Improving immunobinding using oriented immobilization of an oxidized antibody

Recent technical advances in biorecognition engineering and microparticle fabrication enabled us to develop a single-step purification process using magnetic particles (MPs). The process is simple, efficacious, easy to automate, and economical. The method immobilizes the ligand molecule in a particular orientation on commercial MPs that have surface carboxyl groups. Mouse IgG and anti-mouse IgG antibody were the model capture and ligand molecules for this study. The immunobinding efficacy of anti-mouse IgG antibody using “oriented immobilization” was compared with the efficacy of a conventional amine-coupling system that results in random orientation and of another standard method, the biotin–streptavidin system. The oriented immobilization was accomplished by oxidizing the sugar moiety in the CH2 domain of the antibodyʹs Fc and covalently conjugating the moiety to the hydrazine-coated MP. The specific binding affinity of the oriented immobilization process was about 2.5 times that of the amine-coupling system, and selectivity from a binary mixture was about 2 times greater for the oriented immobilization method. Results were nearly identical for the biotin–streptavidin system and the oriented immobilization system, matching the calculated binding stoichiometry between mouse IgG and anti-mouse IgG antibody. The binding improvement over the amine-coupling system shown by assay was confirmed by a separate surface plasmon resonance experiment. In summary, the oriented immobilization method was as effective as the streptavidin–biotin system, yet simpler and cost-effective.