Team:Freiburg/Project/Future Directions
Future Directions
Room for Improvement
The detection of antibodies in a blood sample is a versatile and widely applied method of today's diagnostics. Common methods include ELISA, an immunodiagnostic method based on the interaction of antibodies and corresponding antigens. As test reliability is crucially dependent on native-state proteins 1), ELISA plates require stable conditions for handling and storage. Maintaining these conditions is already quite expensive under European standards but becomes elaborate when working in tropical or arid regions.
Especially in regions, where hospitals are hard to reach and local medical treatment seldom relies on
physicians, it is necessary to reliably distinguish different illnesses as fast and accurate 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 and is additionally functional as a reliable and selective tool in diagnostics.
The DiaCHIP - On the Way Towards Future 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 microfluidic device. At the same time, this device is used for detection of antibodies in a label-free manner. The antigens produced on demand directly before diagnosis are not only less prone to time-related denaturation but also enriched on the array due to a specific surface. Antibodies potentially present in the patient's blood are then observed by a new kind of detection method called iRIf (imaging Reflectometric Interference). The associated measurement device can be scaled down to a size smaller than a shoebox and a price affordable for almost everybody. This portability makes the DiaCHIP useful for mobile applications especially in third world countries or regions of conflict where decisions for the appropriate treatment have to be taken fast and on-site. The potentially low costs of the device render it affordable for organisations with trained personnel such as Doctors Without Borders, replacing lots of bulky equipment and making it easier to operate in solitary areas.
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 array.
Determining the Status of Vaccination
Improving the device in terms of quantification may allow medical professionals to determine a patient's status of vaccination for a whole set of pathogens, thus possibly rendering some vaccination boosts unnecessary.
The DiaCHIP could provide a tool for accurate analysis of the antibody titer of a person, thereby helping to keep up a constant protection against many diseases.
With the iRIf technology antibody-antigen interactions are detected. Nonetheless, there is no possibility of differentiating between antibodies that were already present due to a previous infection or vaccination, or ones that were developed due to an acute infection. A possible approach for dealing with this issue is the labeling of bound antibodies with proteins that bind specifically to one class of antibodies. Antibodies resulting form a previous infection or vaccination belong to the IgG class, those of an acute infection to the IgM class. Protein A may be a candidate for this purpose as it specifically binds to IgG but not to IgM. Additionally, conventional secondary antibodies targeting different classes of human antibodies may be used to cross-validate the results and thereby add an additional layer of reliablility to the test.
Investing further work on the DiaCHIP could make it 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 subtype of a virus. For example, the Human Papilloma Virus (HPV) shows several subtypes (such as HPV-6, HPV-11, HPV-16 or HPV-18) differing in the characteristics of the disease. Knowing the vaccination status of a patient for these different subtypes could help to asses the personal risks for several diseases 2) .
Pre-Pregnancy Testing
The DiaCHIP could additionally be used for pre-pregnancy testing where the evaluation of vaccination statuses for a whole set of diseases like rubella or whooping cough is crucial. In this case, the safety of the unborn child relies on the health status of the mother. Therefore, a lack of vaccination should be detected when a pregnancy is considered. With our DiaCHIP this may be a matter of one drop of blood and only one test, requiring a maximum of two to three hours including the expression of antigens on the slide.
Blood Sample Analysis
Blood samples from blood banks or blood donations can be checked for multiple infectious diseases in the same way. Since the required volume is very small, checks can be performed in short-time intervals and continuous monitoring of blood-quality may be possible.
Furthermore, one could develop a pre-test for critical diseases before donating blood. Like this, donations of infected blood could be prevented from the beginning.
Providing Access to Fresh Protein Arrays for Laboratory Use
Beyond applications in the field of clinics as such, the suggested method of cell-free expression would simplify the preparation of customized protein microarrays on demand as no purification of proteins would be necessary anymore. This could be a major advantage in scientific research as handling and storage of protein arrays still pose 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 3) libraries are a common tool in basic research this can be easily combined with our system of cell-free expression and immobilization of proteins on a glass slide. With this set-up, screenings for potentially interacting proteins can be facilitated and improved by making use of multiplexing applications.
In our iGEM project we were able to detect antibodies specific for two diseases: tetanus and salmonellosis - but these are only two out of 1000 diseases our DiaCHIP will hold in the future.
