Measurement of Poly(ethylene glycol) by Cell-Based Anti-poly(ethylene glycol) ELISA

Polyethylene glycol) (PEG) is increasingly used in clinical and experimental medicine. However, quantification of PEG and PEGylated small molecules remains laborious and unsatisfactory. In this report, we stably expressed a functional anti-PEG antibody on the surface of BALB 3T3 cells (3T3/αPEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The αPEG cell-coated plate bound biotinylated PEG_5K (CH₃-PEG_5K-biotin) and CH₃-PEG_5k-¹³¹I more effectively than did a traditional anti-PEG antibody-coated plate. Competitive binding between PEG (2, 5,10, or 20 kDa) and a known amount of CH₃-PEG₅K-biotin allowed construction of a reproducible competition curve. The αPEG cellbased competition ELISA measured small molecules derivatized by PEG ₂K, PEG₅K, PEG_10k, PEG_20k. and PEG_5k at concentrations as low as 58.6,14.6,3.7,3.7, and 14.6 ng/mL, respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the αPEG cell-based competition ELISA. Finally, we show here that the αPEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG5K, comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH₃- PEG_5K-¹³¹I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.