Description

Diagnosis

Since Lyme disease is most effectively treatable in the early stages of the infection, the team intends to help solve this local problem by creating a diagnostic test that is fast and easy to use. This will allow individuals that have been bitten by ticks to seek treatment earlier.

Lyme disease of Lyme Borreliosis (LB) is caused by the infection of the spirochete Borrelia Burgdorferi, a spiral bacterium carried by Ixodes ticks.

Figure 1 - Digital scanning of the Borrelia Burgdorferi.

The Gap in Current Testing Methods

Current testing methods for Lyme disease can be time-consuming. These methods often rely on testing the patients' blood for biomarkers of the immune response to Borrelia Burgdorferi, which may be elicited between 2 and 30 days after the initial infection. Furthermore, tests such as ELISA and Western blotting techniques require samples to be processed in laboratories often not accessible to smaller cities.

Often when Borrelia Burgdorferi travels from the midgut of the tick to the host, it experiences a temperature change. This causes the bacteria to express a new set of lipoproteins which can vary greatly depending on its environment (1). As a result, it can be difficult for traditional protein-based detection methods to identify the bacteria.

Solution

Filling the Gap

The proteins expressed by Borrelia Burgdorferi are highly variable due to the temperature change between the tick and the host (1). However, when located in the lower temperature midgut of the tick, its surface proteins are constant. Thus, the team believes that testing the tick for Borrelia Burgdorferi will be more effective in addition to its universal compatibility between humans and animals.

Figure 2 - Basic schematic of the QGEM team's biosensor

Among these surface proteins, is the outer surface protein A (OspA) which is present in abundance and has been highly studied. The SUBLyme test offers fast and cost-effective testing by using an antibody fragment called an ScFv (single-chain variable fragment) that specifically targets the OspA protein. Attached to the ScFv is alkaline phosphatase, an enzyme that can take a novel substrate (BCIP) and convert it into a high quantity of fluorescent product (Oxidized NTB) which can be seen by the naked eye as a blue color. This way, a color change can be observed when even minuet quantities of OspA are present.

In our modular protein design, we have included a green fluorescent protein (GFP), which is attached to the alkaline phosphatase by a glycine linker and a TEV protease cut site. The GFP serves as an indicator of correct folding of the ScFv and alkaline phosphate components. If the biosensor has been correctly folded within the E.coli cells producing them, we will see green fluorescence within the cell, and we can then purify the protein, and cleave the GFP tag, making our protein ready for binding.

Although we are performing our proof-of-concept test using a bioreceptor specific for outer surface protein A, our team has created bioreceptors with the same modular design as outline above for many surface proteins present on Borrelia. This way, we can ensure our test is specific for detecting the presence of Borrelia as well as can distinguish between Lyme disease and relapsing fever, another disease caused by the Borrelia genus of bacteria.

References

1. Fingerle, V., Laux, H., Munderloh, U. G., Schulte-Spechtel, U., and Wilske, B. (2000) Differential expression of outer surface proteins A and C by individual Borrelia burgdorferi in different genospecies. Med. Microbiol. Immunol. 189, 59–66

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Team:Queens Canada/Description