Compartmentmentalized immuno-sequencing (cI-Seq) : identification of immune complex interactions

Today, a lot of proteomic research is aimed at discovering disease specific proteins. This requires theavailability of high-throughput, ultra-sensitive protein detection methods. Compartmentalized immunosequencing(cI-Seq) is a proximity-independent immuno-polymerase chain reaction (IPCR) based proteindetection method. Antigen recognition in cI-Seq is mediated by antibody pairs in which one of theantibodies is conjugated to a DNA-probe. The affinity recognition events occur in emulsion droplets inwhich the DNA-probes will be amplified through emulsion PCR (emPCR) and thereafter analyzed usingMassively Parallel Sequencing (MPS). The amplifiable nature of the DNA-probes improves the sensitivityof the detection, while the use of emulsion droplets and MPS increases the multiplex capacity andthroughput. Ultimately, cI-Seq enables analysis and detection even of lowly abundant proteins therebyincreasing the probability of discovering novel disease specific proteins. In this project, conjugation of DNA probes to antibodies was performed through two different approaches;Covalent Conjugation and Conjugation using Biotin and NeutrAvidin. Both of these approaches showedadvantageous and disadvantageous features. However, neither of them succeeded in producing stableconjugates in an efficient and reproducible manner. After conjugation, the DNA-conjugated antibodieswere used in immune complex formation. However, the immune complexes either failed to form or wereformed in an inefficient manner