Monoclonal Antibody-Based Quantitation of Poly(ethylene glycol)-Derivatized Proteins, Liposomes, and Nanoparticles

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• 作者：Tian-Lu Cheng ; Chiu-Min Cheng ; Bing-Mae Chen ; Der-An Tsao ; Kuo-Hsiang Chuang ; Sheng-Wen Hsiao ; Yi-Hung Lin ; and Steve R. Roffler
• 刊名：Bioconjugate Chemistry
• 出版年：2005
• 出版时间：September 2005
• 年：2005
• 卷：16
• 期：5
• 页码：1225 - 1231
• 全文大小：219K
• 年卷期：v.16,no.5(September 2005)
• ISSN：1520-4812

文摘

Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is aproven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitativeanalysis 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 describea new IgG₁ monoclonal antibody (E11) to PEG and show that it can be used in combination withAGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunitsof 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 chainson pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modifiedenzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detectionantibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. Inaddition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG_20,000, PEG_2,000-quantum dots, and PEG_2,000-liposomes at concentrations as low as 20 ng/mL (1.0nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show thatthe sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. Theseantibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.