This work addresses the relative impact of the phenomena of orientation, multipoint attachment, and local density of the immobilized antibody on immunosorbent performance. 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 performance of Emphaze, Affiprep(TM), and Cellulose bead-based immunosorbents. A two-step antibody immobilization method involving permeation of the beaded matrix followed by a fast coupling reaction was used to study the effect of local density of the Mab 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 (η((Ag))) as compared to immobilizations with an 'unmasked' antibody via random coupling. Mabs immobilized with the two-step method gave two to three-fold higher η((Ag)) when compared to immobilizations using conventional procedures under fast reacting conditions. Immobilizations of modified Mabs did not result in an increase in η((Ag)). We have used pepsin digestion of immunosorbents to quantitatively correlate the increases in η((Ag)) to the accessibility of the Fab domains on the immobilized Mab. Factors impacting immunosorbent performance at low, moderate, and high antibody densities have been identified and ranked.
|Original language||English (US)|
|Number of pages||16|
|Journal||International Journal of Bio-Chromatography|
|State||Published - 2000|
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