Quantification of Anti-PEG Antibody

The ELISA to detect anti-PEG IgG and IgM was conducted using a previously developed method.¹⁰ Briefly, the eight-arm PEG-NH₂ (40 kDa, 200 μg mL^–1, JenKem Technology, USA) in PBS was coated onto MaxiSorp 96-well plates (Nunc, Denmark) for 18 h at 4 °C, followed by washing with PBS four times. Plates were blocked with 5% (w/v) skim milk powder in PBS for 22 h, followed by adding serially diluted human plasma in 5% skim milk in duplicate for 1 h at 22 °C. Plates were washed with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, Sigma-Aldrich, USA)/PBS buffer twice and PBS four times prior to addition of an HRP-conjugated antihuman IgG (Dako Agilent, USA) at 1:20,000 dilution or HRP-conjugated antihuman IgM (Jackson ImmunoResearch Laboratories, USA) at 1:10,000 for 1 h at 22 °C. Plates were washed as above and then developed using 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate (Sigma-Aldrich, USA). The reaction was stopped with 0.16 M H₂SO₄ and read at 450 nm. End point titers were calculated as the reciprocal plasma dilution giving signal 2× background using a fitted curve (4-parameter log regression) and reported as a mean of duplicates. Background was detected by adding the diluted plasma samples (at a 1:10 dilution in 5% skim milk) to the non-PEG-coated wells, followed by the same ELISA procedure.