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 analysed the blood serum of a person that was vaccinated against tetanus for the presence of anti-tetanus antibodies. First we confirmed the binding of antibodies present in the blood to our purified tetanus antigen by ELISA. Then we spotted the antigen and GFP, which both were expressed in E.coli and purified by His-tag affinity purification, on a PDITC surface. The much stronger binding of blood components to the tetanus antigen spots compared to GFP spots indicates specific interactions. By confirming that the bound blood components are human antibodies with anti-human, we successfully demonstrated that we are able to detect antibody binding even in complex samples. ▼ Detailed Description.
Figure 2: Western Blot of his-tagged C. tetani antigen with anti His-tag HRP Conjugate. The soluble fraction of the overexpressed protein was used for this Western Blot. The expected molecular weight is 50 kDa. Anti- His Tag HRP conjugated was used in a 1:1000 dilution in blocking buffer.
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 2). 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 that bind specific to our purified tetanus antigen. Therefore, we spotted tetanus antigen and bBSA of the same concentration (12.5 µg/mL) and 2 times 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 Streptavidin (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.
After the validation of the antigen antibody binding for tetanus through ELISA we wanted to see if we can detect this interaction also with iRIf. Therefore we first expressed our Clostridium tetani antigen and GFP in E.coli. Both proteins were purified, using the His tag we fused to them, and spotted on a PDITC surface. The slide with the immobilized proteins was flushed with human blood serum, and anti human antibodies. Strong binding was detected for the tetanus antigen spots, while the GFP spots only exhibited minimal binding (see figure 1A-D). This indicates that components contained in the blood serum bind specifically to the tetanus antigen. The subsequent binding of anti human antibodies to the tetanus antigen spots (figure 2) confirms that the proteins that bound during the blood serum step are antibodies. We therefore successfully demonstrated that it is possible to detect the binding of antibodies from blood serum to antigens, we immobilized on a surface.
With an additional experiment we wanted to see if we, in principle, can differentiate the vaccination status of patients with our system. Therefore we used blood serum samples, taken before and two weeks after a tetanus boost vaccination. A slide with immobilized tetanus antigen and GFP was prepared (see figure 3). It was consecutively flushed with both blood sera. The individual binding of the two sera is seen in figure 3B and 3C. The blood serum taken after vaccination exhibits a much more intense binding to the tetanus spot than the serum taken before binding. This observation is supported by the binding curves obtained by this measurements (see figure 3D). The validation that the exhibited binding, shown by the protein spots during the exposure to blood serum is due to antibodies could not be performed, due to problems with the anti human antibody supply.
While the outcome of this first experiment looked promising for our future application to determine the vaccination status of patients, further experimentation did not confirm these initial results. We were still able to detect binding of both blood sera to the tetanus antigen, but no difference in binding intensity was visible (See figure 4B and C). To test the assumption that the used blood was not fresh enough and therefore lost some of its binding capabilities a fresh blood sample five weeks after vaccination was taken for the experiment shown in figure 4. 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.
Detection of Anti-GFP Antibodies using Cell-Free Expressed GFP on Ni-NTA
Our self-made cell-free expression mix, the DiaMIX, in combination with specific glass surfaces allow the expression and immobilization 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 stable on the surface. This is shown during an iRIf measurement: Antibodies against GFP are flushed over the slide to detect GFP that is present on the slide. The specific binding of anti-GFP antibodies to the control spot (purified GFP) and the cellfree expressed GFP confirms the success of cellfree GFP expression and its immobilization on the surface.
▼ 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).
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.
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.
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.
The quotient pictures shows all intensity shifts that occured during the measurement. Two left spots: GFP spots at which an intensity shift occured during anti-GFP flush. Right spot: anti-HCV antibody (derived from rabbit), at which an intensity shift occured during the flush with anti-Rabbit.
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.
▲ Close BoxEstablishing 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 1.5 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 use, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity 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
A special feature of our DiaCHIP is the on-demand production via cell-free expression. Thereby, a fixed DNA template is copied into our desired antigen, which in turn can bind to our specific surface. Our expression mix called DiaMIX was established from scratch. It is based on an E. coli lysate. In a direct comparison with a commercial product our mix showed high expression rates, indicating its performance capability (fig.1). ▼ Detailed Description.
Our DiaMIX is a complex system consisting of several individually adjusted components that allow us to make use of the cells own production system, without the tedious drawbacks of normal cell-based protein expression.
As a main component of the DiaMIX the E. coli lysate contains the necessary transcription ans translation apperatus. The lysate is complemented by additives needed for energy regeneration, buffering and generation of the protein. An overview of cell-free expression systems and its components can be found on our project pages.
As ultimate evaluation, we compared the DiaMIX to a commercial kit as seen in figure 1.
Both expression systems successfully expressed the applied vector.
Furthermore, the cell-free expression of GFP with our cell-free mix could be verified in an iRIf measurement.
To obtain these final results, a lot of optimization and testing had to be done. Want to know more about how we established the DiaMIX? You can find detailed information about the most important experiments on the cell-free expression results page.
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
