Development of an edema factor-mediated cAMP-induction bioassay for detecting antibody-mediated neutralization of anthrax protective antigen

Zmuda, Jonathan F.; Zhang, Linyi; Richards, Terri; Pham, Quyen; Zukauskas, David; Pierre, Jennifer L.; Laird, Michael W.; Askins, Janine; Choi, Gil H.

doi:10.1016/j.jim.2004.12.022

Cited by 8 publications

(9 citation statements)

References 22 publications

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“…The CHO cellbased assay was based on the work of Zmuda et al (36) and was further optimized. In this assay, the cells and serum samples were treated as described for the LT-TNA, except that the plated cells (40,000 cells/well) were incubated for approximately 22 h before addition of the serum-toxin mixture and that ET (50 ng/ml PA and 160 ng/ml EF, unless otherwise stated) was used instead of LT. To prevent cAMP degradation, the serum-toxin mixture contained 750 M 3-isobutyl-1-methylxanthine.…”

Section: Methodsmentioning

confidence: 99%

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Comparison of Three Anthrax Toxin Neutralization Assays

Ngundi

Meade

Lin

et al. 2010

Clin Vaccine Immunol

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Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays-J774A.1 cell-, RAW 264.7 cell-, and CHO cell-based assays-yield comparable estimates of neutralization activities for sera obtained after vaccination with anthrax vaccines composed of recombinant protective antigen (rPA). In order to compare the assays, sera were assayed alongside a common reference serum sample and the neutralization titers were expressed relative to the titer for the reference sample in each assay. Analysis of sera from rabbits immunized with multiple doses of the rPA vaccine showed that for later bleeds, the quantitative agreement between the assays was good; however, for early bleeds, some heterogeneity in relative neutralization estimates was observed. Analysis of serum samples from rabbits, nonhuman primates, and humans immunized with the rPA vaccine showed that the relative neutralization estimates obtained in the different assays agreed to various extents, depending on the species of origin of the sera examined. We identified differences in the magnitudes of the Fc receptormediated neutralization associated with the J774A.1 cell-and RAW 264.7 cell-based assays, which may account for some of the species dependence of the assays. The differences in the relative neutralization estimates among the assays were relatively small and were always less than 2.5-fold. However, because toxin neutralization assays will likely be used to establish the efficacies of new anthrax vaccines, our findings should be considered when assay outputs are interpreted.

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Section: Methodsmentioning

confidence: 99%

“…Thus, TNA measures the subset of antibodies that are considered functional. Moreover, TNA is considered to be species independent and has been standardized for use with multiple species (10,13,20,36). A species-neutral attribute is important for an assay that is to be used to bridge animal protection data to efficacy in humans.…”

mentioning

confidence: 99%

Comparison of Three Anthrax Toxin Neutralization Assays

Ngundi

Meade

Lin

et al. 2010

Clin Vaccine Immunol

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“…cAMP Induction Assay. The potency of rEF was analyzed by induction of cAMP in cells treated with anthrax edema toxin (PA plus EF) (14). Changes in cAMP concentration after edema toxin treatment were determined using an immunoassay system from Applied Biosystems, cAMP‐Screen Direct System.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, clinical trials are underway to show the safety of these antibody‐based treatments in humans. To support these clinical trials and the FDA licensing process, a robust bioassay was needed to determine the potency of the antibodies in neutralizing PA. To help address this need, an EF‐mediated, cAMP‐induction bioassay was recently developed at Human Genome Sciences, Inc. (14), which possesses a broad dose−response range and is highly accurate. This bioassay is based on the ability of PA to bind and shuttle EF into mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

Production of Biologically Active Bacillus anthracis Edema Factor in Escherichia coli

Cooksey¹,

Sampey²,

Pierre³

et al. 2004

Biotechnol. Prog.

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Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc-dependent metalloprotease that results in death. EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli.

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“…In vitro toxin neutralisation (TN) assays based on murine macrophage cell lines J774A.1 and RAW264.7 are frequently used and cell survival is determined following exposure to LT or to a mixture of LT and an antibody of choice [11,12,13]. A CHO cell-based assay has also been used to assess anti-PA therapeutic monoclonal antibody levels by measuring reduction in ET-induced cAMP levels [14]. …”

Section: Introductionmentioning

confidence: 99%

Development of an in Vitro Potency Assay for Anti-anthrax Lethal Toxin Neutralizing Antibodies

Whiting

Baker

Rijpkema

2012

Toxins

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Lethal toxin (LT) of Bacillus anthracis reduces the production of a number of inflammatory mediators, including transcription factors, chemokines and cytokines in various human cell lines, leading to down-regulation of the host inflammatory response. Previously we showed that the reduction of interleukin-8 (IL-8) is a sensitive marker of LT-mediated intoxication in human neutrophil-like NB-4 cells and that IL-8 levels are restored to normality when therapeutic monoclonal antibodies (mAb) with toxin-neutralising (TN) activity are added. We used this information to develop cell-based assays that examine the effects of TN therapeutic mAbs designed to treat LT intoxication and here we extend these findings. We present an in vitro assay based on human endothelial cell line HUVEC jr2, which measures the TN activity of therapeutic anti-LT mAbs using IL-8 as a marker for intoxication. HUVEC jr2 cells have the advantage over NB-4 cells that they are adherent, do not require a differentiation step and can be used in a microtitre plate format and therefore can facilitate high throughput analysis. This human cell-based assay provides a valid alternative to the mouse macrophage assay as it is a more biologically relevant model of the effects of toxin-neutralising antibodies in human infection.

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Development of an edema factor-mediated cAMP-induction bioassay for detecting antibody-mediated neutralization of anthrax protective antigen

Cited by 8 publications

References 22 publications

Comparison of Three Anthrax Toxin Neutralization Assays

Comparison of Three Anthrax Toxin Neutralization Assays

Production of Biologically Active Bacillus anthracis Edema Factor in Escherichia coli

Development of an in Vitro Potency Assay for Anti-anthrax Lethal Toxin Neutralizing Antibodies

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