Proximity Extension Assay (PEA) is a technique in molecular biology, particularly within proteomics, for detecting and quantifying proteins. It allows for highly sensitive and specific measurement of multiple proteins simultaneously from small biological samples, aiding research into their roles in health and disease.
How Proximity Extension Assay Works
Proximity Extension Assay begins with the interaction of specific antibodies with target proteins in a biological sample. Each antibody is tagged with a short DNA oligonucleotide. These antibody-oligonucleotide complexes are introduced to the sample, where they bind to their corresponding protein targets.
Once two antibody-oligonucleotide complexes bind to the same protein, or to different proteins that are very close to each other, their attached DNA oligonucleotides are brought into close proximity. This arrangement allows the DNA oligos to hybridize. A DNA polymerase enzyme then extends one of these hybridized oligonucleotides, using the other as a template, to form a new, unique DNA sequence.
This newly formed DNA sequence acts as a unique template, indicating the presence of the original target protein. The amount of this newly synthesized DNA is directly proportional to the initial concentration of the protein in the sample. This DNA template is then amplified using quantitative Polymerase Chain Reaction (qPCR), a technique that makes many copies of DNA.
The amplification step increases the signal. The amplified DNA is subsequently detected and quantified, typically through real-time PCR or next-generation sequencing. This final measurement provides quantification of the proteins originally present in the biological sample.
Key Advantages of Proximity Extension Assay
Proximity Extension Assay offers high sensitivity, enabling the detection of proteins even at very low concentrations, often in the femtomolar (fM) range. This capability is useful for identifying biomarkers present in minute quantities, which can be important for early disease detection.
The technique boasts significant multiplexing capability, meaning it can simultaneously measure hundreds of different proteins from a single, small sample. This reduces the amount of biological material needed and saves time compared to analyzing each protein individually. Some platforms can analyze up to 1161 human proteins in a single microliter of plasma.
PEA also provides high specificity due to its unique two-antibody binding requirement and the subsequent proximity-dependent ligation step. For a signal to be generated, two different DNA-tagged antibodies must bind to the target protein in close proximity. This dual recognition mechanism significantly reduces the chances of non-specific binding and false positive signals.
PEA requires only minute amounts of biological samples, such as a few microliters of plasma or serum, making it suitable for situations where sample volume is limited. This is advantageous when working with precious samples, such as cerebrospinal fluid, dried blood spots, or tumor biopsies.
Applications of Proximity Extension Assay
Proximity Extension Assay is used in biomarker discovery and validation, where its sensitivity and multiplexing capabilities help identify and confirm protein biomarkers associated with various diseases. Researchers use PEA to find protein patterns linked to conditions like cancer, cardiovascular disorders, autoimmune diseases, and neurodegenerative disorders.
In drug development, PEA is used to monitor drug efficacy, identify potential drug targets, and understand how drugs interact with biological systems. By measuring changes in protein levels in response to drug treatment, scientists can assess a drug’s impact. The method can also be used to measure binding of small drug molecules to specific proteins, aiding in target engagement studies.
PEA contributes to disease monitoring and prognosis by quantifying specific protein panels over time, which can track disease progression or predict patient outcomes. For instance, it has been adapted to detect anti-SARS-CoV-2 antibodies in blood samples, providing a sensitive and accurate method for community surveillance of virus exposure.
The technique is also valuable in basic biological research for studying complex biological pathways, protein-protein interactions, and cellular responses. PEA can reveal how proteins interact within cells and how those interactions change under different conditions, providing insights into biological processes.
Proximity Extension Assay Compared to Other Methods
Enzyme-Linked Immunosorbent Assay (ELISA) is widely used for detecting single proteins but typically lacks the multiplexing capacity of PEA. ELISA usually measures one protein at a time in each well.
Western Blot, a common laboratory technique, is primarily used for qualitative or semi-quantitative analysis of one or a few proteins. It separates proteins by size, providing information about molecular weight and modifications, but it is less sensitive and quantitative than PEA and not designed for high-throughput multiplexing. Western blotting is also more labor-intensive and time-consuming, often taking 1-2 days to complete.
PEA stands apart by combining the specificity of antibody-based immunoassays with the sensitivity and scalability of DNA amplification. This integration allows for the simultaneous detection of numerous proteins with high accuracy and low sample volume, a combination not readily achieved by ELISA or Western Blot. The DNA amplification step in PEA provides a significant signal boost, enabling detection of proteins at concentrations that might be challenging for other methods.