4.10. Anti-PEG Detection Assays in Human Plasma

Chimeric anti-PEG antibodies (c3.3, an IgG anti-PEG, and cAGP4, an IgM anti-PEG) were obtained from our collaborators (Roffler et al.) who developed them [69,70]. The patient’s serum was assayed for the presence of anti-PEG IgG and IgM in a direct ELISA against immobilized PEG as described previously in details [12]. Briefly, the patient’s serum samples were diluted 25-fold in 2% (w/v) powdered skim milk in PBS (Dulbecco’s phosphate-buffered saline, Thermo Fisher Scientific). Two additional 2-fold serial dilutions were made in dilution buffer (4% human negative serum in 2% skim milk in PBS). Standard curves were obtained by serial dilutions (3-fold) of chimeric anti-PEG antibodies (c3.3 or cAGP4) starting at 2.5 or 2 μg/mL, respectively in dilution buffer. The patient’s serum samples at dilutions of 25, 50, and 100-fold and the antibody standards were incubated in duplicate for 1 h RT in Maxisorp 96-well microplates previously coated with 0.5 μg/well NH2-PEG10K-NH2 then blocked with skim milk 5% in PBS. Unbound antibodies were washed with 0.1% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) in PBS, then PBS only. Horseradish peroxidase (HRP)-conjugated secondary antibodies (goat F (ab′)2 anti-human IgG Fc or goat F (ab′)2 anti-human IgM) were added to the IgG or IgM detection plates, respectively, for 1 h RT. The plates were washed and incubated with ABTS substrate for 30 min RT. The absorbance (405 nm) in the wells was measured using a BioTek Synergy™ 4 Hybrid Microplate Reader (Winooski, VT, USA). Positive responses were defined as samples with absorbance values at least 3 times greater than the mean background absorbance (dilution buffer). The relative concentrations of anti-PEG IgG or IgM in positive samples were calculated by comparison with c3.3-IgG or cAGP4-IgM standard curves, respectively. Positive samples were confirmed by a PEG competition assay as described in [12].