Difference between revisions of "Team:Freiburg/Results"

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<h2>Detection of anti-Tetanus antibodies in human blood serum</h2>
 
<h2>Detection of anti-Tetanus antibodies in human blood serum</h2>
 
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png"></img>
 
<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png"></img>
 
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             <strong>Video 1: Detection of anti-tetanus antibodies in human serum.</strong> The left part shows the slide being flushed by the sample taken after vaccination, the right part shows the negative control sample taken before vaccination. The lowest spot in each part contains the tetanus antigen.
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             <strong>Video 1: Detection of anti-tetanus antibodies in human serum.</strong>
 
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<strong>We tested our DiaCHIP with blood serum to determine the tetanus vaccination (proof of tetanus antibodies)...</strong>
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<strong>We specifically detected anti-Tetanus antibodies in human blood serum.</strong>
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To test the DiaCHIP under real-life conditions, we wanted to analyze the blood serum of a vaccinated person for the presence of anti-Tetanus antibodies. Blood from the same person was taken before vaccination and served as negative control. The <a href="http://parts.igem.org/Part:BBa_K1621003">corresponding antigen</a> was expressed in <i>E. coli</i>, purified by His tag based affinity purification and spotted on a specific Ni-NTA surface.
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To test the DiaCHIP under real-life conditions, we analyzed the blood serum of a vaccinated person for the presence of anti-Tetanus antibodies. We compared blood samples before and after vaccination and could directly detect its effect. To capture the antibodies, the <a href="http://parts.igem.org/Part:BBa_K1621003">corresponding antigen</a> was expressed in <i>E. coli</i>, purified by His-tag affinity purification and spotted on a specific Ni-NTA surface.  
 
 
 
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<div class="kommentar"> hier noch rein bringen, dass ihr euren eigenen cell-free mix hergestellt habt und mit diesem auch GFP expremieren könnt und es im array erfolgreich nachweisen könnt</div>
 
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<h2>
 
Detection of anti-GFP antibodies in rabbit serum using cell-free expressed GFP
 
Detection of anti-GFP antibodies in rabbit serum using cell-free expressed GFP
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png"></img>
 
<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png"></img>
 
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    <strong>Figure 1: Binding of anti-GFP antibodies to cell-free expressed GFP.</strong> Cell-free expressed GFP was spotted on a specific Ni-NTA slide and flushed with serum of a rabbit immunized against GFP. The lowest spot consisting of cell-free expressed GFP shows a significant signal compared to the negative control (middle spot).
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    <strong>Figure 1: Binding of anti-GFP antibodies to cell-free expressed GFP.</strong>
 
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<div class="kommentar"> hier noch rein bringen, dass ihr euren eigenen cell-free mix hergestellt habt und mit diesem auch GFP expremieren könnt und es im array erfolgreich nachweisen könnt</div>
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To show that cell-free expressed proteins can be immobilized on the protein surface while maintaining their antibody binding properties, the expression mix was spotted on a specific Ni-NTA surface after GFP expression. Cell-free expressed GFP was spotted on a specific Ni-NTA slide and flushed with serum of a rabbit immunized against GFP. The lowest spot consisting of cell-free expressed GFP shows a significant signal compared to the negative control (middle spot).
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To show that cell-free expressed proteins can be immobilized on the protein surface while maintaining their antibody binding properties, the expression mix was spotted on a specific Ni-NTA surface after GFP expression.
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Serum of a rabbit immunized with GFP was applied to the DiaCHIP, which resulted in a strong signal at the referring spot evoked by antibody binding events (figure 1). Thus, His-tagged GFP was successfully expressed and immobilized on the surface, still enabling anti-GFP antibodies in a serum sample to bind.
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Find out more about the <a href="https://2015.igem.org/Team:Freiburg/Results/Assembling">preparation of the DiaCHIP</a> by producing a protein microarray from a DNA template using cell-free expression.  
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<div class="accordion">
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<div class="accordion-section">
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<a class="accordion-section-title" href="#accordion-1">Detailed description</a>
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<p>
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Serum of a rabbit immunized with GFP was applied to the DiaCHIP, which resulted in a strong signal at the referring spot evoked by antibody binding events (figure 1). Thus, His-tagged GFP was successfully expressed and immobilized on the surface, still enabling anti-GFP antibodies in a serum sample to bind.
 +
<br>
 +
Find out more about the <a href="https://2015.igem.org/Team:Freiburg/Results/Assembling">preparation of the DiaCHIP</a> by producing a protein microarray from a DNA template using cell-free expression.
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</p>
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<a href="https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Own_Device_Foto.jpg">
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<a href="https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Own_Device_Foto.jpg">
 
<img src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_Own_Device_Foto_preview.jpg"></img>
 
<img src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_Own_Device_Foto_preview.jpg"></img>
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<strong>Figure 2: Functional, but low-cost variant of the measuring device.</strong> Our self-built device consistent of not much more than two lenses and a camera (A) reliably detected the binding of anti-GFP to GFP (B).
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<strong>Figure 2: Functional, but low-cost variant of the measuring device.</strong>  
 
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The measuring device we used in collaboration with the AG Roth (ZBSA) to detect antibody binding events is a really expensive machine, but the physics which it is based on is rather simple. To enable future iGEM teams to profit from this label-free detection method as we did, a cost-efficient and easy to rebuild variant of the device was developed (figure 2A). Figure 2B shows the binding of anti-GFP to GFP as it was established before, measured with our own device.  
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The measuring device we used in collaboration with the AG Roth (ZBSA) to detect antibody binding events is a really expensive machine, but the physics which it is based on is rather simple. Our self-built device consistent of not much more than two lenses and a camera (A) reliably detected the binding of anti-GFP to GFP (B). To enable future iGEM teams to profit from this label-free detection method as we did, a cost-efficient and easy to rebuild variant of the device was developed (figure 2A).  
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</p>
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Look here, to see how you can <a href="https://2015.igem.org/Team:Freiburg/Results/Own_Device">build your own device</a> for label-free detection of antibody binding on a protein microarray needing two lenses, a camera and a little bit of fine feeling (??) sensitivety/sensitiveness/finesse/sure instincts.  
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<div class="accordion">
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<div class="accordion-section">
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<a class="accordion-section-title" href="#accordion-1">Detailed description</a>
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<div id="accordion-1" class="accordion-section-content" style="display:none; padding:15px;">
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<p>
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Figure 2B shows the binding of anti-GFP to GFP as it was established before, measured with our own device.  
 +
Look here, to see how you can <a href="https://2015.igem.org/Team:Freiburg/Results/Own_Device">build your own device</a> for label-free detection of antibody binding on a protein microarray needing two lenses, a camera and a little bit of fine feeling (??) sensitivety/sensitiveness/finesse/sure instincts.  
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Revision as of 15:45, 12 September 2015

""

Ramona: Bilder werden noch ersetzt; Text wartet auf Kommentare!
Nicole: Hab ich es richtig verstanden, dass hier nur eure Highlights and Ergebnissen dargestellt werden sollen (mit verlinkung zu den anderen Ergebnissen)? Was haltet ihr von einer kurzen Einleitung... In the last months we (aimed to) developed a diagnostic tool which enables [...] We succeded to detect anti-tetanus antibodies in human serum, develop/generate our own cell-free mix...
mehr Formulierungen wie: we demonstrated..., we (successfully) tested, we achieved...
In den text eine bessere Reihenfolge rein bringen. Es wird vom blut gesprochen, dann Expression in E.coli und dann wieder Blut.

Detection of anti-Tetanus antibodies in human blood serum

Video 1: Detection of anti-tetanus antibodies in human serum.

We specifically detected anti-Tetanus antibodies in human blood serum. To test the DiaCHIP under real-life conditions, we analyzed the blood serum of a vaccinated person for the presence of anti-Tetanus antibodies. We compared blood samples before and after vaccination and could directly detect its effect. To capture the antibodies, the corresponding antigen was expressed in E. coli, purified by His-tag affinity purification and spotted on a specific Ni-NTA surface.

Detailed description
hier noch rein bringen, dass ihr euren eigenen cell-free mix hergestellt habt und mit diesem auch GFP expremieren könnt und es im array erfolgreich nachweisen könnt

Detection of anti-GFP antibodies in rabbit serum using cell-free expressed GFP

Figure 1: Binding of anti-GFP antibodies to cell-free expressed GFP.

To show that cell-free expressed proteins can be immobilized on the protein surface while maintaining their antibody binding properties, the expression mix was spotted on a specific Ni-NTA surface after GFP expression. Cell-free expressed GFP was spotted on a specific Ni-NTA slide and flushed with serum of a rabbit immunized against GFP. The lowest spot consisting of cell-free expressed GFP shows a significant signal compared to the negative control (middle spot).

Detailed description

Building our very own, low-cost DiaCHIP measuring device.

Figure 2: Functional, but low-cost variant of the measuring device.

The measuring device we used in collaboration with the AG Roth (ZBSA) to detect antibody binding events is a really expensive machine, but the physics which it is based on is rather simple. Our self-built device consistent of not much more than two lenses and a camera (A) reliably detected the binding of anti-GFP to GFP (B). To enable future iGEM teams to profit from this label-free detection method as we did, a cost-efficient and easy to rebuild variant of the device was developed (figure 2A).

Detailed description

Click on one of the images to get further insight how we build up our DiaCHIP