Difference between revisions of "Team:Pasteur Paris/Immunology"

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   <p>
 
   <p>
 
  <FONT size="3pt">
 
  <FONT size="3pt">
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  <a href=« #J1">August 4th, 2016</a></br>
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  <a href="#J1">August 4th, 2016</a>
 
</FONT>
 
</FONT>
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  <FONT size="3pt">
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                          <a href="#J2"><h2>August, 5th, 2016 </a>
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</FONT>
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  <FONT size="3pt">
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                          <a href="#J4"><h2>August, 17th, 2016 </a>
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</FONT>
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  <FONT size="3pt">
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                          <a href="#J5"><h2>August, 25th, 2016 </a>
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</FONT>
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  <FONT size="3pt">
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                          <a href="#J6"><h2>October, 14th, 2016 </a>
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</FONT>
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</p>
 
</p>
 
       </center>
 
       </center>
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<U>Method:</U></br></br>
 
<U>Method:</U></br></br>
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<strong><U>Sample preparation:</U> </strong></br>
+
<U>Sample preparation:</U></br>
 
The sample is tagged using EZ-Link kit. </br></br>
 
The sample is tagged using EZ-Link kit. </br></br>
 
1. Add 500mL of ultrapure water to the dry-blend Phosphate Buffered Saline (PBS).</br></br>
 
1. Add 500mL of ultrapure water to the dry-blend Phosphate Buffered Saline (PBS).</br></br>
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<i> &#8594; The conjugate can be store at 4°C for up to 4 weeks. </i></br> </br> </br>
 
<i> &#8594; The conjugate can be store at 4°C for up to 4 weeks. </i></br> </br> </br>
  
−
<strong><U>Membrane:</U> </strong></br>
+
<U>Membrane:</U></br>
 
1. <strong>Coating:</strong>  put 1 &#181;l  of a non-diluted antibody on the membrane, let the membrane dry for 5 minutes at room temperature. </br> </br>
 
1. <strong>Coating:</strong>  put 1 &#181;l  of a non-diluted antibody on the membrane, let the membrane dry for 5 minutes at room temperature. </br> </br>
 
2. <strong>Saturation:</strong> </br>
 
2. <strong>Saturation:</strong> </br>
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4. <strong>Washing:</strong> Wash the membrane 3 times for 5 minutes on a rocker at room temperature. </br> </br>
 
4. <strong>Washing:</strong> Wash the membrane 3 times for 5 minutes on a rocker at room temperature. </br> </br>
 
5. <strong>Revelation:</strong> Reveal the membrane using approximately 1ml of Pierce ECL Western Blotting substrate. </br> </br> </br>
 
5. <strong>Revelation:</strong> Reveal the membrane using approximately 1ml of Pierce ECL Western Blotting substrate. </br> </br> </br>
 +
 +
<center><img src = "https://static.igem.org/mediawiki/2016/2/26/IMMUNO1_Pasteur_Paris_2016.png" width= 35%  alt=""></center>
 
</p>
 
</p>
 
</div>
 
</div>
 +
 
<div class="text1">
 
<div class="text1">
−
<p>
+
<p>
−
<div id=« J1"><h2><B>August 4th, 2016 : </B></h2></div> </br></br></br>
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<div id="J1"><h2><B>August 4th, 2016 : </B></h2></div> </br></br></br>
 
<h3> <strong>EZ-link binding test: Proof of concept of the technic</strong> </h3>
 
<h3> <strong>EZ-link binding test: Proof of concept of the technic</strong> </h3>
 
<U>Aim:</U>To test the technic, we used our protocol and checked that we were able to detect pure viral protein bind to EZ-link. We used the previous protocol, on the membrane, we coated 4G2, BSA was used as negative control. On the membranes, we incubate the membrane with the following solution. Negative control are membrane A to D, to validate the experiment, membrane E has to be positive. </br> </br>
 
<U>Aim:</U>To test the technic, we used our protocol and checked that we were able to detect pure viral protein bind to EZ-link. We used the previous protocol, on the membrane, we coated 4G2, BSA was used as negative control. On the membranes, we incubate the membrane with the following solution. Negative control are membrane A to D, to validate the experiment, membrane E has to be positive. </br> </br>
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&bull; C: PBS + HRP EZ-link </br>
 
&bull; C: PBS + HRP EZ-link </br>
 
&bull; D: BSA+ HRP EZ-link </br>
 
&bull; D: BSA+ HRP EZ-link </br>
−
&bull; E: YFV E-protein + HRP EZ-link </br> </br>
+
&bull; E: YFV E-protein + HRP EZ-link </br> </br> </br> </br>
−
 
+
<U>Results:</U></br>
 +
<center><img src = "https://static.igem.org/mediawiki/2016/b/b3/Immuno2_Pasteur_Paris.png" width= 40%  alt=""></center></br></br>
 +
<center><img src = "https://static.igem.org/mediawiki/2016/c/cc/Capture_d%E2%80%99%C3%A9cran_2016-10-17_%C3%A0_21.28.58.png" width= 40%  alt=""></center>
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</p>
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</div>
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        <div class="text1">
 +
                <p>
 +
Conclusion: The principe of the technic is validated.
  
 
</p>
 
</p>
 
</div>
 
</div>
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 +
 +
<div id="J2"><h2><B>August, 5th, 2016 : </B></h2></div> </br></br></br>
 +
</div>
 +
 +
<div class="text1">
 +
<p>
 +
<U>Aim:</U> Sensitivity of the detection.</br></br>
 +
To evaluate the sensitivity, we proceed as described before, and we used YFV E protein pure or serially diluted into PBS + BSA 1% to mimic the presence of mosquito proteins in the sample. 3 dilutions were used : 1mg/ml, 10-4mg/ml, 10-8mg/ml.</br></br>
 +
<U>Results:</U> No signal was visible.</br></br>
 +
</p>
 +
</div>
 +
 +
 +
<div id="J3"><h2><B>August, 9th </B></h2></div> </br></br></br>
 +
</div>
 +
 +
<div class="text1">
 +
<p>
 +
<U>Aim:</U> Sensitivity of the detection.</br>
 +
The previous experiment was repeated, we made new reagents and used nitrocellulose membrane in addition to PVDF membrane. </br></br> 
 +
<U>Results:</U> No signal was visible.</br></br>
 +
</p>
 +
</div>
 +
 +
<div id="J4"><h2><B>August, 17th: </B></h2></div> </br></br></br>
 +
</div>
 +
 +
<div class="text1">
 +
<p>
 +
<U>Aim:</U> Sensitivity of the detection</br>
 +
The previous experiment was repeated, we used a new antibody (anti-chik 3E4) together with CHIKV E2 envelope protein. </br></br>
 +
<U>Results:</U> </br></br>
 +
</p>
 +
</div>
 +
 +
 +
<div id="J5"><h2><B>August, 25th: </B></h2></div> </br></br></br>
 +
</div>
 +
 +
<div class="text1">
 +
<p>
 +
<U>Aim:</U> Sensitivity of the detection with mosquito protein.</br>
 +
100 mosquitoes were grinded and CHIKV E2 envelope proteins were diluted into in order to have approximately the same amount of viral proein we would find into one infected mosquito alone and in one infected mosquito together with 100 non-infected mosquitoes in order to mimic the fact that not all mosquitoes trapped will be infected with a pathogen. </br></br>
 +
<U>Results:</U> .</br>.</br>
 +
</p>
 +
</div>
 +
 +
<div id="J6"><h2><B>October, 14th </B></h2></div> </br></br></br>
 +
</div>
 +
 +
<div class="text1">
 +
<p>
 +
<U>Aim:</U> Proof of concept.</br>
 +
In this experiment, we used 8 prototype patches to check their ability to detect pathogens in mosquitoes.
 +
4 patches were used to repeat the previous experiments in real conditions. The 4 patches remaining were used to test infected mosquitoes. Aedes  aegypti were experimentally infected with the vaccinal strain of Yellow fever virus. 14 days after infection, they were grinded in 1,5ml tubes. Tubes were centrifuged and supernatant were inactivated by placing the tubes under a UV lamp for 30 minutes. All steps using infectious materials were performed by a coach in a BSL3. .</br></br>
 +
Samples were then tested on the patch using the same protocol as decribed previously with the patch as a solid support for antibody (in remplacement of membranes). </br></br>
 +
<U>Results:</U></br></br>
 +
Patches are able to detect high amount of viral proteins, the sensitivity seems to be improved for detecting pathogens at a lower level.
 +
</br></br>
 +
</p>
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</div>
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Revision as of 19:52, 17 October 2016