title: Development and optimization of a high-throughput assay to measure neutralizing antibodies against Clostridium difficile binary toxin.
abstract: Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.
authors.affiliation: Department of Vaccine Research, Merck Research Laboratories, West Point, Pennsylvania, USA.
authors.forename: Jinfu
authors.initials: J
authors.lastname: Xie
authors.forename: Melanie
authors.initials: M
authors.lastname: Horton
authors.forename: Julie
authors.initials: J
authors.lastname: Zorman
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Antonello
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Zhang
authors.forename: Beth A
authors.initials: BA
authors.lastname: Arnold
authors.forename: Susan
authors.initials: S
authors.lastname: Secore
authors.forename: Rachel
authors.initials: R
authors.lastname: Xoconostle
authors.forename: Matthew
authors.initials: M
authors.lastname: Miezeiewski
authors.forename: Su
authors.initials: S
authors.lastname: Wang
authors.forename: Colleen E
authors.initials: CE
authors.lastname: Price
authors.forename: David
authors.initials: D
authors.lastname: Thiriot
authors.forename: Aaron
authors.initials: A
authors.lastname: Goerke
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Gentile
authors.forename: Julie M
authors.initials: JM
authors.lastname: Skinner
authors.forename: Jon H
authors.initials: JH
authors.lastname: Heinrichs
mesh_terms: D036002:ADP Ribose Transferases / Q000276:immunology*; D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood*; D001426:Bacterial Proteins / Q000276:immunology*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D057166:High-Throughput Screening Assays / Q000379:methods*; D008253:Macaca mulatta; D014709:Vero Cells
chemical_list: D057134:Antibodies, Neutralizing; D001426:Bacterial Proteins; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium