Site-Specific Immobilization of Monoclonal Antibodies Using Spacer-Mediated Antibody Attachment

Abstract

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 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 0.87 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.

Improved Stabilization of Genetically Modified Penicillin G Acylase in the Presence of Organic Cosolvents by Co‐ Immobilization of the Enzyme with Polyethyleneimine

“…[46] Two hundred milligrams of enzymeagarose derivative were mixed with 1 mL of 1 mM N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) in 10 mM phosphate buffer pH 8.0 and the mixture was incubated for 1 h at room temperature. Then, samples were reduced with a 50 mM ditiothreitol (DTT) at pH 8.0.…”

Affinity membranes for hormone removal from aqueous solutions

“…The antibody volumetric capture is about 0.4% from the antibody up-take and therefore is negligible. The quantity of immobilized antibody is significantly less than the number of active sites available for antibody coupling, which is presumably due to the fact that antibodies have a relatively large dimension about 15 nm [10]. The coupling reaction between the surface and antibody depends on the antibody concentration.…”

“…The methods used for various affinity columns can be directly applied to membrane activation [6,8]. Immobilization of ligands, and we refer here more specifically to antibodies, onto surfaces is well documented [9][10][11]. The two major requirements for the immobilization of ligands onto insoluble matrices for affinity separation are a stable linkage between the matrix and the ligand, and maintaining of specific binding characteristics of the immobilized ligand.…”

TIRF Array Biosensor for Environmental Monitoring

“…Noncovalent attachment of antibodies via binding by Protein A or Protein G or covalent attachment through the carbohydrate side chain limit the attachment site to the Fe region of an antibody; thus there is no attachment in the binding site. Fab antibody fragments have been linked via the thiol group at the opposite end of the molecule from the antigen binding site [139,140]. The methods for preparing antibodies to be immobilized through either the carbohydrate or the thiol groups result in a significant loss of antibody during the processing procedure but generally produce highly functional surfaces.…”