Development of dual capture assays for antibody suspension bead arrays
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Utveckling av dubbel antikropps suspensions array (Swedish)
Plasma and serum are believed to at some point in time contain all proteins of the human body, possibly containing information about every disease state. This makes plasma and serum widely utilized for biomarker discovery. Biomarker discovery is at the momen performed either by identifying proteins from the protein mass (i.e. amino acid sequence) or with affinity reagents or by employing a combination of these two. In the group of Biobank Profiling, Royal Institute of Technology, KTH, a Standard Assay (STA) for affinity based biomarker discovery with Suspension Bead Array has been applied. This STA is based on direct labelling of plasma samples, a method with potentially intrinsic challengens in distinguishing the target-protein signal from noise and background of off-target binding. To circumvent or reduce these problems, an idea based on a capture-recapture of the target protein, entitled Dual Capture Assay (DCA). This concept could enrich the arget protein and reduce the complexity of the sample for the second binding event. In thsi master thesis project, the DCA concept has been investigated in the aspects of elution, incubation times as the reported Median Fluorescent Intensity (MFI). When evaluating DCS, a system mimicking a biomarker discovery setting has been applied by spike-in of a non-human protein into human plasma. These experiments show that DCA create a less complex environment for the second capture, implied by a higher Signal to Noise ratio than for the STA. DCA was then applied in a disease study on Duchenne Muscular Dystrophy samples. In this application, the STA reports higher MFI signals than the DCA but the both assays show the same trend in separation of cases vs. controls, supporting previously achieved data. DCA also shows a reduction to background signals for 7 out of 10 positive controls in human blood while the STA yields high signals for all 10. Together with the other datapresented in this thesis, DCA appears to be a possible approach to complement STA because of the higher Signal to Noise ratios. With further optimizing and efforts in providing more rigid proof of the concept, DCA could probably be implemented in the standard work flow of this group, providing more reliable data due to the capture- recapture of the target protein.
Place, publisher, year, edition, pages
Assay development, microarrays, antibodies, bead arrays, plasma analysis, specificity
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-150485OAI: oai:DiVA.org:kth-150485DiVA: diva2:744718