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 year old blood serum from a rabbit that has been immunized to GFP and from a non-immunized rabbit. We immobilized purified GFP next to a positive and a negative control and performed two measurements.
As a positive control for this experiment, we spotted bBSA whose specific binding partner is streptavidin. To make sure that no unspecific binding events are responsible for the result, we spotted the DiaMIX without DNA template onto the surface as a negative control. One time the DiaCHIP was flushed with serum derived from the non-immunized rabbit and one time with the serum of the immunized rabbit. Figure 1 and 2 show the quotient pictures of these two measurements and figure 3 and 4 the corresponding binding curves.
Comparison of the quotient pictures shows that there occured an increase in layer thickness at the positive control spot and the spot of interest. This indicates that anti-GFP antibodies bind to cell-free expressed GFP as well as spreptavidin binds to bBSA. At the same time, no changes are observed at the negative control spot indicating that the binding of anti-GFP occured specifically to GFP. The respective binding curves visualize the relative light intensity at certain spots over the course of the experiment. The increase of relative light intensity when the DiaCHIP is flushed with anti-GFP is due to their binding to GFP. Such an increase can be seen for the positive control spot and the spot of interest, but not for the negative control spot therby indicating specific binding of anti-GFP to cell-free expressed GFP.
Our result demonstrates, that we can use the DiaCHIP to specifically detect anti-GFP antibodies in rabbit blood serum. Additionally, we showed that the use of a cell-free expression system for production of the protein array is suitable. At least cell-free expressed GFP exhibits antibody binding properties comparable to conventionally purified GFP. This experiment also shows that target proteins can be immobilized out of a complex mixture as the DiaMIX in an amount that is sufficient for antibody detection using our Ni-NTA surface.

Figure 1: Quotient picture from iRIf measurement with serum from not immunized rabbit. The light spot on the left shows the positive control (bBSA), in the middle spot corresponds to the negative control (BSA) and on the right spot GFP was pipetted. The GFP spot shows no signal, illustrating, that in the serum no antibodies against GFP were present.

Figure 2: Quotient picture from iRIf measurement of binding from anti-GFP in rabbit serum to GFP. The light spot on the left shows the positive control (bBSA), the middle spot corresponds to the negative control (BSA) and on the right spot GFP was pipetted. The GFP spot shows a clear signal, indicating that the rabbit has been successfully immunized to GFP.

Figure 3: Change of relative light intensity at a spot related to the background while flushing the slide with rabbit serum of not immunized rabbit. The red line shows the real time binding of Streptavidine to biotinylated BSA. The blue line correlates to the GFP spot where no binding occurs.

Figure 4: Change of relative light intensity at a spot related to the background while flushing the slide with rabbit serum containing anti-GFP. The red line shows the binding of Streptavidine to biotinylated BSA and the blue line shows the binding of anti-GFP from the rabbit serum to GFP.

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 7 (B)). The presence of both proteins was additionally validated by Western Blot with an anti-His conjugated antibody (figure 7 (A)). With this measurement we demonstrated that our system is able to detect a specific antigen-antibody binding.

Figure 5: Quotient picture from iRIf measurement, showing binding of anti-S. Typhimurium to Salmonella Typhimurium antigen spot. The left spot shows the binding of anti-GFP to GFP-His (positive control) and the middle spot corresponds to the negative control (bBSA). On the left spot binding of anit-S. Typhimurium to Salmonella antigen can be seen.

Figure 6: Change of relative light intensity at a spot related to the background while flushing the slide with anti-S. Typhimurium The blue line shows binding of anti-GFP to GFP. The red line shows real time binding of our self purified anti-S. Typhimurium antibody to our aswell self purified Salmonella Typhimurium antigen.

Figure 7: 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 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.

Figure 8: Change of relative light intensity at a spot related to the background during the iRIf measurements The blue line represents the real-time binding of anti-GFP to GFP. The grey line shows the binding of anti-rabbit to antibodies from rabbit and the red line shows the binding of anti-mouse to antibodies from mouse.

Figure 9: Quotient pictures from iRIf measurement, showing the binding of the three different antibodies to their corresponding antigens. On the first slide the binding of anti-GFP to GFP is shown. On the middle slide anti-rabbit antibody binds to antibodies from rabbit. On the left slide anti-mouse antibody shows a binding to antibodies from mouse, but also a slight cross reactivity to the antibodies out of rabbit.

Expression of several antigens

Figure 10: 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 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