Figure 2.

Application of peroxidase-linked anti-IgG nanobodies. (a) A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225. It was then decorated with HRP-conjugated goat anti–mouse polyclonal IgG (5 nM) or anti–mouse IgG1 Fc nanobody TP1107 (5 nM) and detected via ECL. Similarly, a rabbit polyclonal antibody targeting Nup54 was decorated with HRP-conjugated goat anti–rabbit polyclonal IgG or anti–rabbit IgG nanobody TP897 (5 nM). (b) Oxidation of the fluorogenic ELISA substrate Amplex Ultra Red. A dilution series of pure HRP or recombinant anti–mouse IgG1 Fc nanobody TP1107–APEX2 fusion was incubated with Amplex Ultra Red and H₂O₂. Oxidation leads to formation of the fluorescent compound resorufin. The data obtained were fit with a four-parameter logistic regression. The inflection points of the curves can be used to compare attainable sensitivity. A.U., arbitrary units. Error bars, mean ± SD (n = 3).