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 determine if someone is infected or not.

Preliminary Protocol:

1. 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 and mix well.


2. Suspension is passed through 0.22μm PTFE filter to remove cells and non-specific antibodies.


3. Serum antibodies are bound to a Poly-L-Lysine-coated surface (premade eppendorffs), and incubated for ~2hours, mixing occasionally.


4. Remove unbound serum and wash 3x PBS


5. 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, mixing occasionally.


6. Wash 1x PBS.


7. Add prewashed E.coli expressing MIMOTOPE 1 and QUORUM-SENSING 'SENDER' culture [see reagents], incubate for ~1 hour, mixing occasionally.


8. Wash 3x with PBS then add E.coli transformants expressing MIMOTOPE 2 and QUORUM-SENSING 'SENDER', incubate for max ~1 hour, mixing occasionally.


9. Wash 3x with PBS then add medium.


10. Incubate at room temperature or a maximum of 37˚C monitoring for 2-7hours.
    OUTPUT:A green fluorescent response under UV indicates a positive disease diagnosis because antibodies to Mimotope 1 AND Mimotope 2 have been detected by the surface display mimotope cultures.
    Quorum sensing 'Sender' and 'Receiver' act as an AND logic gate to ensure a GFP signal is only produced when antibodies to both Mimotopes are present. This decreases the false-discovery rate of the assay, making it more reliable in the field.
    Importantly, the approach represents a platform technology, since this general approach may be repeated by combining cultures expressing different mimotopes, thus multiple disease phenotypes may be testing simultaneously.

Reagents:

1. 0.9% w/v Phosphate-Buffered Saline, sterilised.
2. 3% Milk solution may be made from milk powder or from whole milk boiled & filtered in Phosphate-Buffered Saline.
3. Eppendorffs are internally coated using 0.01% Poly-L-lysine, incubated at room temperature for 2hours, then washed with sterile water 2-3x. When to be used, the water is removed and the poly-L-lysine allowed to dry (don’t bash!).
4. E. coli cultures should be grown in sterile antibiotic medium [see recipes, NCIMB is an excellent source of advice for this]
5. Sender cultures must be washed using sterile water or PBS immediately before use.