Site-specific immobilization of monoclonal antibodies using spacer-mediated antibody attachment

Site-directed and random coupling of antibodies (Abs) was performed using aminated silica surfaces as substrates. The site-directed coupling linked the Ab, a monoclonal anti-fluorescein IgG1 (9-40), to the surface via 3400 Dalton (Da) poly(ethylene oxide) (PEO) spacers. The hydrazide end groups of these spacers were attached to aldehyde groups in the hinge region of the oxidized Ab to yield surfaces with an Ab concentration of 2.1 pmol/cm(2). The random coupling, in turn, linked the Ab via its available primary amines directly to the glutaraldehyde-activated surface with a yield of 3.8 pmol/cm(2). Despite a nearly twofold difference in Ab concentration, the two surfaces bound similar amounts of the FL-BSA antigen (0.56 vs 0.55 pmol/cm(2)), while their nonspecific uptake of protein was 0.11 vs. 0.21 pmol/cm(2), respectively, reflecting the protein repellent quality of PEO-coated surfaces. Fab' fragments of the 9-40 Ab were also Linked to the same tethers. Here, the succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) difunctional coupling reagent was attached to the PEO-hydrazide via its succinimide end and to the carboxy-terminal thiol of the the Fab' via its maleimide end. The concentration of reactive groups was varied by mixing difunctionalized PEO (PEO - (HZ)(2)) with monofunctionalized polymer (CH3O-PEO-HZ) prior to surface attachment. At 100% PEO-(HZ)(2) the FL-BSA (Ag) binding was 0.77 pmol/cm(2) while the nonspecific binding was 0.058 pmol/cm(2). Progressive dilution of the reactive PEO chains to 25% led to the remarkable binding of 0.57 pmol/cm(2) of Ag with 0.037 pmol/cm(2) of nonspecific binding. Competitive release from these various surfaces showed more favorable kinetics for the Fab' surface with 25% active tethers.