PEGylation is commonly utilized to modify nanoparticles, vaccines, therapeutic proteins, and biomaterials. The increased use of PEG-containing cosmetics and medicines results in anti-PEG immunoglobulins in humans, including IgG, IgM, and IgE types. Recent studies have shown that high-affinity antibodies against PEG can be isolated through immunization. These antibodies have been used in immunoassays and for decorating PEGylated nanoparticles with targeting ligands. However, conventional antibodies have limitations, such as their large size and potential stability issues. Here, we developed a protocol to isolate anti-PEG nanobodies (Nbs) from a yeast library. We prepared ∼60 nm fluorescently labeled PEGylated and non-PEGylated iron oxide nanoworms for negative and positive magnetic selection, followed by multiparameter flow cytometry sorting. The representative Nb clone exhibited highly specific binding to PEGylated liposomes and nanoparticles and was able to compete with commercial antibackbone PEG IgG but not with anti-methoxy PEG IgG for PEG binding. The Nb showed a micromolar dissociation constant, no aggregation, high thermal stability, and reversible structural stability. This work represents a proof-of-concept screening and isolation of anti-PEG Nbs.
Keywords: PEG; affinity; nanobody; nanoparticle; thermal stability; yeast display.