Der folgnde Absatz liest sich sehr holperig. es wäre schön, wenn man da nochmal drüber gehen könnte und ein bisschen präziser formulieren würde. Prinzipiell nochmal hervorheben, dass es darum geht vor ort die Tests durchzuführen (vll noch in die ecke Ärzte ohne Grenzen bringen, von wegen schnellen test in Krisengebieten.
Hab mal dran gebastelt -> bitte korrigieren! (jb 20150909)
The detection of antibodies in a blood sample is a versatile and widely applied method of today's diagnostics 1) 2). Common methods include ELISA-assays which rely on the immobilization of antigens beforehand, requiring stable conditions for handling and storage of the plates as test reliability is crucially dependent on native-state proteins. Maintaining these conditions is already quite expensive under European standards, but becomes elaborate when working in tropical or arid regions.
Especially in those regions, where hospitals are mostly hard to reach and local medical treatment seldom relies on physicians, it is necessary to reliably distinguish between different illnesses as fast as possible. The treatment of potential epidemic pathogens requires an enormous logistic effort and differential diagnosis of a less potent pathogen may save thousands of dollars and prevent fear among people. Thus, it would be beneficial to have a large library of known antigens that is easy and cost-effective to store and to ship, but nonetheless functions as a reliable selective tool in diagnostics.
Our idea of how to tackle this problem is to use the unique properties of DNA as a robust information storage and combine it with cell-free expression inside a detection device. The antigens, produced on-demand directly before diagnosis, are not only less prone to denaturation, but are also enriched on the array in the device by a specific surface. Antibodies potentially present in the patient's blood are then observed by a new kind of detection method called iRIf that can be scaled down to a size as small as a laptop computer.
This portability makes the DiaCHIP useful for mobile applications especially in third world countries or regions of conflict where treatment decisions have to be made fast and on-site. The potentially low costs of the device render it affordable for organisations with trained personal such as "Medicines sans frontiers", replacing lots of bulky equipment and making it easier to get access to remote areas.
Hier vll noch spezifische Sekundärantikörper erwähnen, um zu unterscheiden zwischen impfung und aktuer krankheit.
done (20150909 jb)
Our project keeps many possible applications at hand if we will be able to further optimize the cell-free expression system and the specific binding to the surface for generating a protein chip. Improving the device towards a more quantitative method may allow medical professionals to determine the state of vaccination of a patient for a whole set of antigens, thus rendering some additional immunizations unnecessary.
The DiaCHIP could provide a tool for accurate analysis of the antibody titer of a person, thus helping to keep up a constant protection against many diseases.
As the iRIf technology only detects binding on the surface it is not possible to directly differentiate between existing antibodies due to an infection in the past or fresh antibodies resulting from an acute infection or a vaccination. A possible approach for dealing with this issue is the labeling of bound antibodies with proteins specific for one type of antibody, past infection related IgG or vaccination related IgM. Protein A may be such a candidate as it specifically binds to IgG but not to IgM. Additionally conventional secondary antibodies against both of these types may be used to cross-validate the results and adding an additional layer of reliablility to the test.
With further work on the DiaCHIP it could also be possible to distinguish between vaccines from different manufactures by spotting different epitopes of one antigen on the slide. This offers the opportunity to determine, whether the patient is vaccinated against a certain type of a virus. For expample the human papillomavirus (HPV) shows several subtypes (as HPV-16 and HPV-18 or HPV-11 and HPV-6) differing in the charactersitics of the disease. Knowing the vaccination status of a patient for these different subtypes could help to asses the personal risks for several diseases.
The DiaChip could also be used for pre-pregnancy test, where evaluation of the antibody titers against certain diseases like e.g. rubella or whooping cough is crucial. Here the safety of the unborn child relies on the health status of the mother, therefore missing vaccinations should be detected before considering a pregnancy. With our chip this may be a matter of one drop of blood and a test of two to three hours including the expression of antigens on the slide.
In the same way blood samples from blood banks or blood donations can be checked. And as the volume needed is very small checks can be performed in short-time intervals and nearly continuos monitoring of blood-quality may be possible.
Was die Zeitfrage angeht, so muss man aufpassen, wie der Workflow nachher wirklich ist. Geht man davon aus, dass die Proteine erst noch exprimiert werden müssen, kommen da schonmal 1-2h nur für die expression dazu. Dazu sieht es momentan so aus, dass man alleine 30 min zum blocken des slides braucht. Mit expression würde ich also sagen, dass das mindestens 3-4 h dauert ein Test...
Was man noch anbringen könnte wäre irgendwas bezüglich Grundlagenforschung. Man könnte mit der Methode Interaktoren von Signalproteinen/Transkriptionsfaktoren finden. einfach zellfrei eine cDNA library exprimieren, aufgereinigtes protein-of-interest drüber jagen und rausfinden mit was es interagiert.
Beyond fields of application in clinics as such, the suggested method of cell-free expression would simplify the preparation of protein microarrays on demand as no purification of protein is necessary anymore. This could be a major advantage in scientific research as handling and storage of protein arrays still poses some challenges. With our system of cell-free expression the production of protein arrays could provide an easy method to work with freshly produced protein chips.
As cDNA labraries are a common tool in basic research, combined with cell-free expression and immobilization on the glass slide, screenings for potential interacting proteins can be facilitated and improved through multiplexing applications.
In our iGEM project we were able to detect two diseases (Tetanus and Salmonella), but these are just two out of 1000 diseases our DiaCHIP will hold.
