Abstract: Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA) and a ~32 kDa subunit B (RTB). There are fears of ricin being used as a weapon for warfare and terrorism and, as such, there is an increasing need for the development of immunodiagnostic reagents targeted towards this toxin. This article describes the production and characterization of a panel of six ricin-specific monoclonal IgG antibodies (mAbs), previously selected based upon their ability to inhibit ricin-mediated killing of cultured cells. Subsequent epitope binding analysis using the surface plasmon resonance (SPR) array biosensor (ProteOn XPR36) indicated three distinct, non-competitive binding epitopes (“bins”). The association (ka) and dissociation (kd) rate constants and binding affinities (KD) of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (KD) ranged from 0.1 nM to 9 nM. We present the coding sequences of the variable domains of the six mAbs, the expression, kinetic and cytotoxicity assays for two recombinant Fab (rFab) fragments and demonstrate a rFab affinity improvement by chain-shuffling. Together, these antibodies and constituent rFabs represent a panel of reagents for high-affinity recognition of ricin with potential national security biosensor applications.
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Cummins, M.; Dogovski, C.; Robert, R.; Alderton, M.; Chong, D.; Proll, D.; Pontes-Braz, L.; Raicevic, A.; Hattarki, M.; Nuttall, S.; Dolezal, O. Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications. Antibodies 2014, 3, 215-231.
Cummins M, Dogovski C, Robert R, Alderton M, Chong D, Proll D, Pontes-Braz L, Raicevic A, Hattarki M, Nuttall S, Dolezal O. Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications. Antibodies. 2014; 3(2):215-231.
Cummins, Michelle; Dogovski, Con; Robert, Remy; Alderton, Malcolm; Chong, Damien; Proll, David; Pontes-Braz, Luisa; Raicevic, Anna; Hattarki, Meghan; Nuttall, Stewart; Dolezal, Olan. 2014. "Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications." Antibodies 3, no. 2: 215-231.