Measurement of poly(ethylene glycol) by cell-based anti-poly(ethylene glycol) ELISA.
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/alphaPEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The alphaPEG 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 alphaPEG 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 alphaPEG cell-based competition ELISA. Finally, we show here that the alphaPEG 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.
Chuang
Kuo-Hsiang
KH
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Tzou
Shey-Cherng
SC
Cheng
Ta-Chun
TC
Kao
Chien-Han
CH
Tseng
Wei-Lung
WL
Shiea
Jentaie
J
Liao
Kuang-Wen
KW
Wang
Yun-Ming
YM
Chang
Ya-Chen
YC
Huang
Bo-Jyun
BJ
Wu
Chang-Jer
CJ
Chu
Pei-Yu
PY
Roffler
Steve R
SR
Cheng
Tian-Lu
TL
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D041702:BALB 3T3 Cells; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D051379:Mice; D011092:Polyethylene Glycols / Q000032:analysis* / Q000493:pharmacokinetics*; D044967:Serum / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry
D000906:Antibodies; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols