In this SelectScience webinar, now available on demand, Dr Pieter Kennis from Ablynx explores how nanobodies – small, single-domain antibody fragments that retain full antigen-binding capabilities and the properties of heavy-chain antibodies – and nanobody discovery and screening platforms exploit high-throughput flow cytometry to increase the throughput of nanobody screening campaigns.
While antibodies are set to be increasingly important as therapeutic options for cancer and other diseases, the discovery of fully functional antibodies that lack light chains in Camelidae (camels and llamas) has opened the door to an exciting new generation of therapeutic antibodies known as nanobodies.
Unlike conventional antibodies, the heavy-chain antibodies of Camelidae contain a single variable domain (VHH) and two constant domains (CH2 and CH3) only. Using these functional antibodies as a base, nanobodies have been created and represent the next generation in antibody-derived biologics.
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Q: Do you see differences in results between fresh and frozen cells?
PK: In general, we do not see a lot of differences in the fresh versus the frozen cells. The main difference we observe is in viability, where certain fragile cells can sometimes see a drop in viability after freezing.
Q: Which dead stain would you recommend?
PK: We use PI, TO-PRO-3 and, since the recent purchase of the iQue Screener PLUS, we also use the DAPI stain. Of course, for some purposes, it is also necessary to use a fixable dead stain, and for those ones, we use the Zombie dyes.
Q: For the multiplexing, using the PKH26 and CFSE, which dead stain did you use there? And why didn't you compensate the data?
PK: It is quite difficult to compensate the data, so for the CFSE and the PKH we were able to separate the cell lines sufficiently without the compensation. Regarding the dead stain that we used, since we only had the iQue Screener, we had four colors available. We used one color for the CFSE, one for the PKH26, and then one we used for nanobody detection - a good option for us was PI. However, if the dye was extra bright, it was very difficult to separate the PI from the CFSE label itself. For that, we used single cell line controls.
Q: Do you screen for intracellular proteins, and if so, do the anti-tag antibodies permeate well?
PK: We don't have the need to target intracellular proteins, so there's no need to detect the nanobodies here. However, we did some testing for intracellular proteins and could see antibodies against these, so the detection antibodies can permeate through the cell membrane.
Q: Did you try combining different dyes to increase multiplexing? For example, FL1 and AF6 or 7 in different concentrations?
PK: That is a good question as it is on my to-do list for further optimization. For example, there is the Ethyl 1 and Ethyl 4 staining dyes, the MultiSite ones, and we use both in the experiments. However, if we were to combine them, we would be able to have a multiplex of photocell lines of 25.
Q: What could be improved in the workflow or technology or data analysis to make it better suited for your high throughput screening and analysis?
PK: In our current protocol, there are still a few manual parts. For example, the washing of cells is currently done by centrifugation and flicking the plate upside down, but we are looking for ways to semi-automate this using plate washers or another alternative. This might be one good way to improve the workflow. We are also awaiting the feasibility of increasing the plex by having the beads conjugated with the target proteins. Another way the workflow could be improved is by generating standard cell lines that already express a certain intensity of a dye and use those to transect our target protein. This way, we would not need to stain big batches of the cell lines each time.
Q: How many clones do you test per target or phage display round?
PK: That mainly depends on the project. For some projects, you don't have a lot of outputs, then you really need to screen a lot of clones. But for other projects, it's really straightforward and you may only need to screen 1,000 clones, for example.
Q: Do you typically normalize the nanobody concentrations before doing comparative screening?
PK: For the screening, we do not normalize our nanobody concentration, as we use saturated concentrations of the nanobody. As this is more a yes/no answer, and we don't know the exact concentration, we don't do that.
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