Available online 17 March 2026, 101609

determining the appropriate methods and rational positive thresholds for human pre-existing anti-PEG antibody detection.

discovering of the different binding selectivity and affinity of human pre-existing anti-PEG antibodies.

deciphering the adverse impacts of human pre-existing anti-PEG antibodies on PEGylated liposomes.

Polyethylene glycol (PEG) is a widely used pharmaceutical excipient to improve drug solubility and prolong in vivo circulation duration. Although it was initially considered non-immunogenic, recent studies have confirmed the ubiquitous presence of anti-PEG antibodies pre-existing in human, influencing the efficacy and safety of multiple PEGylated medicines. Due to the contradictions of current anti-PEG antibody detection methods and unclear induction mechanism of human pre-existing anti-PEG antibodies, the actual prevalence, binding features and biological effects of pre-existing anti-PEG antibodies remain elusive, hampering the clinical implementation of personalized PEGylated therapeutics, as well as the rational design of evasion strategies and the development of innovative PEG materials and PEGylated nanocarriers. Herein, we established a robust enzyme-linked immunosorbent assay (ELISA)-based methodology for the detection of human pre-existing anti-PEG antibodies, and defined the positive threshold based on complement activation. Mechanistic analysis revealed that human pre-existing anti-PEG IgG predominantly recognized the PEG backbone, while anti-PEG IgM specifically bound to the methoxy end group. Notably, the weak binding affinity of human pre-existing anti-PEG IgG limited its ability to induce complement activation or promote capture of PEGylated liposomes by mononuclear phagocyte system, in contrast to IgM. Collectively, this study not only provided a reliable detection strategy for human pre-existing anti-PEG antibodies, but also revealed the distinct binding patterns, affinities and biological effects of pre-existing anti-PEG IgM and IgG, with important implications for the safe and rational applications of PEGylated therapeutics.

Pre-existing anti-PEG antibodies

IgM

IgG

Binding affinity

Biological effects

© 2026 The Authors. Published by Elsevier B.V. on behalf of Xi’an Jiaotong University.