound reporter concentration equivalent to the corresponding target abundance in the specimen. Multiplexed same-sample protein expression profiling is achieved by quick sequential release and segregation of all target-bound reporters one-by-one (e.g., into separate wells of a 96-well plate for high-throughput quantitative analysis with a plate reader).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4556981&req=5

f1: Schematic illustration of the multiplexed in-cell immunoassay.The specimen is first incubated with a library of self-assembled 1′Ab/PrA-ssDNA probes and then with a cocktail of complementary ssDNA′-functionalized reporters (e.g., an enzyme alkaline phosphatase, AP). Target labeling occurs through (i) antibody-antigen binding and (ii) ssDNA-ssDNA′ hybridization. As a result, all targets of interest are simultaneously labeled by the same reporter, but each target is linked to a reporter via a displaceable DNA bridge with a unique sequence (e.g., X1-X2, Y1-Y2, or Z1-Z2). This enables the release of subsets of target-bound reporters into solution via sequence-specific DNA bond displacement with a longer complementary ssDNA probe (X3, Y3, or Z3, respectively), yielding unbound reporter concentration equivalent to the corresponding target abundance in the specimen. Multiplexed same-sample protein expression profiling is achieved by quick sequential release and segregation of all target-bound reporters one-by-one (e.g., into separate wells of a 96-well plate for high-throughput quantitative analysis with a plate reader).

Mentions: Herein, we describe a simple and robust methodology that combines versatility of ELISA format with a vast encoding capacity of DNA hybridization for multiplexed same-sample protein expression profiling. While retaining many of the components of conventional and in-cell ELISA platforms for broad compatibility with assay reagents and specimen preparations, an inherently singleplex enzyme-based reporting mechanism is rendered multiplexable by introduction of the DNA-programmed release mechanism that enables selective release of target-bound enzyme reporters into solution for subsequent quantification of the released reporter concentration (Fig. 1). Specifically, all surface-bound target proteins (e.g., within formalin-fixed cells) are first simultaneously encoded with unique single-stranded DNA (ssDNA) sequences via recognition by ssDNA-tagged primary antibodies. In contrast to complex and expensive covalent antibody-ssDNA bioconjugation approaches, a non-covalent self-assembly between intact primary antibodies (1′Abs) and ssDNA-linked adaptor protein A29 (PrA-ssDNA) is employed to yield a flexible and simple route to on-demand preparation of antibody-ssDNA libraries (1′Ab/PrA-ssDNA). Specimen incubation with a cocktail of complementary ssDNA’-tagged alkaline phosphatase reporters (ssDNA’-AP) simultaneously labels all target proteins with AP via partial DNA hybridization. Finally, addition of a longer “displacement” ssDNA probe breaks complementary Ab-AP links via full hybridization with ssDNA’-AP and triggers “release” of a sub-set of reporters associated with a particular target protein encoded by the “displacement” sequence. Segregation of the released AP fraction allows for quantification of its concentration independent of the reporters remaining on the specimen. Sequential release and segregation of all reporter sub-sets thus allows for analysis of multiple protein targets within the same specimen using an identical reporter enzyme. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. Importantly, such measurements employ a well-characterized, sensitive, and robust enzyme reporting mechanism and only require instrumentation commonly available in biomedical laboratories.


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)

Schematic illustration of the multiplexed in-cell immunoassay.The specimen is first incubated with a library of self-assembled 1′Ab/PrA-ssDNA probes and then with a cocktail of complementary ssDNA′-functionalized reporters (e.g., an enzyme alkaline phosphatase, AP). Target labeling occurs through (i) antibody-antigen binding and (ii) ssDNA-ssDNA′ hybridization. As a result, all targets of interest are simultaneously labeled by the same reporter, but each target is linked to a reporter via a displaceable DNA bridge with a unique sequence (e.g., X1-X2, Y1-Y2, or Z1-Z2). This enables the release of subsets of target-bound reporters into solution via sequence-specific DNA bond displacement with a longer complementary ssDNA probe (X3, Y3, or Z3, respectively), yielding unbound reporter concentration equivalent to the corresponding target abundance in the specimen. Multiplexed same-sample protein expression profiling is achieved by quick sequential release and segregation of all target-bound reporters one-by-one (e.g., into separate wells of a 96-well plate for high-throughput quantitative analysis with a plate reader).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Schematic illustration of the multiplexed in-cell immunoassay.The specimen is first incubated with a library of self-assembled 1′Ab/PrA-ssDNA probes and then with a cocktail of complementary ssDNA′-functionalized reporters (e.g., an enzyme alkaline phosphatase, AP). Target labeling occurs through (i) antibody-antigen binding and (ii) ssDNA-ssDNA′ hybridization. As a result, all targets of interest are simultaneously labeled by the same reporter, but each target is linked to a reporter via a displaceable DNA bridge with a unique sequence (e.g., X1-X2, Y1-Y2, or Z1-Z2). This enables the release of subsets of target-bound reporters into solution via sequence-specific DNA bond displacement with a longer complementary ssDNA probe (X3, Y3, or Z3, respectively), yielding unbound reporter concentration equivalent to the corresponding target abundance in the specimen. Multiplexed same-sample protein expression profiling is achieved by quick sequential release and segregation of all target-bound reporters one-by-one (e.g., into separate wells of a 96-well plate for high-throughput quantitative analysis with a plate reader).
Mentions: Herein, we describe a simple and robust methodology that combines versatility of ELISA format with a vast encoding capacity of DNA hybridization for multiplexed same-sample protein expression profiling. While retaining many of the components of conventional and in-cell ELISA platforms for broad compatibility with assay reagents and specimen preparations, an inherently singleplex enzyme-based reporting mechanism is rendered multiplexable by introduction of the DNA-programmed release mechanism that enables selective release of target-bound enzyme reporters into solution for subsequent quantification of the released reporter concentration (Fig. 1). Specifically, all surface-bound target proteins (e.g., within formalin-fixed cells) are first simultaneously encoded with unique single-stranded DNA (ssDNA) sequences via recognition by ssDNA-tagged primary antibodies. In contrast to complex and expensive covalent antibody-ssDNA bioconjugation approaches, a non-covalent self-assembly between intact primary antibodies (1′Abs) and ssDNA-linked adaptor protein A29 (PrA-ssDNA) is employed to yield a flexible and simple route to on-demand preparation of antibody-ssDNA libraries (1′Ab/PrA-ssDNA). Specimen incubation with a cocktail of complementary ssDNA’-tagged alkaline phosphatase reporters (ssDNA’-AP) simultaneously labels all target proteins with AP via partial DNA hybridization. Finally, addition of a longer “displacement” ssDNA probe breaks complementary Ab-AP links via full hybridization with ssDNA’-AP and triggers “release” of a sub-set of reporters associated with a particular target protein encoded by the “displacement” sequence. Segregation of the released AP fraction allows for quantification of its concentration independent of the reporters remaining on the specimen. Sequential release and segregation of all reporter sub-sets thus allows for analysis of multiple protein targets within the same specimen using an identical reporter enzyme. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. Importantly, such measurements employ a well-characterized, sensitive, and robust enzyme reporting mechanism and only require instrumentation commonly available in biomedical laboratories.

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