Difference between revisions of "Team:Freiburg/Results/Diagnostics"
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Revision as of 21:32, 18 September 2015
Diagnostics results
The following section summarizes the most interesting results we obtained this summer establishing our diagnostic tool. On our way to detecting anti-tetanus antibodies in human blood serum we achieved many other results in the field of diagnosis. In addition to the detection of anti-tetanus antibodies, we could identify anti-GFP antibodies with our own cell-free expressed GFP immobilized on our specific surface (Essential Results). Before we achieved these major results we demonstrated that our device is indeed capable of detecting specific antigen-antibody binding and that antibodies can be detected within animal blood serum.
Specific Detection of anti-GFP in rabbit serum
Before our DiaCHIP is finally tested, we wanted to check if we are able to detect antibodies present in a complex solution like blood serum. Luckily we obtained 20 year old blood serum from a rabbit that has been immunized against GFP and a non-immunized rabbit. We immobilized purified GFP next to a positive and a negative control.
The positive control for this experiment was biotinylated BSA whose specific binding partner is streptavidin. Additionally we wanted to make sure that no unspecific binding events are responsible for the obtained result. Therefore we spotted BSA without biotinylation as a negative control.
In the first experiment the DiaCHIP was flushed with serum derived from the non-immunized rabbit, following by a measurement with serum of the immunized rabbit. Figure 1 and 2 show the quotient pictures of these two experiments including figure 3 and 4 the corresponding binding curves.
Comparison of the quotient pictures shows an increase in layer thickness at the positive control spot (bBSA) and the spot of interest (GFP). This indicates that anti-GFP antibodies of the serum bind specifically to GFP as well as streptavidin binds to bBSA. At the same time, no changes are observed at the negative control spot indicating that no unspecific binding occured.
The respective binding curves visualize the relative light intensity at certain spots over time. The increase of relative light intensity is due to the specific binding of anti-GFP to GFP. This binding event was observed while the DiaCHIP was flushed with the blood serum of the immunized rabbit. It produced antibodies against GFP as an immune response against GFP. The increase in the intensity can be seen for the spot of interest, but not for the negative control spot - indicating the specificity of the binding process.
The result demonstrates, that we can use the DiaCHIP to specifically detect anti-GFP antibodies in blood serum, which were built upon an immunization.
Detection of Salmonella Typhimurium Single Chain Antibodies
We obtained the sequence for an immunogenic Salmonella Typhimurium antigen and a corresponding anti-S. Typhimurium antibody from Prof. Dr. Hust's laboratory. Both His-tagged proteins were successfully expressed in E. coli and spotted on a PDITC surface. In an iRIf measurement we analyzed the binding between S. Typhimurium antigen and antibody (figure 5). The measurement was a great success. When the S. Typhimurium antibody was flushed over the chip a distinct shift in the binding curve for the S. Typhimurium antigen spot was detectable, whereas the negative control showed no binding event (figure 6). Moreover, the obtained iRIf result was validated with a standardized method. The purified antigen was analyzed by a 12.5% SDS-PAGE. Afterwards a Western Blot was performed using the self-purified antibody. This antibody is genetically fused to a c-Myc tag that is not present in the antigen. Therefore, we used an anti-c-Myc antibody derived from goat as secondary antibody. For detection via chemiluminescence an anti-goat HRP was used. The conventional method confirmed the binding of the anti-S. Typhimurium antibody to the corresponding antigen (figure 7B). The presence of both proteins was additionally validated by Western Blot with an anti-His conjugated antibody (figure 7A). With this measurement we demonstrated that our system is able to detect a specific antigen-antibody binding.
Specific Detection of Multiple Binding Events
Another important experiment on our way to the establishment of the DiaCHIP was the immobilization of three different proteins on one slide (GFP, a rabbit- and a mouse-derived antibody). In this experiment the slide was sequentially flushed with three different antibodies, each specifically binding to one of the immobilized proteins. In three different outputs, dependent on the antibody, we received a highly specific binding at each spot. The corresponding binding curve shows the changes in relative light intensity at each spot. The occuring binding events are specific, except a slight cross-reactivity of the anti-mouse antibody (figure 8). Quotient pictures additionally visualize distinct binding of the respective antibodies to the corresponding protein (figure 9). This confirmed specific binding events of antibodies to proteins in our setup. With this promising result we were one step further along our diagnostic application.
Expression of several antigens
As we propose a new diagnostic device for the detection of several diseases, we expressed several antigenic peptides in E.coli. Some of these antigens were successfully overexpressed and verified by Western Blot or SDS-PAGE (see labjournal protein purification). In addition to the expressed S. Typhimurium and C. tetani antigen, we were able to overexpress the HIV multi-epitopic antigen as well as a HCV antigen. Figure 10 shows the Western Blot for verifcation of the HIV multi-epitopic antigen using anti-HIV-1 P24 polyclonal antibody.
As ‘Health and Medicine’ is one of the most popular tracks chosen in the iGEM competition, we want to share the sequences encoding for these antigenic peptides with the iGEM community. Thus, future iGEM teams have the opportunity to take advantage of our research, if they are planning to work in the field of diagnostics. BioBricks iGEM Team Freiburg 2015