Difference between revisions of "Team:Freiburg/Results"

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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 befor vaccination. The lowest spot in each part contains the tetanus antigen.
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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.
 
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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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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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 of 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.  
 
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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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.
 
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 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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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>
 
</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.  
 
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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                <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 containing cell-free expressed GFP shows a significant signal compared to the negative control (middle spot).
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="80%"></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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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.  
+
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.  
 
</br>
 
</br>
 
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 (??).  
 
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 (??).  

Revision as of 09:46, 9 September 2015

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Detection of anti-Tetanus antibodies in human blood serum


Video 1: Detection of anti-tetanus antibodies in human serum. The left part shows the slide being flushed by the sample taken after vaccination, the right part shows the negative control sample taken befor vaccination. The lowest spot in each part contains the tetanus antigen.

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 of 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 1). 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.


Figure 1: Binding of anti-GFP antibodies to cell-free expressed GFP. 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 containing cell-free expressed GFP shows a significant signal compared to the negative control (middle spot).

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


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

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.
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