Specific Detection of anti-GFP in rabbit serum

Before our DiaCHIP is finally tested, we wanted to check if we are able to detect antibodies present in a complex solution like blood serum. Luckily we obtained 20 year old blood serum from a rabbit that has been immunized against GFP and a non-immunized rabbit. We immobilized purified GFP next to a positive and a negative control.
The positive control for this experiment was biotinylated BSA whose specific binding partner is streptavidin. Additionally we wanted to make sure that no unspecific binding events are responsible for the obtained result. Therefore we spotted BSA without biotinylation as a negative control. In the first experiment the DiaCHIP was flushed with serum derived from the non-immunized rabbit, following by a measurement with serum of the immunized rabbit. Figure 1 and 2 show the quotient pictures of these two experiments including figure 3 and 4 the corresponding binding curves.
Comparison of the quotient pictures shows an increase in layer thickness at the positive control spot (bBSA) and the spot of interest (GFP). This indicates that anti-GFP antibodies of the serum bind specifically to GFP as well as streptavidin binds to bBSA. At the same time, no changes are observed at the negative control spot indicating that no unspecific binding occurred. The respective binding curves visualize the relative light intensity at certain spots over time. The increase of relative light intensity is due to the specific binding of anti-GFP to GFP. This binding event was observed while the DiaCHIP was flushed with the blood serum of the immunized rabbit. It produced antibodies against GFP as an immune response against GFP. The increase in the intensity can be seen for the spot of interest, but not for the negative control spot - indicating the specificity of the binding process.
The result demonstrates, that we can use the DiaCHIP to specifically detect anti-GFP antibodies in blood serum, which were built upon an immunization.

Figure 1: Quotient picture from iRIf measurement with serum from not immunized rabbit.The quotient picture shows all changes in intensity that occurred during the measurement. Only in case of the the positive control (bBSA) a shift in intensity can be detected. The middle spot corresponds to the negative control (BSA) and on the right spot GFP was spotted. The GFP spot shows no signal, illustrating, that in the serum no antibodies against GFP were present.

Figure 2: Quotient picture from iRIf measurement with serum from not immunized rabbit.The quotient picture shows all changes in intensity that occured during the measurement. In case of the the positive control (bBSA) and the GFP spot a shift in intensity can be detected. No shift in intensity can be seen at the spot of the negative control (BSA). The GFP spot shows an intense signal, illustrating that in the serum of the immunized rabbit antibodies against GFP were present.

Figure 3: Change of relative light intensity plotted over time at the three spots of interest. This measurement was conducted with the 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. The grey line represents the intensity shifts at the negative control spot.

Figure 4: Change of relative light intensity plotted over time at the 3 spots of interest. This measurement was conducted with the rabbit serum of the immunized rabbit. The red line shows the binding of streptavidine to biotinylated BSA. The blue line shows the binding of antibodies against GFP to the GFP spot. The grey line represents the intensity shifts at the negative control spot.

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 7B). The presence of both proteins was additionally validated by Western Blot with an anti-His conjugated antibody (figure 7A). 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 His-GFP- (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 during the measurement 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 as well 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 chemiluminescence our self purified anti-S. Typhimurium antibody to our as well self purified Salmonella Typhimurium antigen.

Specific Detection of Multiple Binding Events

Another important experiment on our way to the establishment of the DiaCHIP was the immobilization of three different proteins on one 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 occurring 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 antibodies bind to antibodies from rabbit. On the left slide anti-mouse antibodies show binding to antibodies from mouse, but also a slight cross-reactivity to the antibodies from 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 weight 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 verification 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