Difference between revisions of "Team:Freiburg/Results"

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We successfully diagnosed anti-Tetanus antibodies in human blood serum
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Detection of anti-Tetanus antibodies in human blood serum
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Check out this video showing how the antibodies in the serum bind our tetanus antigens on the DiaChip
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="100%"></img>
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="100%"></img>
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We took a persons blood before and after a tetanus vaccination and screened for a tetanus infection.  
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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 corresponding antigen TeNT_Hc 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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An immunized person has antibodies against tetanus antigens - we bound spots of our purified tetanus antigens onto the specific surface of our chip. We then incubated the DiaChip with 33 µl of the blood serum for half an hour and measured the results.
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</br>
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Applying the sample taken three weeks after vaccination to the DiaCHIP resulted in a significant signal at the antigen spot, whereas no signal was obtained with the sample assumed to be negative. But convince yourself how great the DiaCHIP performed. The video on the right shows these measurements right next to each other in real-time.  
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Besides tetanus, some other antigens of immunological relevance were taken into account. See all the <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics">results we obtained in terms of diagnostics</a>, including an approach for quantification of the measurements.  
  
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Take a look at the video and see for yourself how good our DiaChip performed.
 
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And remember: that this isn’t an ELISA, we don’t need no expensive secondary Antibodies!
 
 
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We measured the binding of anti-GFP to cell-free expressed GFP in blood serum
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Detecion of anti-GFP antibodies in rabbit serum using cell-free expressed GFP
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We expressed GFP with our own cell-free expression system and bound it to the surface of our DiaChip. We than ran some rabbit blood serum over the chip which we had spiked with anti-GFP antibodies. The results show that our cell-free proteins can be measured even in a complex solution such as blood serum. </p>
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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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</br>
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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 2). 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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</br>
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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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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="100%"></img>
 
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We built our very own, low-cost DiaChip device - and can measure with it!
 
We built our very own, low-cost DiaChip device - and can measure with it!
 
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="80%"></img>
 
<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="80%"></img>
 
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A green LED, two lenses and a regular SLR Camera is basically all you need to build your own iRIf 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. 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 3A). Figure 3B shows the binding of anti-GFP to GFP as it was established before, measured with our own device.
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</br>
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Look here, to see how you can <a href="https://2015.igem.org/Team:Freiburg/Results/OwnDevice">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 (??).  
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Revision as of 09:26, 9 September 2015

""

Detection of anti-Tetanus antibodies in human blood serum


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 corresponding antigen TeNT_Hc was expressed in E. coli, purified by His tag based affinity purification and spotted on a specific Ni-NTA surface.
Applying the sample taken three weeks after vaccination to the DiaCHIP resulted in a significant signal at the antigen spot, whereas no signal was obtained with the sample assumed to be negative. But convince yourself how great the DiaCHIP performed. The video on the right shows these measurements right next to each other in real-time.
Besides tetanus, some other antigens of immunological relevance were taken into account. See all the results we obtained in terms of diagnostics, including an approach for quantification of the measurements.

Detecion of anti-GFP antibodies in rabbit serum using 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.
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 2). Thus, His-tagged GFP was successfully expressed and immobilized on the surface, still enabling anti-GFP antibodies in a serum sample to bind.
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.


We built our very own, low-cost DiaChip device - and can measure with it!


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 3A). Figure 3B 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 build your own device for label-free detection of antibody binding on a protein microarray needing two lenses, a camera and a little bit of fine feeling (??).

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