Effect of antibody orientation on immunosorbent performance

Journal of Molecular Recognition : JMR
A Subramanian, W H Velander

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 binding regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were inactivated 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 (nAg) of 42-48% compared with 18-22% for those made by random coupling. The amount of (Fab)2 released from pepsin digestion of immunosorbents was about 3-4-fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab)2 accessibility to pepsin correlates well with higher functional efficiency (nAg) and serves as a measure of orientation...Continue Reading

References

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Citations

Jan 6, 2006·Analytical and Bioanalytical Chemistry·King-Keung SinReinhard Renneberg
Mar 31, 2009·Current Microbiology·Maria del Mar Garcia-SuarezFrancisco J Mendez
Jul 4, 2009·Analytical Chemistry·Erin M JohnsonVicki H Wysocki
Dec 24, 2008·Analytical and Bioanalytical Chemistry·Lisa-Jo A ClariziaMelisenda J McDonald
Mar 29, 2000·Journal of Biomedical Materials Research·C R Jenney, J M Anderson
May 18, 2016·Toxins·Özlem ErtekinSelma Öztürk
Oct 26, 2011·Analytica Chimica Acta·Thomas MoreauOdile Vandenabeele-Trambouze

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