2.1. Materials
Eight-arm PEG N-hydroxysuccinimide ester with an Mw of 10 kDa (8-arm PEG), 6-maleimidohexanoic acid N -hydroxysuccinimide ester (EMCS), iminothiolane hydrochloride (IT), and 3,3’,5,5’-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (USA). CRM197was kindly provided by Hualan Biological Engineering, Inc. (China). The RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (USA). Horseradish peroxidase-conjugated goat anti-mouse IgG Fc antibody (HRP-IgG), IgG1 Fc antibody (HRP-IgG1), IgG2a Fc antibody (HRP-IgG2a) and IgM Fc antibody (HRP-IgM) were ordered from Abcam (USA). IFN-γ and IFN-β ELISA kits were ordered from Legend (USA). TNF-α and IL-5 ELISA kits were ordered from Invitrogen (USA). All other reagents were of analytical grade.