Quantitative protein detection by circular proximity ligation assay [electronic resource]
Keywords: Chemical Engineering.

Quantitative detection of proteins is essential for diagnosis, monitoring, and personalized treatment of diseases. In a clinical context, it is required to have an assay with high sensitivity (limit of detection below pico-molar concentration), high specificity (low false positive), low sample consumption (micro-liter size) and multiplexing capabilities (> 10). Enzyme-linked immunosorbent assay (ELISA) has mostly served as the clinical 'gold standard' for targeted quantification of single proteins; however, it suffers from poor sensitivity, requires a large sample volume and cannot be multiplexed. To overcome the limitations of ELISA, several approaches have been developed and some of them rely on combining immunoassays with nucleic acid-based amplification. One particularly promising approach within this domain is the proximity ligation assay (PLA). PLA is one of the most sensitive antibody-based methods to quantitatively detect proteins in complex mixtures, where two antibodies conjugated with oligonucleotides (affinity probes) are brought into close proximity in the presence of target antigen to generate a new DNA molecule via a ligation event, which is subsequently amplified and quantified. This thesis describes the development of, circular proximity ligation assay (c-PLA), which enhances specificity and sensitivity compared to traditional PLA, and simplifies the workflow. In contrast to traditional PLA, the affinity probes are employed as bridges enabling the connection of two free oligonucleotides via dual ligation events resulting in the formation of a circular DNA molecule. The addition of an extra oligonucleotide increases specificity and circle formation exhibits selective advantages, as uncircularized DNA can be removed enzymatically. Ligation products are quantified by real time q-PCR. For c-PLA assay development, a synthetic oligo system was used to determine the optimal concentration of each assay component. After assay development, c-PLA was applied to several biomarkers and compared with traditional PLA. c-PLA demonstrated higher specificity and improved performance (limit of detection ranging from low femto-molar to nano-molar). Subsequently, kinetic analysis using surface plasmon resonance and biolayer interferometry showed that the variation in limit of detection is directly dependent on affinity of the antibodies used. The knowledge from affinity analysis can be used as a screening tool to find suitable antibodies prior to conjugating them with oligonucleotides. Furthermore, it can be utilized to optimize antibodies' concentrations for enhancing the assay performance across a wide range of biomarkers. Modeling of c-PLA at equilibrium was performed and the result indicated that the enhanced specificity of c-PLA allows the affinity probe concentration to be increased while maintaining a high signal-to-noise ratio, thereby ensuring assay compatibility for low affinity reagents. This is a major advantage compared to traditional PLA, especially for targets where high affinity probes are not available, allowing not only new analytes to be detected but also enabling greater access to sensitive protein detection assays. In addition, circle formation opens up possibilities for new detection schemes including rolling circle amplification, direct fluorescent readout and DNA sequencing. Circular PLA with its versatility and performance will be the method of choice because of its usefulness in a variety of clinical applications.