Recommending recombinant

Today we're looking at recombinant antibodies, particularly those that have been modified to reduce off-target binding by mutating the Fc domain. The idea here is that the Fc portion of the antibody is what binds to Fc receptors, creating some of the undesired signals in flow cytometry. By mutating portions of the Fc, interactions with Fc receptors can be greatly reduced, potentially eliminating the need for Fc blocking reagents and increasing your signal to noise ratio. If you want to read more about the development and diversity of Fc silencing, here's a review article on that topic.

We would expect advantages from using Fc-silenced recombinant antibodies when we have samples with lots of cells containing Fc receptors, macrophages in particular. This could be when we're trying to stain myeloid cells themselves, or when we're trying to prevent those cells from interfering with our staining on other cells in the mixtures. Let's look at an example of that, and then an example of an unexpected benefit.

Here we are trying to detect the pro-form of IL-1-beta. We've got some mouse peritoneal macrophages, which are incredibly sticky, and we've stimulated them with phorbol esters and ionomycin to kick up cytokine production (not really essential for IL-1, but it doesn't hurt). These cells are covered in all sorts of Fc and scavenger receptors, and they just love to bind antibodies. I've blocked using the 2.4G2 anti-FcgRII/III clone, but that doesn't do much to block the high affinity Fc receptor FcgRI (CD64), which will be present on these cells. Consequently, staining with a normal, non-recombinant rat IgG1 anti-pro-IL-1-beta PE-Cy7 produces a fair amount of non-specific binding, at least based on the fact that all cells move up together as we add more antibody. In contrast, with the recombinant REA human IgG1 anti-pro-IL-1-beta PE-Vio770, we get a bright population of IL-1+ cells separating out without as much shifting up of the entire pool of cells.

Mouse peritoneal macrophages, stained for pro-IL-1-beta

The unexpected bit, for me at least, is that these Fc-mutated antibodies also give higher specificity of staining on cells without Fc receptors. Here's an example looking at mouse CD4 T cells from the lymph node, which is pretty clean and does not have a ton of myeloid cells present. While human T cells can sometimes express Fc receptors, mouse T cells, at least in healthy SPF conditions, do not.

Mouse lymph node CD4+CD3+ viable T cells

There's nothing wrong with the 22F6 Helios clone (from BioLegend in this example). It's quite good. But the REA one produces less background and better separation.

This isn't an isolated example with Helios. The REA for T-bet produces considerably less background than the non-recombinant 4B10 clone. I see similar effects with antibodies for BATF, Bcl-6, BDNF, Eomes and GATA-3. So, I quite like using these for intracellular targets when they're available. While you may see advantages for any target with an Fc-silenced antibody, the impact is likely to be greater for low expression markers or those only expressed on rare cell types.

Not all recombinant antibodies are Fc-silenced. I may be wrong, but I don't see any indication that the ones sold by BioLegend have any Fc engineering (those are good too, though). There are other advantages of using recombinant antibodies though, including increased standardization of production (cheaper), standardization of the fluorophore conjugation process (all the same), and perhaps some benefits in terms of animal welfare.

I have used and can recommend Fc-silenced antibodies from Miltenyi and Proteintech. Fluorophore options from these vendors are not as diverse as some of the major flow companies.

Violet Sabrewing, Costa Rica