The Secret of the Alpaca-Antibody-Advantage

What’s so special about Alpaca antibodies and how were they discovered? Alpacas aren’t typical lab animals, so these are legitimate questions...

 

As is often the case with important discoveries, chance helped scientists at the Free University of Brussels in the late 1980s. As Michael Gross remembers the story: During a practical course, a couple of biology students were to extract antibodies from human blood serum. They were not overly excited, on the one hand because they were concerned that the samples might be contaminated with HIV, on the other hand because this type of experiment had already been done numerous times before and the result was well documented in their text books. Their tutors then offered to sacrifice a few mice instead – not a very popular choice either. Eventually a few liters of frozen dromedary serum were discovered in the lab freezer – this exotic example inspired the students sufficiently to start working on the antibody separation.

 

In addition to the usual distribution of immunoglobins, they also discovered a group of smaller antibodies that did not correspond to anything known to science. This might have ended in obscurity, had not two researchers, Raymond Hamers and Cecile Casterman, investigated the matter more deeply. They did not believe that this species were just degraded variants of the “real” antibodies, but started to characterize them in more detail. Eventually, it became clear that they had discovered a new class of antibodies that were devoid of light chains and had a single antigen recognizing domain. These antibodies were later found in many camelid species, including llamas and alpacas. If you would like to learn more about the captivating story of this discovery, which includes travels to Morocco, a stolen camel and help from a Sheikh, you may read Michael Gross’s book “The birds, the Bees and the Platypuses”.

Based on their structure, these peculiar camelid antibodies have been named Heavy Chain Antibodies (hcAb), as they are composed of heavy chains only and are devoid of light chains. HcAbs are not found in other mammals except in pathological cases. In 1995, Greenberg and colleagues found similar hcAbs in nurse sharks (Greenberg et al., 1995), but evolutionary analysis showed that camelid and shark hcAbs evolved independently (Nguyen et al., 2002). There are many speculations about the evolutionary driving force for the emergence of heavy chain antibodies in such distantly related species. A plausible explanation could be that, unlike conventional (comparably large) antibodies, these small single domain antigen binding fragments allow the targeting of otherwise inaccessible epitopes, e.g. catalytic centers of enzymes (Flajnik et al., 2011).


In the absence of light chains, the fragment-antigen-binding (fab) part of these antibodies is reduced to a single domain, the so called VHH (variable domain of heavy chain antibodies) domain or nanobody. This single domain contains a complete antigen binding site and is the smallest functional antigen binding fragment (around 15 kDa, only one tenth the size of a conventional antibody).

In fact, there are many more advantages of this novel class of antibodies. They can be readily selected and produced in bacteria, ensuring their virtually unlimited supply in consistent quality. In contrast to conventional antibodies, nanobodies are also exceptionally stable, withstanding conditions of extreme temperatures and pH. At ChromoTek we tested nanobody preparations that were more than five years old: they were still functional, with little or no loss of activity. 


Although people soon realized that there were almost limitless applications for this class of antibodies, the development of actually useful reagents proved to be somewhat less straightforward. As probably most of us have experienced themselves, the devil is in the details. After many experiments with different protein modifications, chemical linkers and solid supports, our lab eventually succeeded in generating something useful with these nanobodies. What we created were extremely efficient tools for (co-)immunoprecipitations which we dubbed “Nanotraps”. The first Nanotrap that was developed years before the inception of ChromoTek recognized the green fluorescent protein (GFP). Our “GFP-Trap®” proved to be useful for many researchers, since GFP is used as a tag in many constructs. Not long after the GFP-Trap® was first introduced at conferences, our lab supplied around sixty labs with this fancy new tool. Not least owing to the many enthusiastic comments from these early afficionados, we started up ChromoTek in late 2008. 


Today, we offer a collection of eighty plus products used by more than three thousand labs worldwide. Our best-sellers are the above nanotraps that are known for exceptionally clean and fast IPs and which are sold in different formats (agarose beads, magnetic beads, microwell plates). Numerous protocols are available for a variety of uses, including special applications such as chromosome IPs (ChIP). By the way, of the more than four hundred scientific publications citing these products, more than half of them in high ranking journals with an impact factor of ≥10.

But nanobodies can be much more than just useful and robust laboratory reagents: what is probably most exciting about them is that they are able to bind to their targets within living cells. By coupling nanobodies to fluorescent proteins, Rothbauer et al. (2006) showed that these novel constructs, termed “Chromobodies”, allow to track endogenous intracellular targets in live cells in real time. Instead of looking at still images, cell biologists could now watch complete “movies” and observe the actions of their protein of interest in real time. Moreover, since Chromobodies trace endogenous proteins, there is less of a chance that one sees artifacts due to misfolded or not properly processed fusion proteins. At the time, this was an enormous achievement, since the majority of the scientific community was convinced that it would be extremely unlikely that nanobodies, having several cysteine bridges, would ever fold correctly in the reducing environment of the cell’s cytoplasm and be able to recognize their target. And what made Chromobodies even more attractive was the fact that they often show very fast on-off kinetics, thus reducing a potential (negative) impact on the cellular function of the target.