High-Throughput Kinetic Characterization of Ricin Toxin B Chain and Ovalbumin Antibodies Using Surface Plasmon Resonance

Hybridoma cell lines were developed against ricin toxin B (RTB) chain and ovalbumin. These cells were then grown in culture and purified for characterization and possible placement in the Critical Reagents Program repository. From the cell culture medium, antibodies were purified using protein A pur...

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Bibliographic Details
Main Authors: Warner, Candice R, Collett, Patricia, Carney, James P, Kragl, Frank J, Thompson, Roy, Buckley, Patricia
Format: Report
Language:English
Subjects:
ANTIBODIES
ANTIBODY CHARACTERIZATION
Biochemistry
BIOLOGICAL AGENTS
CHEMICAL AGENTS
CLONES
CONCENTRATION(COMPOSITION)
HYBRIDOMA DEVELOPMENT
KINETICS
LABEL-FREE BIOSENSOR ANALYSIS
LIGANDS
Mechanics
Medicine and Medical Research
OVA(OVALBUMIN)
PBS(PHOSPHATE BUFFERED SALINE)
PHOSPHATES
Plasma Physics and Magnetohydrodynamics
PLASMONS
RICIN
RTB(RICIN TOXIN B CHAIN)
SALINE SOLUTION
SPR(SURFACE PLASMON RESONANCE)
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Summary:Hybridoma cell lines were developed against ricin toxin B (RTB) chain and ovalbumin. These cells were then grown in culture and purified for characterization and possible placement in the Critical Reagents Program repository. From the cell culture medium, antibodies were purified using protein A purification, desalted in 1 phosphate buffered saline using Sephadex G-25 columns, and then filtered with a 0.2 m filter. Twenty-seven purified clones were characterized against their respective immunogens using a Bio-Rad ProteOn XPR36 high-throughput surface plasmon resonance instrument. RTB clones were also characterized against ricin holotoxin. Using an indirect assay format, we estimated kinetic data and toxin reactivity in 14 h. Data were collected and analyzed using Scrubber Pro software. Two clones were found to bind ricin holotoxin in addition to the RTB. Kinetic constants for the ovalbumin clones were also obtained. In the future, this highthroughput screening method can be applied to other purified antibody clones to rank their utility in downstream field assay applications. The original document contains color images. Prepared in cooperation with URS Corp., Abingdon, MD and Science and Technology Corp., Edgewood, MD.