Novel assay formats for improved immunoassay sensitivity in a minaturised CD-microlaboratory
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
In the research and development of therapeutic proteins, accurate and high-performing methods for characterization, identification and understanding of these drugs is desired. GyrolabTM is a high- throughput platform, performing miniaturised immunoassays in a less time consuming way, with low reagent costs. Today Gyrolab offers a well-established method to perform a big variation of miniaturised immunoassays. The great advantage of Gyrolab is the high accuracy when measuring small volumes. Still, improved sensitivity in these assays would open up for a broader usage of Gyrolab, both within the area of immunoassays but also in other research areas where small quantities of molecules needs to be analysed.
The aim of this project was to investigate the ability to run assays with a novel design that differs from the standard method and hypothetically would generate a greater signal and improved sensitivity. A standard run for Gyrolab, is a sandwich-immunoassay in three steps: capture antibody- analyte-detection antibody. Two methods were to be tried, Tyramide Signal Amplification-system, which is an enzyme-mediated system where the detectable signal is generated via an enzymatic reaction. The second method was set up in a number of variants, with additional layers compared to the standard 3-step assay. This assay was built up in a reversed way, thus referred to as the “reversed system”.
None of the systems investigated showed any significant improvement in sensitivity, the main issue being an uncontrollable background noise that hid the specific signal. When the signal to noise ratio is not improved, the sensitivity cannot be any better, since it is not possible to distinguish the signal from the noise. The problem with the background noise has its root from the complexity of the chemistry in the assays, which we were unable to control.
Place, publisher, year, edition, pages
2013. , 37 p.
UPKEM E, 22
microlaboratory, immunoassay, antibodies, CD, optimization
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-221518OAI: oai:DiVA.org:uu-221518DiVA: diva2:709245
Master Programme in Chemistry
Mikael, Widersten, Professor