Sponsored Content by Jackson ImmunoResearch Laboratories, Inc.Jan 17 2019
insights from industryDr. Dave FancyChief Operating OfficeJackson ImmunoResearch
An interview with Dr. Dave Fancy from Jackson ImmunoResearch at SfN 2018, discussing the use of secondary antibodies in neurobiology and the future of nanobodies.
How are secondary antibodies used in neurobiology?
Neuroscientists have been using secondary antibodies for years. They are generally used to provide or amplify the signal from a primary antibody bound to a specific target. For example, if a researcher wants to find out the location of a particular protein within a cell, they can run an IHC or immunofluorescence experiment. This involves first applying a primary antibody, followed by a labeled secondary antibody that can be visualized using an imaging technique such as fluorescence microscopy.
Carl Dupont | Shutterstock
What makes secondary antibodies so attractive to researchers is that they allow researchers to use a primary antibody that's unlabeled or carry out multi-colored labeling, for example, to simultaneously observe a whole host of different proteins. This means researchers can look at multiple proteins in a neuron, in a cell, in a synapse and more; with each color corresponding to a different protein. It also gives researchers the flexibility to choose colors that give the best contrast.
Finally, secondary antibodies give researchers the flexibility to detect proteins and compounds in the presence of other antibodies without cross-reactions. Developing secondary antibodies that are minimally cross-reactive is something we are passionate about at Jackson.
You recently launched Alpaca Secondary Antibodies. Why did you feel it was important to add another antibody type to your extensive collection?
Firstly, alpaca antibodies are another species to the antibodies researchers are used to working with, such as mouse and rabbit. These antibodies, therefore, provide another avenue for multi-labeling experiments and add a whole extra repertoire to the research toolbox.
Also, researchers can take alpaca secondary antibodies and further manipulate them down to the nanobody component (the protein-binding VHH component) which makes them small.
This is particularly useful for super-resolution imaging, where researchers want to see fine spots and a traditional mouse, human or rabbit antibodies have several rotational degrees of freedom. This creates a very large circle of fluorescence in the final image, which makes the image difficult to analyze. Using a nanobody, in theory, allows researchers to decrease the size of this circle and increase imaging resolution to 1 or 2 nanometers of scale.
Our goal is to break our alpaca secondary antibody into smaller and smaller pieces so they can be used for techniques such as CLARITY. In this process, brain samples are made transparent by the removal of lipids, and antibodies are added to visualize specific proteins. However, the process is slow as the antibodies take a long time to re-enter the tissues.
Working with a nanobody or VHH domain, for example, makes this process faster. These components can penetrate hydrogels and tissues much faster than normal antibodies. This development will significantly cut down the amount of time it takes to introduce a secondary antibody to a sample.
Overall, our Alpaca range adds a whole extra layer of specificity that we can take advantage of, while also adding flexibility with an extended tool kit. Over the next few years, we hope to exploit more advantages relating to using the VHH domain, and we're looking forward to the alpaca secondary antibody
What advantages do nanobodies provide over conventional mouse and rabbit antibodies?
There are many advantages to nanobodies. The first is their small size, which allows them to penetrate in vitro systems very quickly and pass through the blood-brain-barrier.
They’re also very stable and easy to engineer. Researchers can use just two PCR primers, and produce their own library of nanobodies. Compare this to working with a normal antibody, which requires pairing up the heavy and light chains – this process is not efficient and very difficult to get right.
Carl Dupont | Shutterstock
Can camelid nanobodies be used in the same experiment as human and mouse antibodies without cross-reactivity? Why is this useful for researchers?
If a researcher makes a nanobody to target a particular protein, more often than not, they are going to see a lot of other proteins. At Jackson, we work hard to reduce the cross-reactivity of our antibodies so that researchers can use them alongside other proteins or other antibodies.
Our process is simple; we create the antibody, purify the nanobody, extract the VHH domain, then check for cross-reactivity. If the antibody doesn't meet our standards, it goes back into the system to be cleaned up. This process can take weeks or even months, but it’s an integral part of producing a high-quality antibody.
Secondary antibodies are a lot tougher to make than primary antibodies because they have to be highly specific for researchers to be able to use them in multi-labeling experiments, and the same will be true for nanobodies.
Antibody advice:
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The length of the binding site in nanobodies is smaller – instead of having a large paratope, nanobodies have a very small one, which is likely to make them more cross-reactive. That said, we developed IgG2 and IgG3 antibodies that showed some cross-reactivity with human antibodies but were able to remove this successfully.
What does the next year look like for Jackson ImmunoResearch?
The next year looks really good for us. There have been many novel techniques developed over the past few years, and we've been building antibodies that work with these. Techniques such as CLARITY or super-resolution microscopy are demanding smaller and smaller antibodies, and we are meeting that demand. Fab fragments are another antibody fragment that we have developed and work well with these techniques. I think they could become popular.
Globally, antibodies are huge, especially for pathology, so our main range of products are doing well. The development of CAR-T cell and immunology-based tools have brought this field to life in recent years - it's the perfect time to be an immunologist. We think the future's bright in the nanobody and antibody world.
About Dr. Dave Fancy
Dave Fancy is the COO of Jackson ImmunoResearch, a position he has held for 5 years. Prior to his role at Jackson, Dave worked as a senior scientist at SDIX, where he focused on the development of polyclonal and monoclonal antibodies for immunoassays.
Dave received a Ph.D. in Organic Chemistry from the University of Texas at Austin in 1997. He then became a postdoctoral research fellow in structural biology at the The University of Texas Southwestern Medical Center, and after two years became an Assistant Professor.
In his current role, Dave leads the development and commercialization of antibody-based products and services for researchers working in the life sciences.
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