Comparability of ELISA and toxin neutralization to measure immunogenicity of Protective Antigen in mice, as part of a potency test for anthrax vaccines.
ABSTRACT Complexities of lethal challenge models have prompted the investigation of immunogenicity assays as potency tests of anthrax vaccines. An ELISA and a lethal toxin neutralization assay (TNA) were used to measure antibody response to Protective Antigen (PA) in mice immunized once with either a commercial or a recombinant PA (rPA) vaccine formulated in-house. Even though ELISA and TNA results showed correlation, ELISA results may not be able to accurately predict TNA results in this single immunization model.
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ABSTRACT: The cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of serum 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 95-99% cytotoxicity. In the presence of 50 ng/mL rPA, both rLF-A and rLF-HMA allowed for similar potencies (Effective Dilution 50%, ED50) 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 potentially be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and assuring a consistent renewable supply.Clinical and vaccine Immunology: CVI 05/2013; DOI:10.1128/CVI.00046-13 · 2.37 Impact Factor
Oral Oncology 07/2011; 47. DOI:10.1016/j.oraloncology.2011.06.087 · 3.03 Impact Factor
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ABSTRACT: Presently, few data exist on the level and duration of anti-protective antigen (PA) IgG in vaccinated livestock. Various adaptation of enzyme-linked immunosorbent assays (ELISAs) have been developed in studies to assess immune response following vaccination, albeit mostly in laboratory rodent models. The quantitative anti-anthrax IgG ELISA in this study describes a method of enumerating the concentration of anti-PA specific IgG present in sera of immunized goats, with the aid of an affinity-purified caprine polyclonal anti-anthrax PA-83 IgG standard. This was compared with the anthrax toxin neutralization assay (TNA) which measures a functional subset of toxin neutralizing anti-PA IgG. The measured concentrations obtained in the standard curve correlated with the known concentration at each dilution. Percentage recovery of the standard concentrations ranged from 89 to 98% (lower and upper asymptote respectively). Mean correlation coefficient (r2) of the standard curve was 0.998. Evaluation of the intra-assay coefficient of variation showed ranges of 0.23-16.90% and 0.40-12.46% for days 28 and 140 sera samples respectively, following vaccination. The mean inter-assay coefficient of variation for triplicate samples repeated on 5 different days was 18.53 and 12.17% for days 28 and 140 sera samples respectively. Spearman's rank correlation of log-transformed IgG concentrations and TNA titres showed strong positive correlation (rs = 0.942; p = 0.01). This study provides evidence that an indirect ELISA can be used for the quantification of anti-anthrax PA IgG in goats with the added advantage of using single dilutions to save time and resources. The use of such related immunoassays can serve as potential adjuncts to potency tests for Sterne and other vaccine types under development in ruminant species. This is the first report on the correlation of polyclonal anti-anthrax PA83 antibody with the TNA in goats.BMC Veterinary Research 12/2013; 9(1):265. DOI:10.1186/1746-6148-9-265 · 1.74 Impact Factor