Difference between revisions of "Team:Pasteur Paris/Immunology"
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First, infected mosquitoes are grinded in order to expose pathogen proteins. Then, mosquitoes proteins and pathogen proteins of the sample are bind to horse-radish peroxidase (HRP). The conjugated proteins are deposited on are the patch precoated with specific antibodies. The patch is then washed, pathogen proteins are retained by antibodies, and peroxidase activity is revealed using a specific substrat. </br></br> | First, infected mosquitoes are grinded in order to expose pathogen proteins. Then, mosquitoes proteins and pathogen proteins of the sample are bind to horse-radish peroxidase (HRP). The conjugated proteins are deposited on are the patch precoated with specific antibodies. The patch is then washed, pathogen proteins are retained by antibodies, and peroxidase activity is revealed using a specific substrat. </br></br> | ||
The principle of that method had to be checked before running experiment. In particular, specificity and sensitivity had to be evaluated. For those tests, we used purified viral proteins (E-protein from Yellow fever virus or E2 protein from chikungunya virus) and specific antibodies (4G2 for E-protein of Yellow fever virus and 3E4 antibody for E2 protein from chikungunya virus). </br></br> | The principle of that method had to be checked before running experiment. In particular, specificity and sensitivity had to be evaluated. For those tests, we used purified viral proteins (E-protein from Yellow fever virus or E2 protein from chikungunya virus) and specific antibodies (4G2 for E-protein of Yellow fever virus and 3E4 antibody for E2 protein from chikungunya virus). </br></br> | ||
| − | We first checked that we were able to bind viral protein with HRP, then that we could detect viral protein on a membrane, either purified or diluted in a Bovine Serum Albumine (BSA) solution in order to mimic mosquitoes proteins. Next step was to test the method when viral proteins were diluted with mosquitoes proteins. Finally, we tested the patches ability to detect viral pathogens from infected mosquitoes. For this last experiment, all steps involving infectious materials were performed by one of the coaches in a BSL3. </br></br></ | + | We first checked that we were able to bind viral protein with HRP, then that we could detect viral protein on a membrane, either purified or diluted in a Bovine Serum Albumine (BSA) solution in order to mimic mosquitoes proteins. Next step was to test the method when viral proteins were diluted with mosquitoes proteins. Finally, we tested the patches ability to detect viral pathogens from infected mosquitoes. For this last experiment, all steps involving infectious materials were performed by one of the coaches in a BSL3. </br></br> |
| + | </p> | ||
| + | </div> | ||
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<div class="text1"> | <div class="text1"> | ||
<p> | <p> | ||
| + | <h2>General protocol: </h2></br></br> | ||
| − | <U> | + | <U>Materials:</U></br> |
| − | + | ||
• PVDF or nitrocellulose membrane</br> | • PVDF or nitrocellulose membrane</br> | ||
• YFV E protein, CHIKV E2 protein</br> | • YFV E protein, CHIKV E2 protein</br> | ||
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• Milk 5% in PBS-Tween</br> | • Milk 5% in PBS-Tween</br> | ||
• EZ-link kit</br> | • EZ-link kit</br> | ||
| − | • Rocker agitator</br></br> | + | • Rocker agitator</br></br></br> |
| − | <U> | + | <U>Method:</U></br></br> |
<strong><U>Sample preparation:</U> </strong></br> | <strong><U>Sample preparation:</U> </strong></br> | ||
The sample is tagged using EZ-Link kit. </br></br> | The sample is tagged using EZ-Link kit. </br></br> | ||
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3. <strong>Binding:</strong> Add the sample in the PBS-Tween 0,5%-milk 5% solution at the appropriate dilution, incubate overnight at 4°C on a rocker. </br> </br> | 3. <strong>Binding:</strong> Add the sample in the PBS-Tween 0,5%-milk 5% solution at the appropriate dilution, incubate overnight at 4°C on a rocker. </br> </br> | ||
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> | + | 5. <strong>Revelation:</strong> Reveal the membrane using approximately 1ml of Pierce ECL Western Blotting substrate. </br> </br> </br> |
| − | + | </p> | |
| + | </div> | ||
| − | 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> | + | <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> | ||
| + | <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> | ||
• A: YFV E-protein + HRP EZ-link </br> | • A: YFV E-protein + HRP EZ-link </br> | ||
• B: YFV E-protein only </br> | • B: YFV E-protein only </br> | ||
