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

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.

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

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.

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).

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.

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. All the other components needed for a fully functional cell-free expression system, like energy sources, amino acids and nucleotides and some buffers are added to the lysate directly before expression is started.
With our own cell-free mix we succeeded in expression of correctly folded GFP and luciferase.

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