Team:Freiburg/Results/Diagnostics
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.
Bin für Fig. 2, mit der Vergrößerung kann mans erkennen und dann ist es informativer als 4 (jb 1309)
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.
Figure 1: Quotion picture indicating changes of layer thickness caused by flushing with the specific antibody (anti-salmonella) and the positiv control (GFP) as well as the negativ control (bBSA).
Figure 2: Relative light intensity at a spot related to the background of anti-Salmonella experiment
Figure 3: Western Blot of S. Typhimurium antigen (DHAD) and S. Typhimurium antibody (anti-DHAD, scFv). (A) Western Blot of His-tagged DHAD as well as the corresponding scFv with anti-His HRP Conjugate. The expected molecular weight of DHAD is 63 kDa and 30 kDa for the scFv, respectively. (B) Western Blot of DHAD with the purified S. Typhimurium scFv was used in a 1:100 dilution. Additionally, the scFv is c-Myc tagged. Anti-c-Myc antibody (1:1000; rabbit) was used in a second step. For detection, the anti-rabbit HRP antibody (1:5000) was used.
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.
Figure 4: Relative light intensity at a spot related to the background
Figure 5: Quotion pictures indicating changes of layer thickness caused by flushing with the indicated antibody
Expression of several antigens
Figure 6. Western Blot of HIV multi-epitopic antigen. In this Western Blot the HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. The anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.
