Improving immunobinding using oriented immobilization of an oxidized antibody.

ABSTRACT

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 CH(2) domain of the antibody's 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.