title: Optimization of an Anti-poly(ethylene glycol) (anti-PEG) Cell-Based Capture System To Quantify PEG and PEGylated Molecules.
abstract: Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process of drug development. Here, we combined different anti-PEG Fab expressing 293T cells as capture cells (293T/3.3, 293T/6.3, and 293T/15-2b cells) with four detective anti-PEG antibodies (3.3, 6.3, 7A4, or 15-2b) to optimize an anti-PEG cell-based sandwich ELISA. Then, we quantified free PEG (mPEG2K-NH2and mPEG5K-NH2) or PEG-conjugated small molecules (mPEG5K-biotin and mPEG5K-NIR797), proteins (PegIntron and Pegasys), and nanoparticles (Liposomal-Doxorubicin and quantum-dots). The combination of 293T/15-2b cells and the 7A4 detection antibody was best sensitivity for free PEG, PEG-like molecules, and PEGylated proteins with detection at ng mL-1levels. On the other hand, 293T/3.3 cells combined with the 15-2b antibody had the highest sensitivity for quantifying Lipo-Dox at 2 ng mL-1. All three types of anti-PEG cells combined with the 15-2b antibody had high sensitivity for quantum dot quantification down to 7 pM. These results suggest that the combination of 293T/15-2b cells and 7A4 detection antibody is the optimal pair for sensitive quantification of free PEG, PEG-like molecules, and PEGylated proteins, whereas the 293T/3.3 cells combined with 15-2b are more suitable for quantifying PEGylated nanoparticles. The optimized anti-PEG cell-based sandwich ELISA can provide a sensitive, precise, and convenient tool for the quantification of a range of PEGylated molecules.
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-sen University , Kaohsiung, Taiwan.
authors.forename: Wen-Wei
authors.initials: WW
authors.lastname: Lin
authors.forename: Yuan-Chin
authors.initials: YC
authors.lastname: Hsieh
authors.forename: Yi-An
authors.initials: YA
authors.lastname: Cheng
authors.affiliation: Graduate Institute of Pharmacognosy, Taipei Medical University , Taipei, Taiwan.
authors.forename: Kuo-Hsiang
authors.initials: KH
authors.lastname: Chuang
authors.forename: Chien-Chiao
authors.initials: CC
authors.lastname: Huang
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Chuang
authors.forename: I-Ju
authors.initials: IJ
authors.lastname: Chen
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-sen University , Kaohsiung, Taiwan.
authors.forename: Kai-Wen
authors.initials: KW
authors.lastname: Cheng
authors.forename: Yun-Chi
authors.initials: YC
authors.lastname: Lu
authors.forename: Ta-Chun
authors.initials: TC
authors.lastname: Cheng
authors.forename: Yeng-Tseng
authors.initials: YT
authors.lastname: Wang
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica , Taipei, Taiwan.
authors.forename: Steve R
authors.initials: SR
authors.lastname: Roffler
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-sen University , Kaohsiung, Taiwan.
authors.forename: Tian-Lu
authors.identifier: 0000-0001-6424-4731
authors.initials: TL
authors.lastname: Cheng
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives*; D004317:Doxorubicin / Q000031:analogs & derivatives / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000032:analysis*; D053758:Nanoparticles / Q000032:analysis; D011092:Polyethylene Glycols / Q000032:analysis*; D045663:Quantum Dots / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D007140:Immunoglobulin Fab Fragments; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; C506643:liposomal doxorubicin; D011092:Polyethylene Glycols; D001710:Biotin; D004317:Doxorubicin; C417083:peginterferon alfa-2b; C100416:peginterferon alfa-2a