Comparing our assay to current ones

The two main problems in the treatment of neglected tropical diseases are the bulk of the equipment required and the need for a ‘cold chain’. The cold chain is the transport chain of drugs and other equipment that needs refrigeration from the manufacturer to the patient. This can include a large number of links with some being heavily restricted by time. If these are allowed to warm up they can lose effectiveness. This need for cooling also extends to storage on-site and often requires large fridges and freezers to be transported to facilitate this storage. Our goal was to eliminate the need for a cold chain as well as eliminating the need for other expensive specialist and bulky equipment.

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.

With this diagram in mind we aim to propose a test that can be used to quickly determine if someone is infected or not. Although other tests such as lateral flow cytometry are also used to detect diseases, with one for HAT currently in development, we feel that our system has some advantages:
It relies on materials often available on site, such as milk for blocking the poly-lysine coated cuvettes or eppendorfs.
E.coli naturally grows faster at temperatures up to 37⁰C meaning effectiveness can actually be improved in warm climates.
As the system is contained within bacteria we have considered the possibility of actually being able to grow more of the diagnosis ‘tool’ on site, providing assay reagents indefinitely and acting as a small, continuous factory.

Our Suggested 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 'RECEIVER', 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.
    
    ASSAY ROBUSTNESS: 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.
    
    PLATFORM TECHNOLOGY: 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.