Monoclonal antibody-based quantitation of poly(ethylene glycol)-derivatized proteins, liposomes, and nanoparticles.
Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.
Cheng
Tian-Lu
TL
Faculty of Biomedical Science and Environmental Biology, MedicoGenomic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan. tlcheng@kmu.edu.tw
Cheng
Chiu-Min
CM
Chen
Bing-Mae
BM
Tsao
Der-An
DA
Chuang
Kuo-Hsiang
KH
Hsiao
Sheng-Wen
SW
Lin
Yi-Hung
YH
Roffler
Steve R
SR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008081:Liposomes / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D049329:Nanostructures / Q000032:analysis*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D008081:Liposomes; D011506:Proteins; D011092:Polyethylene Glycols