Difference between revisions of "Team:Freiburg/Results/Diagnostics"
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Another import result during the establishment of our diagnostic device was the immobilization of three proteins on one single slide (GFP, a rabbit- and a mouse-derived antibody). In this experiment the slide was flushed with three different antibodies, one after the other. In three different outputs dependent on the antibody we recieved a highly specific binding at each spot. The corresponding binding curve shows the relative light intensity at each spot. Every binding event is highly specifc (figure 4). Additionally the quotion pictures showed the distinct binding of the antibody to the related antigen (figure 5). This confirmed the specific binding event of protein-antibody in our setup. With this promising result we were one step further along our diagnostic application. | Another import result during the establishment of our diagnostic device was the immobilization of three proteins on one single slide (GFP, a rabbit- and a mouse-derived antibody). In this experiment the slide was flushed with three different antibodies, one after the other. In three different outputs dependent on the antibody we recieved a highly specific binding at each spot. The corresponding binding curve shows the relative light intensity at each spot. Every binding event is highly specifc (figure 4). Additionally the quotion pictures showed the distinct binding of the antibody to the related antigen (figure 5). This confirmed the specific binding event of protein-antibody in our setup. With this promising result we were one step further along our diagnostic application. |
Revision as of 20:36, 13 September 2015
Julia: Bin grad dabei das hier komplett neu zu machen, nach Plan vom Website-Team. Vergleich bitte von Figure 2 und Figure 4. Sollen die Binding curves einheitlich seien. Das eine ist bearbeitet (poster), das andere ausm labjournal.
find ich gut, bin gespannt (NG)
Diagnostics
The following section summarizes the most interesting results we obtained this summer establishing our diagnostic tool. On our way to the detection of anti-Tetanus in human blood serum we achieved many other results in the field of Diagnosis. Besides the detection of anti-Tetanus antibodies we could identify anti-GFP antibodies in a more or less 20 years old serum of a rabbit that was immunized with GFP (Main Results). Before we achieved these main results we proofed our concept, showing that our device is indeed capable of detecting specific antigen-antibody binding.
Detection of anti-Salmonella
We obtained the sequence for a specific Salmonella Typhimurium antigen and a corresponding Salmonella Typhimurium antibody from the lab of Prof. Dr. Hust. Both His-tagged proteins were successfully expressed in E. coli and spotted on a specific PDITC surface. With the following iRIf measurement we analyzed the binding between antigen and antibody (figure 1). The measurement was a great success, showing a distinct shift in the binding curve for the salmonella antigen when the salmonella antibody is flushed over the chip, whereas the negative control is not bound by the antibody (figure 2). Supplementary we validated the obtained result of the iRIf measurement with a standardized method. The purified antigen was analyzed by a 12.5% SDS-PAGE. Afterwards a Western Blot was performed with the self-purified antibody. This antibody contains a c-myc Tag, which is not present at the antigen. Therefore we used as secondary antibody an anti-c-myc antibody derieved from goat. For the detection an anti-goat HRP was used. Figure 3 (B) shows that also in the standardized method the binding of the Salmonella antibody to the corresponding antigen is detectable. The presence of both proteins were also validated by anti-his conjugated antibody (figure 3 (A)). With this measurement we demonstrated that our system is able to detect a specific antigen-antibody binding.
Specific multiple binding event
Another import result during the establishment of our diagnostic device was the immobilization of three proteins on one single slide (GFP, a rabbit- and a mouse-derived antibody). In this experiment the slide was flushed with three different antibodies, one after the other. In three different outputs dependent on the antibody we recieved a highly specific binding at each spot. The corresponding binding curve shows the relative light intensity at each spot. Every binding event is highly specifc (figure 4). Additionally the quotion pictures showed the distinct binding of the antibody to the related antigen (figure 5). This confirmed the specific binding event of protein-antibody in our setup. With this promising result we were one step further along our diagnostic application.
Expression of several antigens
As we suppose a new diagnostic device for the detection of several diseases we expressed these antigenic peptides in E.coli. Due to time constraints we could only verify those expressed antigen by western blot or SDS-PAGE (see labjournal protein purification). Figure 6 shows the HIV multi-epitopic antigen. For this verifcation we used the anti-HIV-1 P24 polyclonal antibody. ‘Health and Medicine’ as one of the most popular tracks chosen in the iGEM competition we want to share the sequences encoding for these peptides with the iGEM community. Thus, future iGEM teams have the opportunity to take advantage from our research if they are planning to work in the field of diagnostics. BioBricks iGEM Team Freiburg 2015