Chemical and Biomolecular Engineering Research and Publications


Date of this Version

January 2000


This article was published in Internation Journal of Bio Chromatography Vol 5 Issue 1 Pp. 67- 82. © 2000 Copyrights OPA (Overseas Publishers Assosciation, AmsterdamN>V.. Published by Harwood Academic Publishers Imprint a division of Gordon and Breach Publishing Group. Reprints are available directly for the publisher. Photocopying permitted only by license. For Published Version contact the publisher.


This work addresses the relative impact of the phenomena of orientation, multipoint attachment, and local density of the immobilized antibody 011 immunosorbent perfornnance. The masking of the antigen binding domains of monoclonal antibodies (Mab) by Fab Masking Antigens (FMAs) was used as a tool to determine the impact of orientation on the perforn~anceo f Emphaze, Affiprep'M, and Cellulose bead-based immunosorbents. A two-step antibody immobilization inethod involving permeation of the beaded matrix followed by a fast coupling reaction was used to study the effect of local density of the Mnh on immunosorbent efficiency. Lysyl groups on Mabs were covalently modified to reduce the number of reactive primary amines available for attachment to the matrix. Both native and modified Mabs were immobilized and the impact of the reduction in the number of available primary amines on immunosorbent efficiency was studied. Immobilizations performed with an "FMA-masked" antibody resulted in two- to three-fold higher antigen binding efficiencies (q(Ag)) as conipared to immobilizations with an "unmasked" antibody via random coupling. Mabs immobilized with the two-step method gave two- to three-fold higher when compared to immobilizations using conventional procedures under fast reacting conditions. Immobilizations of modified Mabs did not result in an increase in have used pepsin digestion of immmnosorbents to quantitatively correlate the increases in to the accessibility of the Fab domaiiis on the immobilized Mab. Factors impacting immunosorbent performance at low, moderate, and high antibody densities have been identified and ranked.