Effect of Antibody Orientation on Immunosorbent

Authors

Anuradha Subramanian, Department of chemical Engineering,University of Nebraska Lincoln.Follow
William H. Velander, University of Nebraska-LincolnFollow

Date of this Version

September 1996

Comments

This article was published in : Journal of Molecular Recognition Volume 9, Issue 5-6, Date: September/December 1996, Pages: 528-535. © John Wiley & Sons, Ltd. This can be viewed at the publishers site: http://www3.interscience.wiley.com/cgi-bin/jhome/13149 Digital Object Identifier (DOI) 10.1002/(SICI)1099-1352(199634/12)9:5/6<528::AID-JMR296>3.0.CO;2-Q

Abstract

The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen bind-ing regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were in-activated and the FMA was removed. Mab was immobilizeed at low bead-averaged densities of about 0.4-1.1 mg Mab/mL matrix to minimize local density effects. Immunosorbents made using masked Mab (oriented coupling) gave antigen binding efficiencies (nAgo) f 4248% compared with 18-22% for those made by random coupling. The amount of (Fab), released from pepsin digestion of immuncsorbents was about --fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab), accessibility to pepsin correlates well with higher functional efficiency (nAJ and serves as a measure of orientation. In summary, at low Mab density and a 2:l molar rhPC to Mab binding stoichiometry, about 80% or more of the Mab randomly coupled through amino moieties was improperly oriented relative to oriented coupled Mab, which correlated with about 50% of lost Mab func-tionality upon immobilization.