Team:Freiburg/Results/Diagnostics
Julia: Bin grad dabei das hier komplett neu zu machen, nach Plan vom Website-Team.
find ich gut, bin gespannt (NG)
hab die Hasen-serum-Messung noch hinzugefügt. (NW)--> sollte dann Hase nicht weiter oben kommen + kannst du im quotient picture noch beschriftet welcher spot was ist; so wie die anderen? (JD)
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.
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 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 spot 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 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 3 (B)). The presence of both proteins was additionally validated by Western Blot with an 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 at the Salmonella Typhimurium antigen spot. Positive control: GFP-His; negative control: biotinylated BSA.
Figure 2: Change of relative light intensity at a spot related to the background while flushing the slide with anti-S. Typhimurium
Figure 3: Western Blot of S. Typhimurium antigen (DHAD) and anti-S. Typhimurium antibody (anti-DHAD, scFv). (A) Western Blot of His-tagged S. Typhimurium antigen as well as the corresponding scFv using anti-His HRP conjugate. The expected molecular weights of the antigen and the scFv are 63 kDa and 30 kDa, respectively. (B) Western Blot of S. Typhimurium antigen using 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 detection via chemiluminecence anti-rabbit HRP antibody (1:5000) was used.
Specific Detection of Multiple Binding Events
Another important experiment on our way to the establishment of the DiaCHIP was the immobilization of three proteins on one single 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 4). Quotient pictures additionally visualize distinct binding of the respective antibodies to the corresponding 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: Change of relative light intensity at a spot related to the background during an iRIf measurement
Figure 5: Quotient pictures indicating changes of layer thickness caused by binding of the indicated antibody
Specific Detection of anti-GFP in rabbit serum
Before we put our DiaCHIP to a final test by measuring with actual human blood serum, we wanted to make sure, we are able to detect antibodies out of such a complex solution. Luckily we obtained 20 years old blood serum from a rabbit that has been immunized to GFP and also serum from unimmunized rabbit. We immobilized purified GFP next to a positive and a negative control and performed two measurements. One with serum from the not immunized rabbit and one with the serum of the immunized rabbit. Fig. 4 and 5 show the quotient pictures for these two measurements and fig. 6 and 7 the corresponding binding curves. They demonstrate, that the serum of the immunized rabbit did contain antibodies against GFP and we were able to detect them directly out of this complex solution, even after the serum has been stored for such a long time.
Figure 4: Quotient picture of measurement with serum from not immunized rabbit.
Figure 5: Quotient picture of measurement with serum from rabbit that was immunized to GFP.
Figure 6: Binding curve of measurement with serum from not immunized rabbit.
Figure 7: Binding curve of measurement with serum from rabbit that was immunized to GFP.
Expression of several antigens
Figure 8: Western Blot of HIV multi-epitopic antigen. The HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. 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.
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 8 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
