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

Scientific Reports
Jing ShangXiaohu Gao

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 ma...Continue Reading

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Citations

Mar 9, 2017·Chemical Reviews·Jian-Feng LiZhong-Qun Tian

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Methods Mentioned

BETA
enzyme-linked immunosorbent assays
ELISA
flow-cytometry
fluorescence imaging
electrophoresis
biopsies

Software Mentioned

Histelide

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