title: Sensitivity of PEGylated interferon detection by anti-polyethylene glycol (PEG) antibodies depends on PEG length.
abstract: Attachment of poly(ethylene glycol) to proteins can mask immune epitopes to increase serum half-life, reduce immunogenicity, and enhance in vivo biological efficacy. However, PEGylation mediated epitope-masking may also limit sensitivity and accuracy of traditional ELISA. We previously described an anti-PEG-based sandwich ELISA for universal assay of PEGylated molecules. Here, we compared the quantitative assessment of PEGylated interferons by anti-PEG and traditional anti-interferon sandwich ELISA. The detection limits for PEG-Intron (12k-PEG) and Pegasys (40k-PEG) were 1.9 and 0.03 ng/mL for anti-PEG ELISA compared to 0.18 and 0.42 ng/mL for traditional anti-interferon sandwich ELISA. These results indicate that the anti-PEG sandwich ELISA was insensitive to PEGylation mediated epitope-masking and the sensitivity increased in proportion to the length of PEG. By contrast, PEG-masking interfered with detection by traditional anti-interferon sandwich ELISA. Human and mouse serum did not affect the sensitivity of anti-PEG ELISA but impeded traditional anti-interferon sandwich ELISA. The anti-PEG sandwich ELISA was comparable to anti-interferon sandwich ELISA and radioassay of 131I-Pegasys in pharmacokinetic studies in mice. The anti-PEG sandwich ELISA provides a sensitive, accurate, and convenient quantitative measurement of PEGylated protein drugs.
authors.forename: Ta-Chun
authors.initials: TC
authors.lastname: Cheng
authors.forename: Kuo-Hsiang
authors.initials: KH
authors.lastname: Chuang
authors.forename: Michael
authors.initials: M
authors.lastname: Chen
authors.forename: Hsin-Ell
authors.initials: HE
authors.lastname: Wang
authors.forename: Shey-Cherng
authors.initials: SC
authors.lastname: Tzou
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Su
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Chuang
authors.forename: Chien-Han
authors.initials: CH
authors.lastname: Kao
authors.forename: Bing-Mae
authors.initials: BM
authors.lastname: Chen
authors.forename: Long-Sen
authors.initials: LS
authors.lastname: Chang
authors.forename: Steve R
authors.initials: SR
authors.lastname: Roffler
authors.forename: Tian-Lu
authors.initials: TL
authors.lastname: Cheng
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007372:Interferons / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000493:pharmacokinetics
chemical_list: D000906:Antibodies; D011092:Polyethylene Glycols; D007372:Interferons