Unveiling surface protein biomarkers in atopic dermatitis: Advancing with Immuno-qPCR analysis of extracellular vesicles

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers due to their presence in various biofluids and the potential to reflect the physiological and pathological state of their donor cells, and thereby the health status. Traditionally, research has so far focused on characterizing the DNA and RNA content of the EVs, whereas the surface protein composition has remained understudied. This study therefore aimed to develop a method for identifying and quantifying EV-specific membrane proteins by integrating an immunoassay with quantitative PCR (qPCR). This involved initially immobilizing either intact EVs, or lysed EV proteins onto magnetic beads, followed by the formation of immune complexes with antibody-DNA oligo conjugates. Subsequently, the DNA tag has been extended with a universal adapter, enabling sensitive qPCR quantification, with antibody-specific primers. We have successfully adapted the method to a 96-well plate format and demonstrated that it can be used to detect as little as 0.5 μg protein. Using the optimized protocol, we then analyzed serum samples from a patient cohort with atopic dermatitis (AD). Our results indicate that the within this master thesis established immuno-qPCR could be a suitable approach for biomarker discovery in this context. However, additional optimization is necessary to further develop the method into a reliable, robust approach that could be applied in clinical settings, for example in continuous health monitoring or disease progression tracking.