Visualizing Filoviral Nucleoproteins Using Nanobodies Fused to the Ascorbate Peroxidase Derivatives APEX2 and dEAPX

Abstract

Fusions of single-domain antibodies (sdAbs, nanobodies) to enzymatic reporters make convenient molecular probes to detect the presence of an antigen of interest. We have previously fused the monomeric hyperactive ascorbate peroxidase derivative APEX2 to anti-Ebolavirus and anti-Marburgvirus sdAbs to generate immunoreagents useful in detecting nucleoprotein (NP) on western blots, ELISA, and within cells following transfection of NP expression plasmids or following virus infection. Here we present the methods used to overexpress and purify these sdAb-APEX2 fusion proteins, and to employ them as probes in various scenarios with colorimetric and fluorometric signal development. We also introduce a dimeric hyperactive ascorbate peroxidase derivative dEAPX that enables bivalent sdAb probes to be produced while avoiding the need to generate more complex tandem sdAbs, leveraging avidity for improved signal strength. The APEX2 and dEAPX reagents appear interchangeable with any existing detection platform and the methods described here should enable a user to study their antigen of interest by simply swapping out the sdAb for their recombinant affinity reagent of choice.

Keywords: APEX2; Ascorbate peroxidase; Filovirus; Focus forming assay; Microscopy; Nanobody; Nucleoprotein; VHH; Virus-infected cell; Western blot; sdAb.