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 20). 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 a fast aid in therapy choice while limiting costs per target. Today's method are all variations of one basic principle. Today's methods to guide medical professionals in their diagnosis 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 21). 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 22). 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 arrays thus provides information about antigen-antibody-interactions 23). Once more, antibody binding is detected by specific secondary antibodies labeled with a fluorescent dye. When immobilizing antigens on microbeads their identity has to 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 24). These microspheres are mixed and incubated with the 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 microbeads 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/Diagnostics"
m |
|||
Line 21: | Line 21: | ||
</p> | </p> | ||
<p> | <p> | ||
− | Medical diagnostics | + | Medical diagnostics comprise the whole process of tracing the source leading to a patients symptoms. Usually, it is understood as the identification of a pathogen or a malfunction responsible for the illness. <br> |
− | To achieve an efficient disaese treatment, clinical diagnostics are mostly divided into the following four steps: | + | To achieve an efficient disaese treatment, clinical diagnostics are mostly divided into the following four <strong>three?</strong> steps: |
</p> | </p> | ||
<p> | <p> | ||
Line 35: | Line 35: | ||
</p> | </p> | ||
<p> | <p> | ||
− | 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 | + | 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 bedside. The output of a test is immediately available – thus circumventing the usually necessary sending of samples to outside labs. |
The term POC encompasses many possible end-use settings outside of a centralized testing facility like emergency settings, regional health clinics, physicians practices as well as home or mobile use. | The term POC encompasses many possible end-use settings outside of a centralized testing facility like emergency settings, regional health clinics, physicians practices as well as home or mobile use. | ||
− | These tests consist of | + | These tests consist of common devices that are present in everyday 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 introduced 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. |
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. | 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. | ||
</p> | </p> | ||
Line 46: | Line 46: | ||
<p> | <p> | ||
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>. | 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>. | ||
− | As the DiaCHIP is an immunodiagnostics method we will focus on this part of diagnostic and compare commonly applied methods in today's clinics and labs. Immunodiagnostic is based on antigen-antibody interactions, that might be present within the body fluids of a patient. By detecting and identifying key proteins within a patients sample like blood or urine, these tests enable | + | As the DiaCHIP is an immunodiagnostics method we will focus on this part of diagnostic and compare commonly applied methods in today's clinics and labs. Immunodiagnostic is based on antigen-antibody interactions, that might be present within the body fluids of a patient. By detecting and identifying key proteins within a patients 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="#fn__7" id="fnt__7" name="fnt__7">7)</a></sup>. |
− | Immunodiagnostic is realized by immunoassays, which summarize a wide range of formats, allowing quantification and | + | 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__8" id="fnt__8" name="fnt__8">8)</a></sup>. Three prominent examples of immunoassays are lateral flow tests, ELISAs and minituarized immunoassays (microarrays). |
</p> | </p> | ||
Line 67: | Line 67: | ||
<br> | <br> | ||
The enzyme linked immunosorbent assay (ELISA) is seen as the state-of-the-art technique for serological diagnosis. ELISA is based on the interaction of a pathogenic antigen and its corresponding antibodies. | The enzyme linked immunosorbent assay (ELISA) is seen as the state-of-the-art technique for 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. This antigen can bind to a capture antibody that is immobilized on the surface of a microplate well. By incubation with a labelled primary or secondary antibody this antigen-antibody interaction can be detected and measured <sup><a class="fn_top" href="#fn__10" id="fnt__10" name="fnt__10">10)</a></sup>. ELISA is a rapid and sensitive test, most commonly used in serological diagnostics, e.g. for | + | The typically used “sandwich” ELISA requires an antigen with at least two binding sites. This antigen can bind to a capture antibody that is immobilized on the surface of a microplate well. By incubation with a labelled primary or secondary antibody this antigen-antibody interaction can be detected and measured <sup><a class="fn_top" href="#fn__10" id="fnt__10" name="fnt__10">10)</a></sup>. ELISA is a rapid and sensitive test, most commonly used in serological diagnostics, e.g. for Varicella Zoster <sup><a class="fn_top" href="#fn__11" id="fnt__11" name="fnt__11">11)</a></sup>, Hepatitis B <sup><a class="fn_top" href="#fn__12" id="fnt__12" name="fnt__12">12)</a></sup>, Toxoplasmosis <sup><a class="fn_top" href="#fn__13" id="fnt__13" name="fnt__13">13)</a></sup> and Ebola <sup><a class="fn_top" href="#fn__14" id="fnt__14" name="fnt__14">14)</a></sup>. |
<br> | <br> | ||
</p> | </p> | ||
Line 87: | Line 87: | ||
<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__20" id="fnt__20" name="fnt__20">20)</a></sup>. 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 a fast aid in therapy choice while limiting costs per target. | <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__20" id="fnt__20" name="fnt__20">20)</a></sup>. 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 a fast aid in therapy choice while limiting costs per target. | ||
Today's method are all variations of one basic principle. | Today's method are all variations of one basic principle. | ||
− | + | Today's methods to guide medical professionals in their diagnosis 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 <sup><a class="fn_top" href="#fn__21" id="fnt__21" name="fnt__21">21)</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__22" id="fnt__22" name="fnt__22">22)</a></sup>. 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 arrays thus provides information about antigen-antibody-interactions <sup><a class="fn_top" href="#fn__23" id="fnt__23" name="fnt__23">23)</a></sup>. Once more, antibody binding is detected by specific secondary antibodies labeled with a fluorescent dye. | 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 arrays thus provides information about antigen-antibody-interactions <sup><a class="fn_top" href="#fn__23" id="fnt__23" name="fnt__23">23)</a></sup>. Once more, antibody binding is detected by specific secondary antibodies labeled with a fluorescent dye. | ||
When immobilizing antigens on microbeads their identity has to 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__24" id="fnt__24" name="fnt__24">24)</a></sup>. These microspheres are mixed and incubated with the 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 microbeads color and the second the signal strength from the fluorescence labeled secondary antibody, again full information about the interaction is achievable. | When immobilizing antigens on microbeads their identity has to 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__24" id="fnt__24" name="fnt__24">24)</a></sup>. These microspheres are mixed and incubated with the 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 microbeads color and the second the signal strength from the fluorescence labeled secondary antibody, again full information about the interaction is achievable. |
Revision as of 15:57, 11 September 2015
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_wiki_background_2_parted_left.png)
![](https://static.igem.org/mediawiki/2015/f/f4/Freiburg_wiki_background_2_parted_right.png)
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.
What are diagnostics
(Bild Diagnostik das auch in Präsentation erscheint im Intro)
Medical diagnostics comprise the whole process of tracing the source leading to a patients symptoms. Usually, it is understood as the identification of a pathogen or a malfunction responsible for the illness.
To achieve an efficient disaese treatment, clinical diagnostics are mostly divided into the following four three? steps:
- A clinician, interviewing the patient and considering his medical history, risk factors and current problems, proposes a certain differential diagnosis, thus limiting the spectrum of possible diseases.
- This is usually followed by the actual performance of diagnostic tests to reinforce the differential diagnosis and to further limit the list of possible causes.
- 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 bedside. The output of a test is immediately available – thus circumventing the usually necessary sending of samples to outside labs. The term POC encompasses many possible end-use settings outside of a centralized testing facility like emergency settings, regional health clinics, physicians practices as well as home or mobile use. These tests consist of common devices that are present in everyday 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 introduced 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.
What are current diagnostic methods
Four most common centralized laboratory techniques are blood chemistry, immunoassays, nucleic-acid amplification tests and flow cytometry 6). As the DiaCHIP is an immunodiagnostics method we will focus on this part of diagnostic and compare commonly applied methods in today's clinics and labs. Immunodiagnostic is based on antigen-antibody interactions, that might be present within the body fluids of a patient. By detecting and identifying key proteins within a patients sample like blood or urine, these tests enable to distinguish between major classes of diseases, like infectious diseases, metabolic diseases, cardiovascular diseases or cancer 7). 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 8). 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. A membrane or paper strip is used to indicate the presence of proteins markers like antigens or antibodies. Prominent examples consist of: Pregnancy tests, HIV diagnostics in developing countries 9) or blood-glucose tests.
ELISA - sensitive but time consuming, no multiplex:
The enzyme linked immunosorbent assay (ELISA) is seen as the state-of-the-art technique for 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. This antigen can bind to a capture antibody that is immobilized on the surface of a microplate well. By incubation with a labelled primary or secondary antibody this antigen-antibody interaction can be detected and measured 10). ELISA is a rapid and sensitive test, most commonly used in serological diagnostics, e.g. for Varicella Zoster 11), Hepatitis B 12), Toxoplasmosis 13) and Ebola 14).
Minituarized Immunoassay (Microarray) - Lab-On-a-Chip (LOC):
A microarray is a multiplexed Lab-On-a-Chip (LOC) ((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 15)16). Today miniaturized immunoassays are one of the most important analysis platform for proteins17).
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 analysis 18). Various LOC diagnostic modules have been integrated within fluidic chips, providing devices with immense multiplexing facilities and functionality 19).
However, up to this date microfluidics and LOC systems have not yet fulfilled people’s expectations to revolutionize the healthcare industry.
As diagnostic systems are a broad and diverse field of methods and techniques, here only covered in broad terms, we wrote a more detailed introduction for one specific case of immundiagnostics: the detection of autoimmune diseases.
What are the 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. 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 shown to have a great potential regarding future diagnostics 25). Yet, immunoassays based on peptides suffer from instability and storage issues. Furthermore, the evaluation of a microarray based immunoassay is often time consuming and requires skilled personal.
In contrast, a fast diagnosis is essential for an immediate onset of appropriate treatments, 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 expanses 26) are due 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 of the developing countries 27).
Thus, a large part of the substantial progress that has been made in the public health and POC sector has only been advantageous to the more developed part of the world.
According to the WHO 28) 2.5 out of 6 billion people lack basic sanitation, 2 billions do not have access to electricity and more than 1 billion lacks 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 senistive enough for the use outside of air-conditioned laboratories, may be a huge leap forward to improving the 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 29).
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 30)! However, the currently available developed-world standards for diagnosis of infectious diseases often prove 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! 31). 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. hier evtl in Fahrenheit angeben
- 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 Dia-Chip contribute a solution to these problems
Our approach basically combines three promising techniques in one Dia-Chip 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 immense multiplexing is possible with microfluidic based LOC systems. Copying from DNA template (Link): Storing and handling problems of conventional peptide based microarrays are circumvented by directly producing our peptide array from a DNA array. 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 peptide information. This allows us to offer different combinations of antigens, providing the optimal detection system for most needs. iRIf detection method (Link): This emerging detection method enables a fast, sensitive and label free detection of binding processes. Binding of the first, 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.
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 basical feasibility of such a device with our own rebuild (Link). 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.