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− | <div class="content_box">
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− | <!-- EDIT1 SECTION "Today's Applications of Immunodiagnostics" [1-206] -->
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− | <h2 class="sectionedit2">The Field of Diagnostics</h2>
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− | <p>
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− | (Bild Diagnostik das auch in Präsentation erscheint im Intro)
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− | 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. <br>
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− | To achieve an efficient disease treatment, clinical diagnostics are mostly divided into the following steps:
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− | <ul>
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− | <li>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.
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− | <li>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.
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− | <li >Finally, this leads to a treatment consisting of medication, surgery, hospitalization or discharge.
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− | </li>
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− | </ul>
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− | Interest in the so called Point-Of-Care (POC) diagnostics increased dramatically <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup><sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup> 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.
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− | 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.
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− | 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 <sup><a class="fn_top" href="#fn__3" id="fnt__3" name="fnt__3">3)</a></sup>, thereby confirming the need for such applications.
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− | 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 <sup><a class="fn_top" href="#fn__4" id="fnt__4" name="fnt__4">4)</a></sup> <sup><a class="fn_top" href="#fn__5" id="fnt__5" name="fnt__5">5)</a></sup> POC tests become inevitable. <br>
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− | 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.
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− | <!-- EDIT2 SECTION "What are diagnostics" [207-3044] -->
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− | <h2>Current Diagnostic Methods</h2>
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− | <p>
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− | The four most common centralized laboratory techniques are blood chemistry, immunoassays, nucleic-acid amplification tests and flow cytometry <sup><a class="fn_top" href="#fn__6" id="fnt__6" name="fnt__6">6)</a></sup>.
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− | As the <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/System" title="DiaCHIP">DiaCHIP</a> 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 <sup><a class="fn_top" href="#2" id="fnt__7" name="fnt__2">2)</a></sup>.
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− | 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 <sup><a class="fn_top" href="#fn__6" id="fnt__8" name="fnt__6">6)</a></sup>. Three prominent examples of immunoassays are lateral flow tests, ELISAs and minituarized immunoassays (microarrays).
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− | </br>
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− | <div class="image_box left">
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− | <span class="kommentar_stefan"> Bilder wären super. (stefan)</span>
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− | (Bild lateral flow test?!)
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− | <p>
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− | <strong>Lateral flow test - simple and rapid, no multiplex, limited sensitivity</strong>:
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− | 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 <sup><a class="fn_top" href="#fn__3" id="fnt__9" name="fnt__3">3)</a></sup> 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.
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− | </br>
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− | <div class="image_box left">
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− | (Bild vom ELISA)
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− | <p>
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− | <strong>ELISA - sensitive but time consuming, no multiplexing</strong>:
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− | </br>
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− | 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.</br>
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− | 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 scales<sup><a class="fn_top" href="#fn__7" id="fnt__10" name="fnt__7">7)</a></sup>. ELISA is a very sensitive and specific test, most commonly used in serological diagnostics, e.g. for <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#Zoster">Varicella Zoster</a> <sup><a class="fn_top" href="#fn__8" id="fnt__11" name="fnt__8">8)</a></sup>, Hepatitis B <sup><a class="fn_top" href="#fn__9" id="fnt__12" name="fnt__9">9)</a></sup>, Toxoplasmosis <sup><a class="fn_top" href="#fn__10" id="fnt__13" name="fnt__10">10)</a></sup> and Ebola <sup><a class="fn_top" href="#fn__11" id="fnt__14" name="fnt__11">11)</a></sup>. Depending on the assay protocol used, a whole ELISA can be carried out within some hours to one day.
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− | <br>
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− | </p>
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− | </br>
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− | <strong>Minituarized Immunoassay (Microarray) and Lab-On-a-Chip (LOC)</strong>:
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− | </br>
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− | 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)).
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− | <br>
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− | Many scientists see LOC-based methods to be the most likely technological driver to fundamentally transform the Point-Of-Care diagnostic industry <sup><a class="fn_top" href="#fn__12" id="fnt__15" name="fnt__12">12)</a></sup><sup><a class="fn_top" href="#fn__13" id="fnt__16" name="fnt__13">13)</a></sup>. Today miniaturized immunoassays are one of the most important analysis platforms for proteins<sup><a class="fn_top" href="#fn__2" id="fnt__17" name="fnt__2">2)</a></sup>.</br>
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− | 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 <sup><a class="fn_top" href="#fn__14" id="fnt__18" name="fnt__14">14)</a></sup>. Various LOC diagnostic modules have been integrated within fluidic chips, providing devices with immense multiplexing facilities and functionality <sup><a class="fn_top" href="#fn__3" id="fnt__19" name="fnt__3">3)</a></sup>. </br>
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− | 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.
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− | </br>
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− | 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 <i>et al.</i> (()). 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.
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− | <div class="accordion">
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− | <div class="accordion-section">
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− | <a class="accordion-section-title" href="#accordion-1">Autoimmune diseases require sophisticated diagnostics</a>
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− | <div id="accordion-1" class="accordion-section-content" style="display:none; padding:15px;">
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− | <p>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 <sup><a class="fn_top" href="#fn__15" id="fnt__20" name="fnt__15">15)</a></sup>.</br> 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.
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− | </br>
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− | </br>
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− | Today's methods to guide medical professionals in their diagnoses of autoimmune diseases are all variations of the same basic principle:
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− | 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 <sup><a class="fn_top" href="#fn__16" id="fnt__21" name="fnt__16">16)</a></sup>. 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 <sup><a class="fn_top" href="#fn__17" id="fnt__22" name="fnt__17">17)</a></sup>. Scaling down this approach results in immobilization of the antigens on protein microarrays or microbeads.
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− | </br>
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− | 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 <sup><a class="fn_top" href="#fn__18" id="fnt__23" name="fnt__18">18)</a></sup>. Once more, antibody binding is detected by specific secondary antibodies labeled with a fluorescent dye.
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− | </br>
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− | 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 <sup><a class="fn_top" href="#fn__19" id="fnt__24" name="fnt__19">19)</a></sup>. 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.
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− | </div><!-- end accordion-section-content -->
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− | </div> <!-- end accordion -->
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− | <div class="content_box">
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− | <!-- EDIT5 SECTION "How can our DiaCHIP contribute a solution to these problems" [16352-17909] -->
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− | <h2 class="sectionedit6">Outlook</h2>
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− | <p>
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− | 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 <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#AIDS" title="AIDS">HIV</a> or <a clas="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#Syphilis" title="Syphilis">Syphilis</a> (Yager et al., 2006).
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− | </br>
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− | </br>
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− | 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 <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Results/Own_Device" title="own_device">own rebuild setup</a>.
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− | 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.
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