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. -> Simon macht das
Bin für Fig. 2, mit der Vergrößerung kann mans erkennen und dann ist es informativer als 4 (jb 1309) im letzten Abschnitt stimmt der Zeilenabstand noch nicht (rj 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 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 in a more or less 20 year old serum of a rabbit that was immunized with GFP (Main Results). Before we achieved these main results we demonstrated that our device is indeed capable of detecting specific antigen-antibody binding.
Detection of Salmonella Typhimurium Single Chain Antibodies
We obtained the sequence for an immunogenic target="_blank"Salmonella Typhimurium antigen and a corresponding target="_blank"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 1). 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 was detectable, whereas the negative control showed no binding event (figure 2). 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 with the self-purified antibody was performed. This antibody contains a c-Myc tag that is not present in the antigen. Therefore we used as secondary antibody an anti-c-Myc antibody derieved from goat. For chemilumineszenz detection an anti-goat HRP was used. Also in the conventional method the binding of the S. Typhimurium antibody to the corresponding antigen is detectable (figure 3 (B)). The presence of both proteins was also validated by an Western Blot with 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: Quotient picture indicating changes of layer thickness caused by antibody binding. Positive control: GFP-His; negative control: biotinylated BSA.
Figure 2: Relative light intensity at a spot related to the background in the anti-S. Typhimurium iRIf measurement
Figure 3: Western Blot of S. Typhimurium antigen (DHAD) and S. Typhimurium antibody (anti-DHAD, scFv). (A) Western Blot of His-tagged S. Typhimurium antigen 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 S. Typhimurium antigen with the purified S. Typhimurium scFv; the purified antibody was used in a 1:100 dilution. The scFv is c-Myc tagged. Anti-c-Myc antibody (1:1000; rabbit) for the detection of c-Myc tagged scFv was used in a second step. For chemilumineszenz detection anti-rabbit HRP antibody (1:5000) was used.
Specific multiple binding event
Another important 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 received a highly specific binding at each spot. The corresponding binding curve shows the relative light intensity. Each binding event is highly specifc (figure 4). Additionally, the quotient pictures showes a distinct binding of the antibody to the related protein (figure 5). 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.
Figure 4: Relative light intensity at a spot related to the background
Figure 5: Quotient pictures indicating changes of layer thickness caused by binding of the indicated antibody
Specific Detection of Multiple Binding Events
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.
