Proximity Extension Assay for Pharmacodynamics and Biomarker-Profiling in Clinical Studies

Soluble inflammatory mediators are crucial in regulating immune responses and can significantly influence the progression of many diseases. Traditional cytokine analysis methods, which rely on single-plex assays, limit our immunological and clinical understanding. In contrast, cytokine multiplexing allows for the simultaneous measurement of several cytokines in a small sample volume, facilitating a more comprehensive understanding of the immunological and biological processes involved in drug action and disease progression.

While advantageous, cytokine multiplexing faces several challenges. Cross-reactivity between antibodies can lead to inaccurate results, and matrix effects from the sample can interfere with the assay’s performance. Additionally, the complexity of analyzing multiple calibration curves and maintaining stringent quality controls can be labor-intensive and time-consuming. The high cost of multiplexing kits and equipment can also be a significant barrier for widespread diagnostic use and pharmacodynamic assessments.

In our laboratory, we have established the Proximity Extension Assay (PEA) technology, which enables the simultaneous analysis of 92 or 45 inflammatory mediators from just 1µL of biological sample, such as plasma, serum, cell culture supernatant, or even precious materials like spinal fluid. PEA employs matched pairs of antibodies, each labeled with DNA oligonucleotides. When both antibodies bind to the same analyte, the oligonucleotides come into proximity, hybridize, and are extended by PCR. The resulting DNA barcodes are quantified by microfluidic qPCR, allowing for the determination of relative or absolute protein expression. This dual antibody recognition, supplemented with DNA hybridization, is designed to increase specificity and sensitivity.

We compared the well-established MesoScale multiplexing immunoassays with PEA technology using untreated blood samples and samples stimulated to express high or low cytokine levels. The expression levels of Interleukin (IL)-6, IL-8, and TNF-α were found to be very similar across both technologies and all donors analyzed. However, PEA demonstrated a broader dynamic range compared to MesoScale multiplexing.
Initial validation results of the PEA using the 92-analyte platform illustrate reasonable intra- and inter-assay variance. In this presentation, we will explain the PEA technology, share our initial validation experiences, including sample dilution results, and highlight potential drawbacks and experimental efforts associated with this relatively new technology. Finally, we will discuss its suitability for pharmacodynamics assessment in clinical studies.