Team:Aberdeen Scotland/Project/Assay

From 2014.igem.org

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<img src="https://static.igem.org/mediawiki/2014/5/5c/Test_backpack.jpg" alt="Diagnostics Backpack">
<img src="https://static.igem.org/mediawiki/2014/5/5c/Test_backpack.jpg" alt="Diagnostics Backpack">
<p>With this diagram in mind we aim to propose a test that can be used to quickly decide 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 decide if someone is infected or not.</p>
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<p>Preliminary Protocol:</p>
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<h3>Preliminary Protocol:</h3>
<ol>
<ol>
<li>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.</li>
<li>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.</li>
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<li>Wash 3x PBS then add LiTat1.5-(R) culture, incubate for max ~1 hour, rocking.</li>
<li>Wash 3x PBS then add LiTat1.5-(R) culture, incubate for max ~1 hour, rocking.</li>
<li>Wash 3x PBS then add medium.</li>
<li>Wash 3x PBS then add medium.</li>
 +
<li>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.</li>
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</ol>
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<h3>Reagents:</h3>
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<ol>
 +
<li>0.9% w/v Phosphate-Buffered Saline, sterilised.</li>
 +
<li>3% Milk solution may be made from milk powder or from whole milk boiled & filtered in Phosphate-Buffered Saline.</li>
 +
<li>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!).</li>
 +
<li>E. coli cultures should be grown in sterile antibiotic medium [see recipes, NCIMB is an excellent source of advice for this]</li>
 +
<li>Sender cultures must be washed using sterile water or PBS immediately before use.</li>
</ol>
</ol>
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Revision as of 23:32, 14 August 2014

Team:Aberdeen Scotland/Project/Assay - 2014.ogem.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:

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:

  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 put on rocker for ~30mins.
  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, rocking.
  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, rocking.
  6. Wash 1x PBS.
  7. Add prewashed LiTat1.3-(S) culture [see reagents], incubate for ~1 hour, rocking.
  8. Wash 3x PBS then add LiTat1.5-(R) culture, incubate for max ~1 hour, rocking.
  9. Wash 3x PBS then add medium.
  10. 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:

  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.


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