Browsing by Author "Drikic, Marija"
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- ItemOpen AccessDetecting total immunoglobulins in diverse animal species with a novel split enzymatic assay(2019-10-28) Drikic, Marija; Olsen, Steven; De Buck, JeroenAbstract Background Total immunolobulin G concentration is a useful, albeit underutilized, diagnostic parameter for health assessments of non-domestic animal species, due to a lack of functional diagnostic tools. Traditional assays, including enzyme-linked immunosorbent assay or radial immunodiffusion, require development of specific reagents (e.g., polyclonal antisera and appropriate protocols) for each animal species, precluding wide and easy adoption in wildlife welfare. As an alternative, bacterial virulence factors able to bind IgGs in antigen-independent manner can be used. To further simplify the diagnostic procedure and increase the number of species recognized by an assay, in this study a recently developed Split Trehalase immunoglobulin assay (STIGA) with bIBPs as a sensing elements was used to detect antibodies in 29 species from 9 orders. Three bacterial immunoglobulin binding proteins (protein G, protein A and protein L) were incorporated into STIGA reagents to increase the number of species recognized. Results IgG concentrations were detected through glucose production and produced signals were categorized in 4 categories, from not active to strong signal. Activation was detected in almost all tested animal species, apart from birds. Incorporation of Protein G, Protein A and Protein L allowed detection of IgGs in 62, 15.5 and 6.9% of species with a strong signal, respectively. Assays combining 2 bacterial immunoglobulin binding proteins as sensing element generally gave poorer performance than assays with the same bacterial immunoglobulin binding proteins fused to both trehalase fragments. Conclusions STIGA assays have potential to be further developed into an easily adoptable diagnostic test for total amount of IgGs in almost any serum sample, independent of species.
- ItemOpen AccessNovel split trehalase-based biosensors for the detection of biomarkers of infectious diseases(2019-05-15) Drikic, Marija; De Buck, Jeroen M.; Gilleard, John Stuart; Dong, Tao G.; Turner, Raymond Joseph; Savchenko, Alexei; Campbell, Robert D.Infectious diseases remain a serious public health challenge in the field of human and veterinary medicine. For instance, infectious diseases were among the leading causes of mortality worldwide in 2016 according to the WHO. In veterinary medicine, infectious diseases have a significant negative impact on animal productivity and welfare leading to decreased profits and increased health-associated costs. Furthermore, the WHO estimates that 61% of diseases affecting the human population are zoonotic. Therefore, programs to control and eradicate these diseases are of crucial importance. Correct diagnosis, supported by robust diagnostic tests, is an essential first step in achieving these goals. An ideal diagnostic test needs to be accurate and robust while being easily accessible and widely-available. Although different studies over the years have aimed to develop such a diagnostic test, there have not been many that have managed to enter into clinical practice. The most successful class of biosensors currently on the market is the glucometer used to monitor blood glucose. Glucometer's sensitivity and specificity, as well as its performance in clinical samples, have been mainly perfected. Measuring devices have been miniaturized and their production costs optimized. In this thesis, we developed and characterized a novel biosensor able to detect antibodies and other infectious disease markers. The biosensor is based on the protein complementation principle and uses an E. coli glycolytic enzyme, trehalase (TreA), as a reporter. Trehalase converts trehalose to glucose, which can then be detected by a glucometer. TreA was split into two non-functional fragments, and each fragment was fused to the sensing element that was specific for the targeted analyte. In the presence of the analyte, the sensing elements bind to it and induce the dimerization of the TreA which then becomes active. In the second chapter, we demonstrated that conditional complementation of the trehalase fragments leading to trehalose hydrolysis and glucose production could be used to detect antibodies, bacterial cells, small molecule, protein-protein interaction, and protein aggregation. We also demonstrated the retention of activity of split TreA in undiluted clinical samples like blood or milk. In the third chapter, we attempted to increase the sensitivity and shorten the time for signal detection of the TreA biosensor by introducing split inteins. We placed the inteins within the previously developed biosensor, but we were not successful in achieving analyte-mediated dimerization of the two biosensor components. In the fourth chapter, we applied this TreA biosensor to develop and validate a new diagnostic tool for the quantification of the total amount of immunoglobulins in bovine colostrum and serum (named STIGA). We demonstrated that STIGA performs more efficiently in quantifying total immunoglobulins in colostrum and calf serum than other diagnostic tools (Colostrometer and Brix) that are used on a farm. Therefore, it is a suitable detection assay to establish colostrum quality and calf immune status in the field. We also proposed a modified format of STIGA for this on-farm application and demonstrated that its performance remained high. In the fifth chapter, we explored the applicability of a modified STIGA with the anticipation to detect immunoglobulins in different animal species. We analyzed 29 animal species and proved that the same detection protocol could be applied with success to the majority of animal species. In conclusion, we engineered a trehalase-based biosensor that requires minimal sample preparation and can be integrated with existing glucometers or sensors, which offers a versatile and convenient method for point-of-care applications.