title: Development and optimization of a novel assay to measure neutralizing antibodies against Clostridium difficile toxins.
abstract: Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on labor-intensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.
authors: Department of Vaccine Research, West Point, PA, USA.
authors: Jinfu
authors: J
authors: Xie
authors: Julie
authors: J
authors: Zorman
authors: Lani
authors: L
authors: Indrawati
authors: Melanie
authors: M
authors: Horton
authors: Keri
authors: K
authors: Soring
authors: Joseph M
authors: JM
authors: Antonello
authors: Yuhua
authors: Y
authors: Zhang
authors: Susan
authors: S
authors: Secore
authors: Matthew
authors: M
authors: Miezeiewski
authors: Su
authors: S
authors: Wang
authors: Anthony D
authors: AD
authors: Kanavage
authors: Julie M
authors: JM
authors: Skinner
authors: Irene
authors: I
authors: Rogers
authors: Jean-Luc
authors: JL
authors: Bodmer
authors: Jon H
authors: JH
authors: Heinrichs
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D057205:Automation, Laboratory / Q000379:methods; D001426:Bacterial Proteins / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000276:immunology*; D006224:Cricetinae; D003584:Cytological Techniques / Q000379:methods*; D004768:Enterotoxins / Q000276:immunology*; D008297:Male; D008647:Mesocricetus; D009500:Neutralization Tests / Q000379:methods*; D012097:Repressor Proteins / Q000276:immunology*; D014709:Vero Cells
chemical_list: D057134:Antibodies, Neutralizing; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D012097:Repressor Proteins; C105615:TcdC protein, Clostridium difficile; C041348:tcdA protein, Clostridium difficile
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