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1.
Clin Vaccine Immunol ; 20(4): 517-25, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23389929

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.


Subject(s)
Antibodies, Neutralizing/immunology , Bacterial Proteins/immunology , Bacterial Toxins/immunology , Clostridioides difficile/immunology , Cytological Techniques/methods , Enterotoxins/immunology , Neutralization Tests/methods , Repressor Proteins/immunology , Animals , Automation, Laboratory/methods , Chlorocebus aethiops , Cricetinae , Male , Mesocricetus , Vero Cells
2.
Toxicon ; 47(4): 445-52, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16499940

ABSTRACT

Reconstitution of lyophilized antivenom is usually accomplished by gentle swirling for sometimes as much as 45min. Gentle resuspension is employed in order to avoid foaming the antivenom, which could have a variety of consequences including unfolding and denaturation of the protein, thus rendering it inactive. However, foaming of antivenom might cause only a small portion of the total protein to unfold or the protein may refold as foaming subsides. In this report, the effects of intentional severe foaming of three antivenom preparations are tested. Samples of gently resuspended antivenoms were subject to severe foaming by mechanical means, then tested for precipitation by examining the amounts of protein remaining in solution, for structural changes by circular dichroism spectroscopy and for activity changes by ELISA. In all cases, either no or minimal changes in antivenom properties were observed. These results indicate that severe intentional foaming does not substantially affect the properties of the antivenoms. It may be possible to employ more vigorous resuspension methods without affecting the efficacy of the antivenom. This could lead to a significant decrease in the time between envenomation and administration of antivenom.


Subject(s)
Antivenins/chemistry , Circular Dichroism/methods , Protein Folding , Snake Venoms , Protein Denaturation
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