Team:Aberdeen Scotland/Project/Assay
From 2014.igem.org
Assay
Comparing our assay to current ones
A typical assay as employed in the field currently looks something like this:
What we aim to to with our assay is to reduce all to that the a small portable system. Meet the Diagnostics Backpack:
With this diagram in mind we aim to propose a test that can be used to quickly decide if someone is infected or not.
Preliminary Protocol:
- Blood samples are taken from patients suspected to be suffering from Human African Sleeping Sickness (Trypanosomiasis), a blood parasite derived from the bite of an infected Tsetse fly. Blood collected is mixed with untransformed E. coli, this removes non-specific binding. Put blood and non-mimotope E. coli in tube together put on rocker for ~30mins.
- Suspension is passed through 0.22μm PTFE filter to remove cells and non-specific antibodies.
- Serum antibodies are bound to a Poly-L-Lysine-coated surface (premade eppendorffs), and incubated for ~2hours, rocking.
- Remove unbound serum and wash 3x PBS
- The surface is blocked to remove regions of non-specific binding with 3% w/v (pre-boiled & filtered) milk solution or 3% milk powder, incubated for ~1hour, rocking.
- Wash 1x PBS.
- Add prewashed LiTat1.3-(S) culture [see reagents], incubate for ~1 hour, rocking.
- Wash 3x PBS then add LiTat1.5-(R) culture, incubate for max ~1 hour, rocking.
- Wash 3x PBS then add medium.