Olink PEA Technology: Principle and Data Analysis Workflow
In high-plex protein detection, Olink, leveraging its proprietary Proximity Extension Assay (PEA) technology, has become a widely adopted platform for biomarker discovery and population-based cohort studies. The PEA method integrates dual-antibody specific recognition, nucleic acid-based tagging, and highly sensitive signal amplification, enabling high analytical throughput without sacrificing quantitative accuracy. In this report, we provide a systematic overview of the underlying principles of Olink PEA technology and outline the commonly used analytical workflows for Olink data, facilitating efficient interpretation and application of this platform in biomedical research.
Principles of Olink PEA Technology
1. Core Concept of Olink PEA Technology
(1) The PEA assay employs pairs of antibodies conjugated to unique oligonucleotide tags.
(2) When both probes bind the same target protein, the oligonucleotides are brought into close proximity, enabling DNA polymerization to generate an amplifiable DNA reporter sequence.
(3) The resulting DNA amplicon is subsequently quantified by qPCR or high-throughput NGS, providing highly sensitive and low-background signal readout.
2. Advantages of Olink PEA Technology
(1) High specificity: dual-antibody binding minimizes non-specific background.
(2) High throughput: a single reaction enables profiling of hundreds to thousands of proteins.
(3) Low sample input: only 1-3 μL of plasma or serum is required per analysis.
(4) Wide dynamic range: protein abundance measurements span up to 10 orders of magnitude.
3. Classification of Olink PEA Detection Platforms
(1) qPCR-based Target and Focus panels, supporting medium to low-plex studies involving dozens to hundreds of proteins.
(2) NGS-based Explore and Explore HT platforms, enabling high-plex analyses covering more than 3,000 proteins.
Overview of the PEA Experimental Workflow
1. Sample Processing for Olink PEA Technology
(1) Serum, plasma, or other biofluids are preprocessed according to standardized protocols.
(2) Batch effects are strictly controlled to ensure consistency in downstream analyses.
2. Reaction Steps of Olink PEA Technology
(1) Oligonucleotide-conjugated antibody probe pairs are incubated with samples to bind target proteins.
(2) Upon dual binding, the proximal oligonucleotide tags serve as templates for DNA polymerization, generating amplifiable DNA sequences.
(3) The resulting DNA templates are subsequently amplified and quantified by qPCR or high-throughput NGS.
3. Signal Readout of Olink PEA
(1) qPCR-based detection: relative quantification is derived from Cq (quantification cycle) values.
(2) NGS-based detection: quantification is achieved through sequencing tag counts with UMI correction, enabling expanded dynamic range and increased throughput.
Olink Data Analysis Workflow
1. Raw Data Processing
(1) Cq matrices (qPCR) or sequencing count matrices (NGS) are generated from instrument readouts.
(2) Technical artifacts, including blank wells and internal controls, are filtered to remove assay noise.
2. NPX (Normalized Protein eXpression) Calculation
(1) Olink’s standard normalization algorithm adjusts protein signals to correct for batch effects.
(2) Log2-scaled NPX values are obtained as relative protein abundance measures, enabling direct comparison across samples.
3. Downstream Analysis
(1) Differential analysis: between-group comparisons to identify candidate biomarkers.
(2) Pathway enrichment: functional network analysis using KEGG and Reactome.
(3) Multi-omics integration: joint integration with transcriptomic and metabolomic datasets.
Applications of Olink PEA Technology and Support from MtoZ Biolabs
1. Applicable Research Scenarios
(1) Cohort studies: high-plex protein profiling in large population-based cohorts.
(2) Drug development: biomarker discovery in pharmacodynamic, toxicological, and mechanistic studies.
(3) Clinical translation: validation of early diagnostic biomarkers.
2. Service Advantages of MtoZ Biolabs
(1) Comprehensive support for Olink Target, Explore, and Explore HT platforms.
(2) Integrated workflow support from experimental design and quality control to bioinformatic analysis.
(3) Delivery of publication-ready datasets suitable for direct use in manuscript preparation and project applications.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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