Team:Aboa/Description
Project Description
An immunoassay is a biochemical test that utilizes the capability of antibodies to recognize and specifically bind other biomolecules. They are commonly used tools for analyzing the concentration of biological substances in various fields, such as health care or enviromental biology.
The aim of our project was to improve the sensitivity of immunoassays by orienting the capture antibodies uniformly to improve the binding capacity of the capture surface.
Immunoassays
Immunoassays are widely used tools to help diagnose and manage diseases as well as detect toxins in the environment. One of the most recognizable examples of such tools is the immunologic pregnancy test which has been standard starting from the 80's. It's function is based on antibodies which detect human chorionic gonadotropin, a hormone produced in high amount by the placenta during the first weeks of pregnancy.
Immunoassays are based on the antibody's capability of specifically binding to a certain biomolecule. In an immunoassay capture antibodies are immobilized on to the surface of a biodetector. As a sample (e.g. blood or urine) is introduced to the biodetector the capture antibodies attach to the analyte binding it to the surface. Then everything that did not bind can be washed away and a labeled antibody can be introduced to the biosensor. This antibody with a label attached to it binds to the same analyte allowing the visual detection of the analyte. Usually the label is a fluorescent molecule, enzyme or radioisotope and it can be detected with various methods. One downside to using traditional technologies to immobilize the binding antibodies leads to a “mess”, where the antibodies are randomly attached to the test surface hindering their ability to bind the antigens. 1 (Fig 1)
Figure 1. Randomly immobilized binder antibodies on a test surface. When the binder antibodies are immobilized randomly, not all of them can bind their analytes because the paratopes are blocked. Modified from 1 .
Correcting antibody orientation
To increase the sensitivity of the immunoassays we are coating a test surface with as many correctly positioned antibodies as possible and this way increase the amount of bound antigens. In order to achieve this we are going to site-specifically modify the binder antibodies using expanded genetic code to incorporate a non-canonical amino acid (ncAA) into the tail of the antibody 2 .
To add an additional amino acid into the antibody, we are adding the amber stop codon (TAG or UAG) into the sequence of the antibody. The modified antibody is then expressed in a host that has all of its amber codons replaced with other stop codons. Also its TAG codon recognizing RF1 (release factor 1) has been removed. The ncAA that we are inserting into the antibody is p-Azido-L-phenylalanine which is known to react rapidly with an alkyne group forming a covalent bond 3 . (Fig 2) Because this reaction is also bio-orthogonal, it does not occur between any natural groups present in the protein or the organism 4 .
Figure 2. Site specifically orientated binder antibodies. Because the azide group of the p-azido-L-phenylalanine reacts bio-orthogonally with an alkyne group forming a covalent bond, the antibody with the amino acid in its tail is forced to the correct position allowing the paratopes to interact with the analytes. Modified from figure 3 .
Expanded genetic code
Expanded genetic code is a technique where one of an organism’s codons, for example a stop-codon, is reprogrammed to code for a ncAA 2 .
This includes replacing a desired codon throughout the genome with an analogous codon and adding an aminoacyl-tRNA synthetase recognizing the replaced codon to be able to introduce a ncAA instead 2 .
Inspiration behind the idea
One of the main focus areas of the Unit of Biotechnology in University of Turku is diagnostic development and since most of our team members are studying there we knew from day one, that we are going to do something related to diagnostic tests. We began on searching ideas by interviewing health care professionals about their demand for diagnostic tests. Soon we found out that there is a need for more sensitive point-of-care tests for cardiac troponins. You can read our search for the idea from our Integrated Human Practices.
After we established our idea to be about increasing the sensitity of immunoassays, we had consider what we were actually going to do since the development of antibodies and finding new biomarkers is a research project of many years. Then we started to think about the random orientation of capture antibodies and how we could correct it. One of our team member had been reading about expanded genetic code and the possibilities it provides for the field of biomolecule conjugations, it really did not take too long to come up with a simple and minimalistic, yet powerful idea of site-specific conjugation of antibodies in immunoassays. This technology had already been used for immobilization of other proteins 5 and modification of therapeutic antibodies 6 . So why not combine these two in a very intuitive way?
The basic principle of using antibodies in diagnostic tools has been a cornerstone of the whole industry for decades now. Regrettably the whole concept of antibody immobilization is a stochastic process which means that the physicochemical model of an immunoassay is at its best, a good guess.
Therefore the optimal performance of any immunoassay could be reached with a minimalistic approach that will orient the antibodies correctly without using any other proteins such as streptavidin to bind antibodies into a sensor surface. This approach could be achieved by using two extremely convenient techniques: click-chemistry and genetic code expansion. This would lead to a more defined biosensors and open up new possibilities in optimizing them to suit the future needs of diagnostics 1 .
References
1. Welch, N.G., Judith, A.S., Muir, B.W. & Pigram, P.J. (2017) Orientation and characterization of immobilized antibodies for improved immunoassays (Review). Biointerphases doi: 10.1116/1.4978435
2. Chin, J.W. (2017) Expanding and reprogramming the genetic. Nature 550: 53-60.
3. Trilling, A., Beekwilder, J. & Zuihof, H. (2013) Antibody orientation on biosensor surfaces: a minireviews. The Analyst 138: 1619-1627.
4. Lang, K. & Chin, J. (2014) Bioorthogonal Reactions for Labeling Proteinss. ACS Chemical Biology 9: 16-20.
5. Raliski, B., Howard, C. & Young, D. (2014) Site-Specific Protein Immobilization Using Unnatural Amino Acids. Bioconjugate Chemistry 25: 1916-1920.
6. Huang, Y. & Liu, T. (2018) Therapeutic applications of genetic code expansion. Synthetic and Systems Biotechnology 3: 150-158.