title: Characterization of the native form of anthrax lethal factor for use in the toxin neutralization assay.
abstract: The cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.
authors: Emergent BioSolutions, Inc., Gaithersburg, Maryland, USA.
authors: Hang
authors: H
authors: Lu
authors: Jason
authors: J
authors: Catania
authors: Katalin
authors: K
authors: Baranji
authors: Jie
authors: J
authors: Feng
authors: Mili
authors: M
authors: Gu
authors: Janet
authors: J
authors: Lathey
authors: Diane
authors: D
authors: Sweeny
authors: Hannah
authors: H
authors: Sanford
authors: Kavita
authors: K
authors: Sapru
authors: Terry
authors: T
authors: Patamawenu
authors: June-Home
authors: JH
authors: Chen
authors: Alan
authors: A
authors: Ng
authors: Zenbework
authors: Z
authors: Fesseha
authors: Stefanie
authors: S
authors: Kluepfel-Stahl
authors: Jacob
authors: J
authors: Minang
authors: David
authors: D
authors: Alleva
mesh_terms: D000818:Animals; D000881:Anthrax / Q000276:immunology / Q000517:prevention & control*; D057134:Antibodies, Neutralizing / Q000097:blood*; D000942:Antigens, Bacterial / Q000235:genetics; D000992:Antitoxins / Q000097:blood*; D001427:Bacterial Toxins / Q000235:genetics; D002470:Cell Survival; D005260:Female; D008252:Macaca fascicularis; D008264:Macrophages / Q000187:drug effects; D008297:Male; D051379:Mice; D009500:Neutralization Tests / Q000379:methods*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; D000992:Antitoxins; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin
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