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 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.
Kang
Jung Hye
JH
Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University, Ansan 426-791, South Korea.
Choi
Hyo Jin
HJ
Hwang
Sang Youn
SY
Han
Sang Ho
SH
Jeon
Jun Yeoung
JY
Lee
Eun Kyu
EK
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D001666:Binding Sites, Antibody; D008280:Magnetics; D051379:Mice; D010084:Oxidation-Reduction; D020349:Surface Plasmon Resonance
D000906:Antibodies