Good afternoon,My name is Maarten MerkxI am an associate proffessor in protein Engineering at the department of Biomedical Engineering in Eindhoven. Today I want to tell you about a new type of reporter enzymes that we developed in our group in Eindhoven that allow one to detect the presence of specific antibodies directly in solution.
Why are we interested in antibodies?Antibodies are the proteins that are generated by our immuunsystem to recognize foreign invaders such as bactaria and virused. The presence of specific antobodies therefore is an excellent way for the detection of infcetious diseases, but also to study auto-immune diseases in which case our immunne system attacks some of our own proteins.
To explain the principle of our new antbody detection assay, we first need to look at the strcuture of antibodies.All antibodies have this veru characteristic Y-shape structure, which consists of 2 heavy chains and 2 light chains. Each antibody has two identical antgen binding sites, which are locatetd ta th end of the two arms and formed at the interface between a heavy and a light chain.
This part, or actually the loops at this interface, is different for each antibody, whereas the rest of the antibody and the overall structure are very similar. By chnaging these loops our immuunsystem is able to generate an antibody for almost any foreign antigen it encounters.
Classical method, but everybody that has ever done this assay knows that it involves a lot of washing and waiting. Now sometimes this unleashes unexpected creativity, but we still wondered whether we cannot make this antibody detection more simple
How can we integrate the detection of the antibody and signal generation. The basic idea behind our invention is to take advantage of this very characteristic Y-shaped structure that is shared by all antibodies and the presence of the identical antigen binding sites.
So here is our idea. The sensor consists of a reporter enzyme € which is linked via long and flexibel linker to an inhbitor domain. In the absence of the antibody target, the inhbitor binds to the enzyme and its activity is inhibited. Now if we introduce two peptide epitopes at the ends of the linker close to the reporter enzyme and the inhibitor domain, these two epitopes will preferably bind to the two antigen binding sites and in this way the interaction between enzyme and inhibitor will be inhibited.
As a reporter enzyme we chose b-lactames, activity can be detected using a simple colorimetric assay, and it has a very well charcaterizedinhbitor protein BLIP, her you see the structure of the complex, that blocks the active site.
Initial design we used epitopes against an anti-HIV1 antibody. You can see here that the enzyme activity is indeed inhbited compared to bet-lactamase itself, but adidtion of antibody did not result in enzyme activiation.
We suspected that this was because the interaction between enzyme and inhibitor was too strong. Ze we systematically started to mutate the interface between enzyme and inhibitor. Combining single mutation on beta lactamse with different mutations on the inhbitor, yielded several variants that showed a nice activation in te presence of antibody
Combining single mutation on beta lactamse with different mutations on the inhbitor, yielded several variants that showed a nice activation in the presence of antibody
So here you see how easy it is to do this assay. The substrate of the reaction is yellow.If you add sensor but there is now target antibody, the color remains yellow, but in the presence of antibody it turns red. Easly detected by eye.
The assay is also quite sensitive. Here you see tha enzyme activity as a function of antibody concentration, afifnity is 200 pM, allowing easy detection of antibodies at concentrations of around 50 pM. You can also see here that other antibodies do not elicit a response
The true beauty of the concept is that it is very generic. Development of the first sensor required quite a lot of work, but if we want to make a reporter for another antibody all we need to do is replace the peptide epitope for that antibody. We have shown this already for several antibodies e.g. also against Dengue virus, which is a very important tropical infectious disease.
Oneremaiming problem is that for many antibodies, good epitopes are not known. To address this issue we also created fluorescent sensors based on the same principle. These consist of two fluorescent protein domains that stick together. In this state enegryu that is anbsrobed by the cyan fluorescnet protein is transferred to yellow domain giving rise to yellow light. In the presence of the antibody the distance between the antibodies if increased and energy can longer be transferred giving rise to cyan emission. So the color of the fluorescence allows one to detect antibody binding.
Currently we are creating a library of these fluorescent antibodies that contains all possible combinations of 7 amino acids. With this library of 1 billoin different sensor proteins we should be able to identify the best peptide epitope for a new antibody, e.g. using fluorescence assisted cell sorting.
So what is our dream, where do we want to go with this technology. We think our antibody reporter enzyme would be very suitable for developing low cost point of care antibody tests win which one could e.g. use a mobile phone to moninor the activity of the reporter enzyme. In particular interesing at the docter’s office, in remoet areas in the developing world, but also e.g. for veterinary applications.
The last slide is to say thank you, thanks to these 4 guys in my group who did the actual work, thanks to the ERC who provided financial support and thank you for your attention.
HVE 2014 Pecha Kucha: Maarten Merckx
ELISA in solution
no washing, less waiting
Multiplex bead-based assays (2000>)
wash incubation wash
molecular recognition signal transduction signal generation
Library with all 7 amino acid
linear epitopes: 207 = 1.3 x 109
• Improve/characterize existing epitopes
• Rapidly evolve specific sensor protein for
Fluorescence Assisted Cell Sorting (FACS)
Consolidator Starting Grant 2011
Proof of Concept Grant 2013
M i s h a
G o l y n s k i y
R e m c o
A r t s
S t i j n
A p e r
S a m b a s h i v a
B a n a l a
Golynskiy et al (2010) ChemBioChem, 11: 2264-2267
Banala et al (2013) ACS Chem. Biol. 8:2127-2132
Banala et al (2013) Org. Biomol. Chem.
Banala et al patent application filed