Team:Freiburg/Results/irif
Results: SirRif
Comparison between specific and unspecific surface
To determine the specificity of our Ni-NTA surface we compared it to an unspecific PDITC surface. We spotted His-GFP-lysate and untagged GFP-lysate as well as purified His-GFP on the surfaces and measured the fluorescence intensity. The graph below shows the mean fluorescence for all spots.The fluorescence intensity for the His-GFP lysate on the Ni-NTA surface is 4 times higher than on the PDITC surface, while the values for the purified His-GFP are nearly the same. The mean intensity for the untagged GFP-lysate is in the range of the background for the Ni-NTA surface and just slightly higher for the PDITC surface, which shows, that the GFP cannot bind to Ni-NTA without a His-tag. The low intensity for the lysates on PDITC indicates, that all lysate proteins including the GFP are bound to the surface.
Simultaneous detection of different proteins
Because the DiaChip has to be able to detect many different diseases simultaneously we showed with this experiment, that the detection of different proteins on one slide without any cross reactivity is possible. Therefore we spotted mouse, goat and rabbit proteins on one slide and measured the binding to their respective antibodies. In the video these three antibodies are flushed over the slides one after the other. The resulting binding curves show, that the anti-mouse binds specifically to the mouse derived protein, but not to the proteins from goat and rabbit and vice versa.
Tetanus iRIf-Measurement
Some of our team members needed to refresh their tetanus vaccination, so we asked their doctor to take a sample of their blood right before and two weeks after their vaccination. We spotted our purified Tetanus antigen along with a positive (GFP) and a negative (Salmonella antigen) control on a glass slide with a PDITC surface and measured it in iRIf. In the videos below you can see the results for both, the serum before (left) and after vaccination (right). First each slide was flushed with anti-GFP, so that the positive control lightens up and then with the serum. In the serum before vaccination nearly no signal can be observed while in the serum two weeks after vaccination there are a lot of antibodies that bind to our tetanus antigen. The binding curves also show a clearly increase of the optical thickness on the tetanus spots.
ELISA Tetanus
To validate the interaction between Tetanus Antibodies in the blood of our freshly vaccinated team member with our self-expressed and purified Tetanus antigen, we also did an ELISA test. We spotted the Tetanus Antigen (12.5 µg/mL), bBSA (12.5 µg/mL) as a positive control and as negative controls PBS. The wells with Tet-antigen, bBSA and one of the wells with only PBS were blocked for 1 h with 5 mg/mL BSA in PBS. After that to Tetanus and the unblocked PBS first the blood Serum (after vaccination), then anti-human out of mouse and finally HRP-labeled anti-mouse was added and each time incubated for 1 h. The well with bBSA and the second negative control were incubated with HRP-Strep (0.5 µg/mL) for 1 h. After each step the wells were washed with PBS. TMB was pipetted into every well. The graph below shows the absorption over time. One can see that the signal for the antibodies in the blood serum bound to the tetanus antigen was even higher than the signal for the positive control.
Salmonella iRIf-Measurement
We expressed and purified a Salmonella antigen together with a single strain antibody that binds to it. Due to the fact, that we purified this antibody ourselves, it has a His-tag, so we couldn’t spot the antigen on a Ni-NTA surface because the antibody would also bind rather to the surface than to the antigen. On PDITC slides though we could see a binding of the Salmonella antibody to the antigen by using the antibody lysate as well as the purified antibody. Again we spotted the antigen next to a positive control (GFP) and a negative control (random stuff). In the video the binding of anti-GFP can be observed first, then the binding of purified anti-Salmonella and at last the binding of anti-Salmonella lysate.
Cell free expressed GFP on Ni-NTA detected with rabbit serum
Now that we established a specific surface and showed, that the cell free expression of GFP works, we wanted to check if we are able to detect our cell free expressed GFP directly with antibodies in the serum of a rabbit that was immunized against GFP. Therefore we used the following spotting pattern:
| # | Spot |
|---|---|
| 1+2 | Cell free expressed His-GFP |
| 3 | Purified His-GFP (expressed in E.coli) |
| 4+5 | Cell free mix without DNA (neg.) |
| 6-7 | His-mCHerry lysate (expressed in E.coli) |
| 8-9 | His-GFP lysate (expressed in E.coli) |
| 10 | BSA (neg.) |
| 11 | bBSA (unspecific binding) |
To make sure the rabbit serum didn’t bind unspecific ally to GFP the slides were first flushed with pre immunization rabbit serum, where - as expected - no binding could be observed. When flushing the slide with the post immunization serum the cell free expressed GFP showed a clear signal, as well as the purified GFP and the signal got even stronger when anti-rabbit was flushed over, which bound to the rabbit anti-GFP. The bBSA spot showed a binding to Strep-Cy5. This interaction usually is really strong, but the binding signal was pretty weak compared to the GFP binding as you can see in the binding curves. This shows us, that there wasn’t as much bBSA on the surface as GFP, so the specific binding of the surface is a lot stronger than the unspecific to proteins without a His-tag, like bBSA.
Binding of anti-GFP to GFP
As soon as we had our first surface ready to test in iRIf, we tried to detect the interaction of anti-GFP to spotted GFP of different concentrations. We got three purified GFPs with a stock solution of 0.5 mg/mL each from different groups and tried all of them to see, which one is working best for us. We spotted according to the following pattern:
| GFP 1, 1:10 diluted | GFP 1, 1:30 diluted | GFP 1, 1:100 diluted |
| GFP 2, 1:10 diluted | GFP 2, 1:30 diluted | GFP 2, 1:100 diluted |
| GFP 3, 1:10 diluted | GFP 3, 1:30 diluted | GFP 3 ,1:100 diluted |
| bBSA (o.5 mg/mL) | BSA (10 mg/mL) |
The quotient pictures show a clear binding of anti-GFP for each concentration. When Strep-Cy5 not only the bBSA spot which was used as a positive control lightens up but also the signal of all the GFP spots increases. As the used GFP antibody is biotinylated, this was expected and confirms that the anti-GFP really bound to the GFPs.
Binding of anti-GFP to cell free expressed GFP
After we validated the successful expression of GFP with our self-established cell free mix by observing the increase of fluorescence at 488 nm in the plate reader and were even able to verify this in a western blot, we decided to immobilize our cell free expressed GFP on our Ni-NTA surface and try to detect it in iRIf. The proteins were spotted as shown below.
| 1-3 | Cell free expressed His-GFP |
| 4-6 | Cell free mix without DNA (neg) |
| 7-8 | His-GFP lysate |
| 9 | Untagged GFP lysate (unspecific binding) |
| 10 | bBSA (unspecific binding) |
| 11 | Purified GFP (1 mg/mL) |
The anti-GFP bound strongly to the cell free expressed GFP, which shows, that we can detect cell free expressed proteins directly out of the lysate. Also this result shows again the specificity of our surface. The His-GFP lysate gave a really strong signal, while the untagged GFP lysate gave nearly no signal at all as well as the bBSA.
Halo surface
Searching for the surface that fits best to our needs we tried out the Halo-Tag-Ligand system that implements a covalent bond. The Halo-Tag binds to chloralkanes which are immobilized to the surface. There are different possibilities how this Ligand can be build up (see Methods surchem). The Ligand that worked best for us, was a 3-chloropropylsilane which we directly immobilized on plasma activated iRIf slides. To test the slides we spotted our self-expressed Halo-GFP and Halo-mCherry as well as purified Halo-GFP as a positive control and purified untagged GFP as a negative control which we both got from a research group from Osnabrück. As an additional negative control we spotted bBSA.
The iRIf measurement showed that the Halo-tagged GFPs were successfully immobilized on the surface. Unfortunately there was a lot of unspecific binding, so that the negative control nearly bound as much to the surface as the positive control. Because of this we decided to continue with the Ni-NTA surface we established.
