Team:Freiburg/Results
Vll etwas doofer Kommentar, aber die "Detailed Description" buttons sehen aus wie links. Ich hab da erst gar nicht drauf geklickt, da ich dachte da werde ich wo ganz anders hingeleitet. Wenn jemand ne kluge Idee hat, wie man andeuten kann, dass da ein drop-down menü drunter liegt, wre das cool (ps1209) Besser?! (jb1209)
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... Stefan: Ihr sprecht hier immer von spotted, dabei wird aber nicht klar wie ihr das gemacht habt. Vielleicht sollte man immer dazu schreiben spotted by hand. Was denkt ihr dazu?
Main Results
Detection of anti-Tetanus antibodies in human blood 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.
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. Hier noch mehr Results beschreibung? Wie viel Blut, welche Verdünnung, welche Flussraten? Bindekurve? Ganztifizieren des Signals könnte vor allem für dieses Exp sehr interessant sein. (Stefan)
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.
Auch, dass euer Mix low-cost ist, und trotzdem auf dem Niveau (oder besser) eines kommerziellen kits liegt - Community gedanke etc. (ps12092015)
Detection 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. 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.
We got hold of a more or less 20 years old serum of a rabbit that was immunized with GFP. This gave us the perfect opportunity to test our device with a more complex solution than antibody-dilutions.
For this experiment we used a specific Ni-NTA surface and spotted cell-free expressed GFP on the surface.
For verification of correct expression of GFP with our Dia-Mix we performed a Western Blot analysis (fig. 1). With this we could prove that the cell-free expression was successful.
To obtain the specific surface an iRif slide was plasma activated and incubated with APTES and PDITC. Afterwards the Ni-NTA layer was added via incubation of the slide in Ni-NTA solution over night. The slide was blocked with blocking solution (?) and cell-free expressed GFP with a His tag ( + other GFPs?) and a positive control as well as a negative control (His-mCherry) were spotted onto the surface.
The serum of the GFP-immunized rabbit was then analysed in an iRif measurement. Binding of the GFP-antibodies present in the rabbit serum can clearly be seen, as the respective spots show a strong signal (fig. 1). A parallel measurement with serum of a rabbit that was not immunized (fig.2) does not show any binding events at the GFP spot.
With this experiment we could show that it is possible to analyse complex fluids like serum. In addition to that we demonstrated that our cell-free expressed GFP with a His-tag worked at binding partner of GFP-antibodies. 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.
Building our very own, low-cost DiaCHIP 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.
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 (??) sensitivety/sensitiveness/finesse/sure instincts.
Specific surfaces
For immobilization of the antigens on the chip we developed our own specific Ni-NTA surface. All expression constructs have a His-tag fusd to the coding sequence, resulting in antigens that can bind specifically to our surface. Compared to an unspecific surface (PDITC) we could show that this Ni-NTA surface allows accurate binding of target proteins and prevents unspecific binding from other proteins of the cell-free mix. ▼ Detailed Description.
Own cell-free mix
The copying mechanism we are using to assemble a protein array from a DNA array template is based on cell-free expression. During our project we successfully established our own cell-free expression system from scratch, starting with an E. coli lysate. With our own cell-free mix we succeeded in expression of correctly folded GFP and luciferase. Comparison of the Dia-Mix with a other cell-free expression kits proved that our own cell-free expression mix is even better than a commercial kit! ▼ Detailed Description.
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Explore the DiaCHIP
For establishing our device, we optimized all steps from the immobilization of DNA to the top of the flow-chamber until the specific binding of the target proteins on the glass slide. This way, we generated protein arrays we could use to detect different antibodies in human and rabbit serum with the iRIf-detection method.
Click on the images below to explore our experiments from expression to detection!
Assembling the DiaCHIP
Diagnosis of Antigens
Click on one of the images to get further insight how we build up our DiaCHIP
