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/aPEG cells) to develop competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The aPEG cell-coated plate bound biotinylated PEG5K (CH3-PEG5K-Biotin) and CH3-PEG5k-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 aPEG cell-based competition ELISA measured PEG2k PEG5k,PEGiok, PEG20K and PEG5K-derivatived small molecules at concentrations as low as 58.6 ng/ml, 14.6 ng/ml, 3.7 ng/ml, 3.7 ng/ml and 14.6 ng/ml, respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the aPEG cell-based competition ELISA. Finally, we show here that the aPEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG5K, comparable to those determined by direct measurement of radioactivity in blood after intravenous injection -PEG5k-131 I to mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules
