In the research and diagnostic labs, you often find yourself looking for a needle in a haystack. In these cases, you may be looking for 10 activated enzyme molecules within a mix of a 1.000.000 unactivated ones, all diffusing around in a well-mixed 80 mg/mL protein cocktail called blood plasma. This may complicate things a bit.
You could of course choose to leave it alone and go do something else, or you can go on to develop a highly specific and sensitive new bioassay! I know just the guy that knows how to do that. In our lab, we make nanobodies to build new diagnostic assays. Nanobodies are recombinant antibody fragments of Llama’s. When we (humans) make antibodies, their capacity to recognize targets depends on multiple peptide chains. This is shown in the left panel of the figure on the left by the intersections between the green and blue chains (derived from this site about protein structure). However, camelids (including Llama’s) also make another kind antibodies. These antibodies can recognize their targets through a single domain (right panel). When you immunize Llama’s and take some of their blood later, it is possible to generate a copy of the Llama’s antibody repertoire that is memorized in the genome of their B-cells. Through a sophisticated method called phage display, we can select specific antibodies that meet our demands. You could even select your antibody to recognize an active enzyme, while it does not recognize the inactive form. That’s a great way to find those needles back – with molecular magnets!
Around Christmas 2009, I immunized two Llama’s with a selection of proteins that are found in the human blood coagulation system and some very helpful colleagues helped me by preparing phage display libraries. Now, we are selecting away to make all kinds of nanobodies for the development of new bioassays. As I have an interest in the contact system and plasminogen activation system, we will try to get tools to quantitatively follow all of these factors. As first and most interesting target, we started to developed a method to measure activated Factor XII (as my Veni project was titled: “A Dangerous Enzyme Revealed”, we needed something to reveal it with). In the end, we had selected two nanobodies that could recognize activated Factor XII and distinguish it from its resting state. After setting up a capture assay to ‘fish’ activated Factor XII from blood plasma, we found something surprising: during its activation, Factor XII transforms from a first activated state to a second activated state! Exactly as we had hypothesized in our “Dangerous Enzyme Revealed” model! We got lucky with these nanobodies, that’s for sure! We published this recently in a very nice methodological article1 that became part of the theme issue on Prekallikrein activation in Thrombosis and Haemostasis.
Right now, we are ‘attacking’ other proteins, including plasma kallikrein, high molecular weight kininogen, Factor XI and platelet Glycoprotein Ib.
1 De Maat et al. Thromb Haemost 2013 Sep;110(3):458-68.