Team:Aberdeen Scotland/Project/Assay
From 2014.igem.org
(Difference between revisions)
Line 69: | Line 69: | ||
<div class="main_content"> | <div class="main_content"> | ||
<p>A typical assay as employed in the field currently looks something like this:</p> | <p>A typical assay as employed in the field currently looks something like this:</p> | ||
- | <img src="https://static.igem.org/mediawiki/ | + | <img src="https://static.igem.org/mediawiki/parts/8/80/ASSAY_1.jpg"><br> |
<p>What we aim to to with our assay is to reduce all to that the a small portable system. Meet the Diagnostics Backpack:</p> | <p>What we aim to to with our assay is to reduce all to that the a small portable system. Meet the Diagnostics Backpack:</p> | ||
- | <img src="https://static.igem.org/mediawiki/ | + | <img src="https://static.igem.org/mediawiki/parts/0/0b/ASSAY_2.jpg" alt="Diagnostics Backpack"> |
<p>With this diagram in mind we aim to propose a test that can be used to quickly determine if someone is infected or not.</p> | <p>With this diagram in mind we aim to propose a test that can be used to quickly determine if someone is infected or not.</p> | ||
<h3>Preliminary Protocol:</h3> | <h3>Preliminary Protocol:</h3> |
Revision as of 23:12, 17 October 2014
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 determine 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 and mix well.
- 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, mixing occasionally.
- 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, mixing occasionally.
- Wash 1x PBS.
- Add prewashed LiTat1.3-(S) culture [see reagents], incubate for ~1 hour, mixing occasionally.
- Wash 3x PBS then add LiTat1.5-(R) culture, incubate for max ~1 hour, mixing occasionally.
- Wash 3x PBS then add medium.
- Incubate at room temperature or a maximum of 37˚C monitoring for 2-7hours. A green fluorescent response under UV indicates a positive disease diagnosis. This may be repeated by combining cultures expressing different mimotopes, multiple disease phenotypes may be testing simultaneously.
Reagents:
- 0.9% w/v Phosphate-Buffered Saline, sterilised.
- 3% Milk solution may be made from milk powder or from whole milk boiled & filtered in Phosphate-Buffered Saline.
- 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!).
- E. coli cultures should be grown in sterile antibiotic medium [see recipes, NCIMB is an excellent source of advice for this]
- Sender cultures must be washed using sterile water or PBS immediately before use.