Team:Freiburg/Results
Essential Results
Our aim was to develop a novel diagnostic tool enabling fast, simultaneous and label-free detection of antibodies in human blood sera. We successfully generated our self-established cell-free expression system based on an E. coli lysate, which yielded an expression efficiency comparable to a commercial kit. To immobilize the expressed proteins, we established a surface specifically binding our target proteins. This system was used to produce a protein array that can be flushed with a serum sample. The binding of antibodies is detected label-free by an interferometric method called iRIf (imaging reflectometric interference) in real-time. Moreover, we rebuilt the detection device in a simplified and cost-efficient manner and thus, made it available for future iGEM Teams. Using the DiaCHIP, we did not only show the presence of antibodies in distinct solutions but also verified them in complex samples as human blood serum.
Detection of Anti-Tetanus Antibodies in Human Blood Serum
To test the DiaCHIP under real-life conditions, we analyzed the blood serum of a vaccinated person for the presence of anti-tetanus antibodies. We compared blood samples from before and after vaccination and could directly detect its effect. To capture the antibodies, the corresponding antigen was expressed in E. coli, purified by His-tag affinity purification and spotted onto a PDITC surface. By comparing the spotted pattern with the localization of signal as detected by iRIf, the system's specificity even for complex samples was shown. ▼ Detailed Description.
The antigen used for detection of anti-tetanus antibodies is derived from the tetanus neurotoxin and commonly used for in vitro testing. For preparation of the DiaCHIP, this antigen had to be overexpressed in E. coli and purified from whole cell lysate. To verify the successful purification of the antigen, Western Blot analysis was performed (figure 1). The usage of an antibody against the N-terminal His-tag revealed a protein with a molecular weight of about 50 kDa. This correlates with the expected molecular weight of the antigen 1).
We performed an ELISA to check, whether the blood samples we obtained contain antibodies specific to our purified tetanus antigen. Therefore, we spotted tetanus antigen and bBSA of the same concentration (12.5 µg/mL) and 2x PBS as negative controls. The tetanus antigen and one PBS well were incubated with 5 mg/mL BSA, blood serum, and HRP-labeled anti-human for 1 h, respectively. The bBSA and the other PBS well were incubated with HRP-labeled Streptavidine (0.5 µg/mL) for 1 h (positive control). After each step the wells were washed with PBS. To visualize the binding, Tetramethylbenzidine was added and the absorption at 650 nm was measured in a plate reader over time. The graph in figure 3 shows that the signal for tetanus is even higher than the positive control, which illustrates the successful binding of antibodies from the blood to tetanus antigens.
The purified tetanus antigen as well as GFP (positive control) and BSA (negative control) was immobilized on a PDITC surface (see figure 4A). Look here to read more about the establishment of this surface. The slide was first flushed with not immunized blood serum (serum (-))followed by anti GFP and immunized blood serum (serum(+)). Flushing the same slide with both blood sera improves the comparability of the resulting iRIf signal for the individual binding events. To exclude that the order in which the sera are flushed over the slide influences the ratio of the binding signals we repeated the experiment with switched flushing sequence.
The human serum samples we obtained have been taken before and five weeks after a patient received a vaccination boost against tetanus. Analysis of the samples was performed using the same array set-up. Figures 2 and 4 show quotient pictures representing changes in the thickness of the layer covering the slide while it was flushed with the respective samples. The negative control spots do not indicate any binding events, while the binding of anti-GFP to the positive control spot verifies that both experiments are reliable. The comparison of the antigen spots shows that we specifically detected anti-tetanus antibodies in human serum in response to vaccination. No binding event was detected by flushing the array with the sample assumed to be negative (figure 2), whereas a distinct spot is visible after flushing it with the positive sample (figure 4). Both measurements in real-time are shown in parallel in video 1.
The increase in relative light intensity at distinct spots over the course of the experiment is visualized in figure 5 and can be correlated with the amount of antibodies binding to the respective antigen spot. This indicates that the DiaCHIP enables quantification of antibody titers in addition to detecting their presence.
Besides tetanus, other antigens of immunological relevance were taken into account. See all the results we obtained in terms of diagnostics.
Auch, dass euer Mix low-cost ist, und trotzdem auf dem Niveau (oder besser) eines kommerziellen kits liegt - Community gedanke etc. (ps12092015)
Detection of Anti-GFP Antibodies Using Cell-Free Expressed GFP on Ni-NTA
Our self-made cell-free expression, the DiaMIX, in combination with specific glass surfaces allows the expression and purification of target proteins in a simple manner. The mix, after expressing His-tagged GFP, was spotted onto a Ni-NTA coated glass slide. Even after several washing steps it could be shown that the proteins maintain their antibody binding properties. A GFP antibody solution was flushed over the slide and a specific binding could be detected in iRIf. ▼ Detailed Description.
We could successfully show that the cell-free expression of GFP-His results in functional GFP proteins that are bound by anti-GFP antibodies during an iRIf measurent. The DNA coding for GFP fused to a 10x His-tag was added to the DiaMIX and expression was performed for 2 h. The DiaMIX containing cell-free expressed GFP-His was then spotted on an iRIf slide with a Ni-NTA surface by hand (figure 1). The spots were incubated on the slide over night.
This specific surface allows binding of the expressed GFP-His via the His-tag while all the other proteins present in the DiaMIX are not bound to the surface. This step was followed by the blocking and washing protocol to prevent further unspecific binding. The iRIf slide was then flushed with a anti-GFP antibody solution, to analyze the binding to the cell-free expressed GFP-His. As a positive control we spotted a purified GFP-His onto the surface as well as DiaMIX that did not contain any DNA as negative control (figure 2).
The measurement shows a high signal for the cell-free expressed GFP-His and the positive control as can also be seen in the respective binding curve shown in figure 3.
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression. ▲ Close Box
Building Our Low-Cost DiaCHIP Measuring Device
Commercial systems using the iRIf technology (imaging reflectomretric interference) are mostly bulky and expensive machines even though the components they are based on are rather simple. So we decided on building our own device consisting of not much more than two lenses, an LED and a camera. With this system we were able to reliably detect the binding of anti-GFP to GFP, thus confirming a detection sensitivity in the range of protein-protein interactions. To enable future iGEM Teams to profit from this device as we did, we provide all plans and parts necessary to rebuild it on this website for everyonbe to download and use. ▼ Detailed Description.
To test our device we aimed at reproducing measurements that we were previously able to perform in our commercial device. For this reason we focused on using two antibodies: anti-GFP and polyclonal anti-rabbit. GFP and rabbit derived anti-HCV (Hepatitis C Virus) antibodies were used as antigens in this experiment. Note that the anti-HCV antibodies only served as binding partners for the anti-rabbit antibodies, since no HCV proteins were used in this experiment. The antigens were spotted onto an iRIf slide whose binding layer consisted of an APTES/PDITC surface. The spots on the slide were produced by pipetting 3.5 µl [1 mg/ml] rabbit-anti-HCV protein and 3.5 µl [1 mg/ml] GFP onto the slide and incubated overnight. After incubation the slide was blocked for 30 min in BSA solution. A Canon 50D camera was used to record the measurement and was set to take one picture every 5 seconds. The exposure time was set in order for the pixels in the image to be approx. 80% of maximum light saturation before the solution was flushed onto the chip. The antibody solutions were pipetted into the flow-chamber without the use of any microfluidic device. Instead, a syringe was loaded with 660 µl [5 µg/ml] anti-GFP antibody solution (diluted in PBS) and connected to the input pipe of our device. The content of the syringe was then slowly released via this pipe into the chamber of the device by gently dispensing the solution from the syringe. When the whole volume ran over the chip, the process was repeated with 660 µl [5 µg/ml] anti-rabbit antibody solution in the same way. The injection of both solutions was performed during approximately 45 minutes.
Video 1 shows the results of the measurement. Both, binding of anti-GFP and anti-rabbit to the corresponding antigen spots, could be observed. Due to the fact that the experiment was performed on our demonstration device which was built with a transparent casing, fluctuations in surrounding light had a strong, detrimental influence on the measurement quality. To minimize the influence of the unstable surrounding light, the resulting pictures had to be averaged over 10 pictures each. This in turn lead to a more stable light situation, however the signal strengh dropped as a consequence. Figure 3 shows a quotient picture of the measurement where the light situation was temporarily appropriate. The problem of surrounding light scattering into the device can of course be overcome using a non-transparent casing. An evaluation of how well our device performs in comparison with the professional device, was not performed, mainly due to time constraints. However our results hint that we get comparable results for strong binding such as GFP/anti-GFP, as can be seen in figure 4.
Establishing a Highly Specific Ni-NTA Surface
Figure 1: Cell-free expressed GFP-His spotted on a Ni-NTA slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free expression mix not containing DNA.
Since there is a variety of proteins present in the cell-free mix a specific surface on the future protein array is essential. That is why we established our Ni-NTA surface for the immobilization of the antigens on the chip. All expression constructs contain a His-tag fused to the coding sequence, resulting in antigens that can bind specifically to our Ni-NTA surface. Compared to an unspecific surface (PDITC) we could show that this Ni-NTA surface allows efficient binding of the target protein and prevents unspecific binding of other proteins that are present in the cell-free mix. ▼ Detailed description.
For cell-free protein expression lots of different proteins, like RNA-polymerases, ribosomes and other E. coli proteins, are essential. So during the process of cell-free expression all these proteins are, besides the target protein, also present in the microfluidic chamber. To have a sufficient amount of target protein immobilized on the chip we established a specific surface chemistry on the glass slide. After testing several tag systems we established a Ni-NTA surface because it worked best for us. Using a Ni-NTA covered glass slide (figure 1) we could increase the amount of bound cell-free expressed GFP-His compared to an unspecific surface (figure 2). In both experiments ____2.5 µg?!?____ of self purified GFP-His was used as positive control. The cell-free expression mix without DNA is supposed to result in no target protein and served as negative control. The cell-free reactions were performed for 2 hours at 37°C. The samples were pipetted by hand onto the surfaces and incubated on the slide overnight. In the iRIf device, the optical detection method we used, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity on the slide where the spotted protein was located represents a binding event. Due to less unspecific binding of proteins to the surface, the interaction of anti-GFP antibodies with cell-free expressed GFP-His is higher on the Ni-NTA surface.
▲ Close BoxProducing Our Cell-free Expression Mix
The mechanism we are using to copy a DNA array template into a protein array on demand is based on cell-free expression. During our project we successfully established our cell-free expression system, the DiaMIX, from scratch, starting with an E. coli lysate. With this mix we succeeded in expressing correctly folded GFP and luciferase. Comparison of the DiaMIX with a commercial cell-free expression kit revealed the great potential of our system! ▼ Detailed Description.
Our DiaMIX was prepared based on a protocol of Kigawa et al.
It is a complex system containing many different enzymes and chemicals for special purposes. An overview of cell-free expression systems and its components can be found on themethodology page.
The evaluation is shown in figure 1 and is based on triplicates. The data was normalized to the mean value of the measurement of air. The uncertainty was calculated using standard deviation. ÜBERARBEITEN JE NACH GRAPH
picture; Figure 1: Cell-free expression of GFP using the DiaMIX and a commercial kit. The reaction was performed in a volume of 50 µl each and monitored every minute at 37°C over a period of two hours. Excitation at 480 nm, emission at 520 nm.
Both expression systems were shown to successfully express the applied vector. There is a clear enhancement of light emission observable at 520 nm for both mixes in comparison to the respective negative control. The expression of GFP using the DiaMIX seems to be slightly better than by using the commercial kit. It is important to mention that the commercial kit is optimized to express linear templates. However, as the basis protocol for our mix was optimized for circular templates, we used a circular vector for the experiment.
Furthermore, as already shown above, we successfully verified the expression of GFP with our cell-free mix in an iRIf measurement.
To obtain these final results, a lot of optimization and testing had to be done. If you want to know more about how we established the DiaMIX for the DiaCHIP, you can find detailed information about the most important experiments on the cell-free expression results page.
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Explore the DiaCHIP
For establishing our device, we optimized all steps from the immobilization of DNA on the silicone of the flow-chamber to the specific binding of the target proteins on the glass slide. This way, we generated protein arrays we could use to detect different antibodies in human and rabbit serum with the iRIf detection method.
Click on the images below to explore our experiments from expression to detection!
Assembling the DiaCHIP
Diagnosis of Antigens
Click on one of the images to get a further insight on how we build our DiaCHIP
