Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications

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 ricinspecific 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 (k a ) and dissociation (k d ) rate constants and binding affinities (K D ) of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (K D ) 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. OPEN ACCESS Antibodies 2014, 3 216

Improving the biophysical properties of anti-ricin single-domain antibodies

“…To accomplish this metric, we started with previously described high-affinity sdAbs specific for three distinct epitopes on ricin [26] , [27] , and implemented a multi-step process for obtaining binders with improved melting temperature and solubility. Ricin is a potent toxin listed as a select agent by the CDC, and the ability to detect ricin remains a high priority [28] , [29] , [30] , [31] . The potential offered by these thermal stabilized sdAb reagents to eliminate the cold-chain makes them a highly attractive alternative to conventional monoclonal antibody and scFv binding reagents.…”

Fluorescent microsphere immunochromatographic assays for detecting bone alkaline phosphatase based on biolayer interferometry-selected antibody

“…In the competitive assay, only one antibody was required, the anti-BAP antibody possessing highest binding affinity with BAP should be used for immunization to achieve a higher competitive inhibition ratio. 40,41 Compared with other anti-BAP antibodies, the ab17272 exhibited the minimum K D . Therefore, the anti-BAP mAb (ab17272) was selected for the FM-LFIA.…”