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Project

Bioassay development towards ultrasensitive affinity-based detection of influenza A nucleoprotein

The field of microorganisms has always been of huge research interest because of their enormous impact on mankind. In this respect, early diagnosis followed by the correct treatment, is often crucial to increase the effect of the therapy and thus the chances of combating the disease. However, fast diagnostic tests are not always available or do not meet other important requirements, such as sensitivity, specificity, hands-on time, time-to-result and cost. Hence, intensive research is performed to fulfill this need, which is illustrated by the global in vitro diagnostics market that is expected to reach 75.1 billion US dollars by 2020.

While non-communicable illnesses are the current number one cause of death worldwide, infectious diseases also have remarkable imprint with, for instance, 13 million deaths registered in 2013. Among all pathogens causing infectious diseases, the influenza virus alone has a huge burden because it is responsible for the yearly flu epidemics, provoking high morbidity and even mortality. Timely diagnosis (at the point-of-care) is crucial for starting the correct therapy and reducing the effect of the infection. However, the current fast diagnostic tests for the detection of influenza do not meet the required needs to be highly sensitive, easy-to-use, cheap and robust. Therefore, in this dissertation, biorecognition elements against influenza A nucleoprotein, both aptamers and antibodies, were selected and thoroughly characterized for the implementation in novel bioanalytical tools with the aim to progress beyond the state-of-the-art in affinity-based testing of influenza. Affinity-based tests have been chosen here as they failed so far to compete with molecular diagnostics in terms of sensitivity, although they present a number of other attractive advantages, the most important being less complex and more affordable.

The first part of this work was focused on the selection of aptamers against influenza A nucleoprotein. Aptamers got their fame over the years as promising alternative for antibodies. A magnetic bead-based SELEX was used as a method accompanied with counter SELEX before every round, which enabled the removal of DNA sequences aspecifically interacting with the bead surface and selection of aptamers with low nanomolar affinity.

Subsequently, the potential of the EvalutionTM platform was explored for kinetic characterization of aptamer candidates in a high throughput fashion. Two unique properties of the system proved to be very valuable in this study: (1) barcoded microparticles, enabling multiplex analysis and (2) a fully automated microfluidic environment, which allows running up to 16 channels simultaneously. The platform was benchmarked with the well-established Biacore analytical system, revealing dissociation constants of the aptamers in the low nanomolar range.

As antibodies often remain the biorecognition molecules of choice, seven commercial antibodies against influenza A nucleoprotein were screened for their binding performance on two well-established platforms: ELISA and SPR. The study showed markedly different behavior of the antibodies under different assay conditions and between platforms. Two antibodies had overall good reactivity with the target on both platforms, although they did not exhibit the lowest dissociation constant. This stressed that the dissociation constant is not the only parameter to take into account when developing a bioassay.

The ability of the EvalutionTM platform to perform high throughput multiplex analysis makes it a valuable tool for straightforward bioassay development. Therefore, three antibodies were tested in a sandwich-based assay for their binding to nucleoprotein using EvalutionTM. This study demonstrated that the platform: (1) allows fast (up to 80 min) multiplex analysis of antibody performance, (2) reaches detection limits in the same range as a standard ELISA and (3) is compatible with testing in complex sample matrix. This showed the potential of the platform to be used as an alternative tool for bioassay development besides ELISA and SPR systems.

In the final part, one antibody pair was implemented on an in-house developed digital ELISA platform for the ultrasensitive detection of influenza A nucleoprotein in buffer and nasopharyngeal swabs. A detection limit of 4 ± 1 fM and 10 ± 2 fM was obtained, respectively, reaching the sensitivity of the currently available fast molecular tests. Hence, it can be concluded that digital ELISA has a huge potential for the development of extremely sensitive affinity-based diagnostic tests.

Date:1 Oct 2011 →  18 Apr 2017
Keywords:influenza virus, diagnostics, bioassay development, aptamers
Disciplines:Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering, Diagnostics, Laboratory medicine, Medicinal products
Project type:PhD project