For choosing the right kind of therapy for a patient suffering from an autoimmune disease, knowing what kind of antibodies are targeting his own cells is crucial. This task is severely complicated by the fact, that through a pathway of inter- and intramolecular epitope spreading the variability of autoantibodies is greatly increased 15). Therefore, a diagnostic assay not only has to test for one kind of target but for a whole spectrum. This leads to a high demand for multiplexed detection systems that may detect all autoantibodies at once, thus providing fast aid in therapy choice while limiting costs per target and amount of sample material. Today's methods to guide medical professionals in their diagnoses of autoimmune diseases are all variations of the same basic principle: Autoantibodies from a patient’s blood sample are captured by antigens derived from HEP-2 or liver cell lines and immobilized on a solid support 16). In the Line Immunoassay (LIA) approach antigens are applied as thin lines on cut nylon membranes. These membrane stripes are then flushed with a sample to be analyzed and processed as in conventional immunoblotting 17). Scaling down this approach results in immobilization of the antigens on protein microarrays or microbeads. For protein microarrays, antigens are spotted using the same techniques as in manufacturing of DNA microarrays. The identity of each antigen is encoded by its location on the array. The detection pattern on the array thus provides information about antigen-antibody-interactions 18). Once more, antibody binding is detected by specific secondary antibodies labeled with a fluorescent dye. When immobilizing antigens on microbeads their identity can be encoded by different techniques. In Laser-microbead-arrays microspheres of up to 100 different laser-reactive colors are coated with one type of antigen each, thus keeping the identity information 19). These microspheres are mixed and incubated with a serum sample and a fluorescence labeled secondary antibody before analysis with dual laser flow cytometry. As the first laser reads the identity information from the microbead's color and the second the signal strength from the fluorescence labeled secondary antibody, again full information about the interaction is achievable.
Difference between revisions of "Team:Freiburg/Project/Diagnostics details"
| Line 245: | Line 245: | ||
Core characteristics of the device are its simplicity, the low cost of the components and the fast outcome of the results. Even though it is still in an experimental stage, some improvements may render it easy to handle, even for untrained users. | Core characteristics of the device are its simplicity, the low cost of the components and the fast outcome of the results. Even though it is still in an experimental stage, some improvements may render it easy to handle, even for untrained users. | ||
</p> | </p> | ||
| − | + | ||
| Line 333: | Line 333: | ||
Guerrant et al., 2001.Practice guidelines for the management of infectious diarrhea. Clinical Infectious Diseases, Vol. 32. doi: 10.1086/318514</a></div> | Guerrant et al., 2001.Practice guidelines for the management of infectious diarrhea. Clinical Infectious Diseases, Vol. 32. doi: 10.1086/318514</a></div> | ||
</div> | </div> | ||
| − | + | </div> | |
</html> | </html> | ||
<!-- Labjournal content ends here --> | <!-- Labjournal content ends here --> | ||
{{Freiburg/wiki_content_end}} | {{Freiburg/wiki_content_end}} | ||
Revision as of 02:08, 17 September 2015
Diagnostics Today
Today's Applications of Immunodiagnostics
In the poorest countries of the world, more than half the deaths are due to infectious diseases – in the wealthiest countries it is less than 5%. Literatur einfügen.
The Field of Diagnostics
(Bild Diagnostik das auch in Präsentation erscheint im Intro)
Medical diagnostics comprise the whole process of tracing the source leading to a patient's symptoms. Usually, it is understood as the identification of a pathogen or a malfunction responsible for the illness.
To achieve an efficient disease treatment, clinical diagnostics are mostly divided into the following steps:
- A clinician, interviewing the patient and considering his medical history, risk factors and current problems, proposes a certain differential diagnosis, thus pre-limiting the spectrum of possible diseases.
- This is usually followed by performance of diagnostic tests (normally in a central laboratory) to confirm the differential diagnosis and to clearly identify or at least further limit possible causes of the symptoms.
- Finally, this leads to a treatment consisting of medication, surgery, hospitalization or discharge.
Interest in the so called Point-Of-Care (POC) diagnostics increased dramatically 1)2) in the last decades. POC diagnostics (or bedside diagnostics) are diagnostic tests that can be performed directly at a patient's side or even bedside. The output of such a test is immediately available – thus circumventing the usually necessary sending of samples to external labs.
The term POC encompasses many possible end-use settings outside of a centralized testing facility like emergency settings, regional health clinics, medical practices as well as home or mobile use.
These tests consist of common devices that are present in everydays life, such as AB0-testing, blood glucose testing, blood gas and electrolytes analysis, pregnancy testing and cholesterol screening. For the near future an increase in the amount of products for POC diagnostic is predicted 3), thereby confirming the need for such applications.
In the face of aging populations, spreading of infectious diseases especially in the developing world, biohazard threats and increasing numbers of autoimmune diseases and allergies in the developed world 4) 5) POC tests become inevitable.
But these devices also are convenient methods for improving everyday life: a drop of blood could easily reveal the physical cause of feelings of discomfort, such as a the lack of certain metabolic substances. A simple diet suggestion thus may increase life quality and health in one go.
Current Diagnostic Methods
The four most common centralized laboratory techniques are blood chemistry, immunoassays, nucleic-acid amplification tests and flow cytometry 6). As the DiaCHIP is an immunodiagnostic method we will focus on this part of diagnostics and compare it to commonly applied methods in today's clinics and labs. Immunodiagnostic is based on antigen-antibody interactions, which might be present within the body fluids of a patient. By detecting and identifying key proteins within a patient's sample like blood or urine, these tests enable to distinguish between major classes of diseases, like infectious diseases, metabolic diseases, cardiovascular diseases or cancer 2). Immunodiagnostic is realized by immunoassays, which summarize a wide range of formats, allowing quantification and monitoring of small molecules, large proteins and even whole pathogens 6). Three prominent examples of immunoassays are lateral flow tests, ELISAs and minituarized immunoassays (microarrays).
Lateral flow test - simple and rapid, no multiplex, limited sensitivity: The lateral flow test is commonly known as strip test. It is rather complicated in its setup, but extremely easy to use, as only a drop of liquid has to be added to get an easy interpretable result within minutes. The test confirms its validity and the presence or absence of the target molecule through the appeareance of colored stripes. Prominent examples are pregnancy tests, but also drug-abuse tests, HIV diagnostics in developing countries 3) or blood-glucose tests. Stripe tests can be seen as the gold standard for Point of Care devices - easy to store, use and easy to read out.
ELISA - sensitive but time consuming, no multiplexing:
The enzyme linked immunosorbent assay (ELISA) is seen as the state-of-the-art technique for highly sensitive serological diagnosis. ELISA is based on the interaction of a pathogenic antigen and its corresponding antibodies.
The typically used “sandwich” ELISA requires an antigen with at least two binding sites and a pair of antibodies binding these sites. First a capture antibody is immobilized on the surface of a microplate well. After incubation with the sample and the binding of the respective antigen, the seconardy antibody is added. Either this secondary antibody or a third one, binding the second, yields a signal enhancement, mostyl by enzyme coupled reactions. This procedure increases the sensitiviy 10,000 fold down to pg/mL scales7). ELISA is a very sensitive and specific test, most commonly used in serological diagnostics, e.g. for Varicella Zoster 8), Hepatitis B 9), Toxoplasmosis 10) and Ebola 11). Depending on the assay protocol used, a whole ELISA can be carried out within some hours to one day.
Minituarized Immunoassay (Microarray) and Lab-On-a-Chip (LOC):
Lab on a Chip (LOC) refers to the idea, that many processes in the lab can be improved and automated by miniaturizing them on or into a chip. A microarry is a multiplexed or multiprallel LOC-device((https://en.wikipedia.org/wiki/Lab-on-a-chip|Wikipedia Article on Lab-on-a-chip)).
Many scientists see LOC-based methods to be the most likely technological driver to fundamentally transform the Point-Of-Care diagnostic industry 12)13). Today miniaturized immunoassays are one of the most important analysis platforms for proteins2).
The development of Lab-On-a-Chip systems is closely linked to the emergence of microfluidics. Microfluidic techniques use small, compact, low-power and mass-producible chips which are designed for small samples sizes and rapid and sensitive analyses 14). Various LOC diagnostic modules have been integrated within fluidic chips, providing devices with immense multiplexing facilities and functionality 3).
However, up to this date complex microfluidics and LOC systems have not yet fulfilled people’s expectations to revolutionize the healthcare industry, even though more simplistic lateral flow assays are a huge succes.
For diagnostic systems, a broad and diverse field of methods and techniques is available. As this is far more than we are able to describe here, we want to refer to the overview article of Roth et al. (()). Further on, we will focus on immunodiagnostics as this is the main application field for the DiaCHIP. To start, we will introduce one specific field of immunodiagnostics: the detection of autoimmune diseases.
Limitations of Currently Available (Immunodiagnostic) Tests
Even though current diagnostic methods provide reliable information on a broad range of diseases, there are still applications where current methods suffer from various restrictions. The commonly used ELISA only provides a limited capacity for multiplexing (as only one interaction per well may be detected) and takes at least several hours and large amouns of sample as well as antibodies (about 0.3 µg antibody per data point ((G. Roth, personal communication)). Lateral flow tests are much faster but can only detect one molecule of interest and are known to perform poorly in terms of sensitivity. Miniaturized immunoassays (microarrays) combined with microfluidic bioanalysis have been shown to have a great potential regarding future diagnostics 2). Yet, immunoassays based on peptides suffer from poor peptide purity (and thus high unspecificity), instability and storage issues. Furthermore, the diagnostics by microarray based immunoassays is scarcely used for rare disease cases after conventional ELISA tests have proven no result. In contrast, a fast diagnosis is essential for an immediate onset of an appropriate treatment, that can be critical for the patients' health and life. Moreover, improved diagnostics are not only required regarding the health of a patient: 70% of healthcare expenses 18) are linked to diagnostic tests. Therefore, improvements in diagnostic technologies have the potential to drastically reduce the overall healthcare costs while increasing health as such. Diagnostic tests are usually developed for the utilization in air-conditioned laboratories with refrigerated storage of chemicals, a constant supply of calibrators and reagents, highly trained personal and rapid transportation of samples. This setting is not available for most developing countries 20). Thus, most of the substantial progress that has been achieved in the public health and POC sector has only been advantageous to the more developed part of the world. According to the WHO 24), 2.5 out of 6 billion people lack basic sanitation, 2 billions do not have access to electricity and more than 1 billion lack basic healthcare services and clean drinking water. Moreover, 50% of all deaths in the most impoverished developing countries are a result of infectious diseases, whereas in the wealthiest developed countries this concerns less than 5%. Therefore, transforming existing technologies into mobile applications, robust and sensitive enough for the use outside of specified laboratories, may be a huge leap forward to improving general health all over the world. Outbreaks and spreading of potential epidemic diseases or sexually transmitted infections can be controlled by rapid diagnosis and appropriate treatment 6).
A need for such technologies is urgent: 500 million people between the age of 15 to 49 are infected with curable sexually transmitted infections like chlamydia, gonorrhea, syphilis or trichonomiasis each year 21)! However, the currently available infrastructure for diagnosis of infectious diseases often prove to be too slow and expansive to be practicable for third world countries. For example the identification of pathogens of an infectious diarrhoe takes 2-4 days – even in the best developed-world laboratory! 22). To be used in low-resource settings future diagnostic methods have to have certain properties as outlined below:
- Rapidity/Speed - a fast diagnosis reduces the time until the beginning of treatment, preventing the spread of epidemic diseases and reducing the severity of a disease.
- Simplicity - the necessary handling should be as easy as possible.
- Low-costs - POC diagnostics need to be affordable for the utility at home or in developing countries.
- Clearness - The output of POC tests needs the clarity and simplicity as a yes/now answer.
- Storability under extreme conditions - As defined conditions may not be required, the device has to be stable under extreme conditions, with temperatures ranging from 10 to 40°C (50 to 104°F).
- Multiplexed test - Covering a broad spectrum of possible diseases, ideally in one device, allows for differential diagnosis even in the case of different diseases with similar symptoms.
How can our DiaCHIP contribute to the solution to these problems?
Our approach basically combines three promising techniques in one DiaCHIP device, offering a great potential to improve future diagnostics. Miniaturized immunoassays combined with microfluidics: Miniaturized immunoassays enable for immense multiplexing. By immobilizing hundreds of different antigens, it is possible to screen a patient’s sample for hundreds of potential antibodies and related diseases. With small volumes of reagents and samples, a rapid delivery of results with fast turnover times and enormous multiplexing is possible with microfluidic based LOC systems. Microarray Copying (generate proteins from DNA templates): Storing and handling problems of conventional peptide based microarrays are circumvented by directly producing our protein array from a DNA array via cell-free expression. As DNA is stable within a large range of temperatures, pH values and other environmental conditions, it proves to be the ideal molecule for storing protein information. This allows us to offer different combinations of antigens, providing the optimal detection system for most needs and producing them on demand. iRIf detection method: This emerging detection method enables a fast, sensitive and label free detection of binding processes. Binding of the serum originated, antibody can be detected directly, omitting the incubation of the sample with a second detection antibody, thus making the detecting cheaper and faster. Nevertheless, the signal can be further amplified with a secondary antibody, if needed or specificity of binding remains unclear. But even then only this second antibody without any fluorescent or enzymatic labeling is nedded.
Outlook
The future of diagnostics may lie in home-care devices based on microfluidic lab-on-a-chip systems. These are supposed to perform assays at a sensitivity, specificity and reproducibility similar to those of central laboratory analyzers. However, the user only needs to insert one drop of blood. Especially people in developing countries could perform routine testing to detect the presence of infectious pathogens like influenza or sexually transferable diseases like HIV or Syphilis (Yager et al., 2006). The DiaCHIP device may be scaled down to a size suitable for smaller medical practices or mobile applications. Thereby it complements the existing techniques that on the one hand are small and handy, but only able to detect a limited spectrum of antibodies and on the other hand are so huge, that an efficient use is only possible in clinical facilities. We showed the basic feasibility of such a device with our own rebuild setup. Core characteristics of the device are its simplicity, the low cost of the components and the fast outcome of the results. Even though it is still in an experimental stage, some improvements may render it easy to handle, even for untrained users.
References
