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

摘要

Poly(ethylene 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/(alpha)PEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The (alpha)PEG cell-coated plate bound biotinylated PEG_(5K) (CH_(3)-PEG_(5K)-biotin) and CH_(3)-PEG_(5K)-~(131)I 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 CH_(3)-PEG_(5K)-biotin allowed construction of a reproducible competition curve. The (alpha)PEG cell-based competition ELISA measured small molecules derivatized by PEG_(2K), PEG_(5K), 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 (alpha)PEG cell-based competition ELISA. Finally, we show here that the (alpha)PEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG_(5K), comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH_(3)-PEG_(5K)-~(131)I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.