Mechanism of Proximity Extension Assay in Olink Analysis

Olink analysis is a highly sensitive protein detection platform with broad applications in biomarker discovery, clinical research, and drug development. One of its core technologies is the Proximity Extension Assay (PEA), which utilizes antibody recognition combined with DNA extension to detect hundreds of proteins in complex biological samples simultaneously.

 

Detailed Mechanism of Proximity Extension Assay

The core mechanism of Olink analysis—the Proximity Extension Assay—relies on DNA-encoded antibodies to recognize and quantify target proteins. Its uniqueness lies in the specific binding of antibodies to the target protein and the subsequent signal amplification via DNA extension, allowing for highly sensitive and specific protein detection.

 

1. Antibody Binding and Proximity Effect

The first step of PEA involves the binding of DNA-labeled antibodies to the target protein. Each protein target is recognized by two specific antibodies, each tagged with a distinct DNA probe. When these antibodies bind simultaneously to the target protein, the DNA probes come into close proximity due to spatial constraints.

 

2. DNA Probe Hybridization and Extension

Upon binding of the antibodies to the target protein, the DNA probes hybridize through complementary sequences. This proximity triggers the DNA polymerase-mediated extension reaction. The extension process significantly amplifies the signal while increasing detection specificity. Unlike conventional antibody-based methods, this dual-probe hybridization and extension reaction dramatically reduces background noise, improving detection accuracy.

 

3. qPCR Signal Detection and Quantification

After the proximity extension is completed, the newly formed DNA fragments are amplified, typically using quantitative PCR (qPCR). This amplification further boosts the previously generated hybridization signal, enabling accurate quantification of low-abundance proteins. Through the quantification of amplified DNA fragments, researchers can precisely determine the concentration of target proteins in the sample.

 

4. Advantages of Signal Amplification

The core advantage of PEA lies in its signal amplification mechanism. This not only allows for the detection of low-abundance proteins but also significantly enhances the specificity and sensitivity of the assay. Since the hybridization and extension reactions only occur when both antibodies bind to the target protein simultaneously, false positives due to single antibody binding or non-specific interactions are effectively minimized.

 

5. Multiplex Detection and Data Analysis

Another major feature of PEA is its multiplexing capability. In a single reaction, Olink's platform can simultaneously analyze hundreds of different protein targets. Each target's DNA sequence is unique, allowing for the independent detection of multiple proteins during the qPCR process. This multiplexing ability makes PEA particularly powerful in large-scale biomarker discovery studies.

 

The Proximity Extension Assay, as the core mechanism of the Olink platform, is revolutionizing proteomics research with its high sensitivity, specificity, and multiplex detection capabilities. This mechanism plays a pivotal role not only in the discovery of disease biomarkers but also in providing an effective method for detecting low-abundance proteins in complex samples.