The 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. At 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, if the antigen was present in the sample. This procedure increases the sensitivity about 10,000 fold down to pg/ml scales
8)
.
ELISA is a very sensitive and specific test, most commonly used in serological diagnostics, e.g. for Varicella Zoster
9)
,
Hepatitis B
10)
,
Toxoplasmosis
11)
or Ebola
12)
.
Depending on the assay protocol used, a whole ELISA can be carried out within some hours to one day.
Difference between revisions of "Team:Freiburg/Design"
Line 74: | Line 74: | ||
</div> | </div> | ||
+ | <div class="content_box"> | ||
+ | <!-- EDIT3 SECTION "What are current diagnostic methods" [3045-11352] --> | ||
+ | <span id="Limitations" class="anchor"></span> | ||
+ | <h2>Requirements for Future Diagnostic Devices</h2> | ||
+ | |||
+ | <div class="image_box left"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/7/7a/Freiburg_sanduhr.jpg" width="120px"> | ||
+ | </div> | ||
+ | |||
+ | <p> | ||
+ | As a lot of diseases can nowadays be treated quite well, fast diagnosis is essential for an immediate onset of appropriate treatment. | ||
+ | It depicts a critical factor for the patient's health and life and significantly determines the wellbeing. | ||
+ | Moreover, improved diagnostics are not only required regarding the health of a patient: 70% of healthcare expenses are linked to diagnostic tests | ||
+ | <sup><a class="fn_top" href="#fn__16" id="fnt__16" name="fnt__16">16)</a> | ||
+ | </sup>. | ||
+ | Therefore, improvements in diagnostic technologies have the potential to drastically reduce overall healthcare costs while at the same time increasing health as such. | ||
+ | </br> | ||
+ | </br> | ||
+ | Diagnostic tests are usually developed for use 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 | ||
+ | <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a> | ||
+ | </sup>. | ||
+ | Thus, most of the substantial progress achieved in the public health and Point-of-Care sector has only been advantageous to the more developed part of the world. | ||
+ | </br> | ||
+ | </p> | ||
+ | |||
+ | <div class="image_box right"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/8/81/Freiburg_DEATHne.png" width="200px"> | ||
+ | </div> | ||
+ | |||
+ | <p> | ||
+ | According to the WHO 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 | ||
+ | <sup><a class="fn_top" href="#fn__19" id="fnt__19" name="fnt__19">19)</a> | ||
+ | </sup>. | ||
+ | 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% | ||
+ | <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup>. | ||
+ | </br> | ||
+ | </br> | ||
+ | Therefore, transforming existing technologies into protable applications is a leap forward to improve general health all over the world. These applications should be robust and sensitive enough for the use outside of equipped laboratories. Outbreaks and spreading of potential epidemic diseases or sexually transmitted infections could be controlled by a rapid diagnosis and immediate onset of appropriate treatment | ||
+ | <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup>. | ||
+ | </p> | ||
+ | |||
+ | <div class="image_box left"> | ||
+ | <div class="thumb2 tcenter" style="width:300px"> | ||
+ | <div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/d/db/Freiburg_STDs.png" title="https://static.igem.org/mediawiki/2015/d/db/Freiburg_STDs.png"> | ||
+ | <img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/d/db/Freiburg_STDs.png" width="250"/></a> | ||
+ | <div class="thumbcaption"> | ||
+ | <div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/d/db/Freiburg_STDs.png" title="vergrößern"> | ||
+ | <img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <p> | ||
+ | A need for such technologies is urgent: 500 million people between the age of 15 and 49 are infected with curable sexually transmitted infections like chlamydia, gonorrhea, <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#Syphilis" title="syphilis">syphilis</a> or trichomoniasis each year | ||
+ | <sup><a class="fn_top" href="#fn__20" id="fnt__20" name="fnt__20">20)</a> | ||
+ | </sup>! | ||
+ | However, the infrastructure currently available for the diagnosis of infectious diseases often proves to be too slow and expensive to be practicable for third world countries. | ||
+ | This can be illustrated with the identification of pathogens of an infectious diarrhea taking 2-4 days – even in the best developed laboratories of the world | ||
+ | <sup><a class="fn_top" href="#fn__21" id="fnt__21" name="fnt__21">21)</a> | ||
+ | </sup>. | ||
+ | </p> | ||
+ | |||
+ | <p> | ||
+ | According to the problems and needs discussed, devices for future diagnostics should meet the following requirements: | ||
+ | |||
+ | <ul> | ||
+ | <li> <strong>Speed</strong> - a fast diagnosis reduces time until the beginning of treatment, preventing the spread of epidemic diseases and reducing the severity of a disease | ||
+ | </li> | ||
+ | </br> | ||
+ | <li> <strong>Simplicity</strong> - the handling necessary to perform the test should be as easy as possible | ||
+ | </li> | ||
+ | </br> | ||
+ | <li> <strong>Low-cost</strong> - Point-of-Care diagnostics need to be affordable in developing countries | ||
+ | </li> | ||
+ | </br> | ||
+ | <li > <strong>Unambiguity</strong> - the output of Point-of-Care tests requires the clarity of a yes/no answer | ||
+ | </li> | ||
+ | </br> | ||
+ | <li> <strong>Storage under sub-optimal conditions</strong> - since defined conditions may not be available, the device has to be stable under extreme conditions concerning temperature, pH or humidity for example | ||
+ | </li> | ||
+ | </br> | ||
+ | <li> <strong>Multiplexing</strong> - covering a broad spectrum of possible diseases in only one device allows for a differential diagnosis with the need for only one sample of the patient | ||
+ | </li> | ||
+ | </br> | ||
+ | </ul> | ||
+ | </p> | ||
+ | </div> | ||
<div class="content_box"> | <div class="content_box"> | ||
Line 306: | Line 396: | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
Revision as of 20:17, 18 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 world's poorest countries more than half of the deaths are due to infectious diseases – in the wealthiest countries it is less than 5% 1) .
Requirements for Future Diagnostic Devices
![](https://static.igem.org/mediawiki/2015/7/7a/Freiburg_sanduhr.jpg)
As a lot of diseases can nowadays be treated quite well, fast diagnosis is essential for an immediate onset of appropriate treatment. It depicts a critical factor for the patient's health and life and significantly determines the wellbeing. Moreover, improved diagnostics are not only required regarding the health of a patient: 70% of healthcare expenses are linked to diagnostic tests 16) . Therefore, improvements in diagnostic technologies have the potential to drastically reduce overall healthcare costs while at the same time increasing health as such. Diagnostic tests are usually developed for use 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 2) . Thus, most of the substantial progress achieved in the public health and Point-of-Care sector has only been advantageous to the more developed part of the world.
![](https://static.igem.org/mediawiki/2015/8/81/Freiburg_DEATHne.png)
According to the WHO 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 19) . 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% 1). Therefore, transforming existing technologies into protable applications is a leap forward to improve general health all over the world. These applications should be robust and sensitive enough for the use outside of equipped laboratories. Outbreaks and spreading of potential epidemic diseases or sexually transmitted infections could be controlled by a rapid diagnosis and immediate onset of appropriate treatment 2).
A need for such technologies is urgent: 500 million people between the age of 15 and 49 are infected with curable sexually transmitted infections like chlamydia, gonorrhea, syphilis or trichomoniasis each year 20) ! However, the infrastructure currently available for the diagnosis of infectious diseases often proves to be too slow and expensive to be practicable for third world countries. This can be illustrated with the identification of pathogens of an infectious diarrhea taking 2-4 days – even in the best developed laboratories of the world 21) .
According to the problems and needs discussed, devices for future diagnostics should meet the following requirements:
- Speed - a fast diagnosis reduces time until the beginning of treatment, preventing the spread of epidemic diseases and reducing the severity of a disease
- Simplicity - the handling necessary to perform the test should be as easy as possible
- Low-cost - Point-of-Care diagnostics need to be affordable in developing countries
- Unambiguity - the output of Point-of-Care tests requires the clarity of a yes/no answer
- Storage under sub-optimal conditions - since defined conditions may not be available, the device has to be stable under extreme conditions concerning temperature, pH or humidity for example
- Multiplexing - covering a broad spectrum of possible diseases in only one device allows for a differential diagnosis with the need for only one sample of the patient
The Field of Diagnostics
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 the 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.
![](https://static.igem.org/mediawiki/2015/3/3a/Freiburg_Patient.jpeg)
Interest in the so called Point-Of-Care (POC) diagnostics increased dramatically
2)
3)
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 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 is predicted
4)
,
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
5)
6)
,
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 may thus increase life quality and health in one go.
Current Diagnostic Methods and Limitations
The four most common centralized laboratory techniques are blood chemistry, immunoassays, nucleic-acid amplification tests and flow cytometry
2)
.
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. Immunodiagnostics are 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
3)
.
Immunodiagnostics are realized by immunoassays, which summarize a wide range of formats, allowing quantification and monitoring of small molecules, large proteins and even whole pathogens
2)
.
Three prominent examples of immunoassays are lateral flow tests, ELISAs and miniaturized immunoassays (microarrays).
![](https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Diagnostics_ELISA.jpg)
ELISA - sensitive but time consuming, no multiplexing:
The commonly used ELISA (enzyme linked immunosorbent assay) only provides limited capacity for multiplexing as only one specific disease can pe detected per well. It takes several hours and large amounts of sample material as well as antibodies (0.05-1.2 µg antibody per well
7)
) to test for all the possible diseases fitting the patient's symptoms.
![](https://static.igem.org/mediawiki/2015/6/62/Freiburg_Schwangerschaftstest.jpeg)
Lateral flow test - simple and rapid, no multiplexing, limited sensitivity
Lateral flow tests are much faster to perform but can only detect one molecule of interest at once. Moreover, they are known to perform rather poorly in terms of sensitivity (!!!!!!!!!!!!LINK!!!!!!!!!!!!!!).
Miniaturized Immunoassay (Microarray) and Lab-on-a-Chip (LOC)
Miniaturized immunoassays (microarrays) combined with microfluidic bioanalysis have been shown to hold great potential regarding future diagnostics
3)
.
In comparison to ELISAs and lateral flow tests for, they already hold the possibility for multiplexing.
Addressable Laser Bead Immunoassays Laser and microsphere based immunoassays obtain the possibility of multiplexing but require hightech equipment and trained personnel. They are based on the immobilization of antigens on microbeads and subsequent anlaysis using laser technology.
Even though current diagnostic methods provide reliable information on a broad range of diseases, they still encounter various restrictions.
As already mentioned, time and the possibility of multiplexing are some crucial issues regarding future diagnostics.
For instance, ELISAs do not provide a possibility for multiplexing and performing the test lasts at least some hours.
Especially for the methods mentioned at last, a lot of expensive equipment is needed for analyzing the results.
This leads either to conflicts regarding involved costs or due to the size of the devices that are mostly far from portable.
This restricts the use to developed countries and special facilities.
Besides those complications, all the methods described above rely on a solid material with immobilized proteins on it.
Anyhow, proteins are difficult to handle as they are rather unstable.
The storage of proteins therefore requires particular conditions that are not easy to provide in some parts of the world.
How Can Our DiaCHIP Contribute to Solving These Problems?
Our approach, the DiaCHIP, basically combines three promising techniques in one device, offering great potential to improve future diagnostics.
Miniaturized Immunoassays Combined with Microfluidics
Miniaturized immunoassays enable immense multiplexing. By immobilizing hundreds of different antigens it is possible to screen a patient’s sample for the existence of many potential antibodies. This allows customized combinations of antigens, thereby providing the optimal detection system for the respective application.
For the microfluidic device only small volumes of reagents and samples are necessary making it cost-efficient additionally to saving time.
Storage and handling problems of conventional peptide based microarrays are circumvented by directly producing our protein array on the basis of a DNA array template via cell-free expression. As DNA is stable within a large range of temperatures, pH values and other environmental conditions it proves to be an ideal molecule for storing protein information.
iRIf Detection MethodThis emerging detection method enables a fast, sensitive and label-free detection of binding processes. In our setup, antibodies present in the patient's blood bind to the antigens produced via cell-free expression and can be detected in real-time. Incubation steps with secondary detection antibodies (that have to be labelled in some way) are for example rendered unnecessary, thus making detection cheaper and faster. Nevertheless, if the signal should be intensified, flushing the array with a secondary antibody can be performed. This would extend the time needed for testing by a maximum of half an hour.
Small Detection DeviceTo enable many people to benefit from this promising detection method we rebuilt this device ourselves. In contrast to the professional device it is now even smaller than a shoebox and built of simple components.
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 analysis.
However, the user should only need to apply one drop of blood.
Especially people in developing countries could perform routine testing to detect the infection with pathogens like influenza or sexually transmittable diseases like AIDS or syphilis
2)
.
The DiaCHIP may be scaled down to a size suitable for smaller medical practices or portable applications.
Thereby, it complements the existing techniques that are either small and handy but only able to detect a limited spectrum of antibodies or are so huge that an efficient use is only possible in clinical facilities.
We showed the basic feasibility of such a device with our rebuilt setup and the successful detection of anti-tetanus antibodies in human blood serum.
Core characteristics of the self-built device are its simplicity, the low cost of the components and the rapid generation of a final evaluable result.
Even though the DiaCHIP is still at an experimental stage, some improvements may render it easy to handle even for untrained users.
On this page, we only focused on diagnostics as this is the main application we suppose. Nevertheless, the methodology behind the DiaCHIP can be used for further applications.