Comparability of ELISA and toxin neutralization to measure immunogenicity of Protective Antigen in mice, as part of a potency test for anthrax vaccines

Parreiras, Patrícia Martins; Sirota, Lev; Wagner, Leslie; Menzies, Sandra L.; Arciniega, Juan

doi:10.1016/j.vaccine.2009.05.045

Cited by 13 publications

(13 citation statements)

References 28 publications

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“…This conclusion appears to have resulted in the use of the standard concentrations of 50 and 40 ng/ml for rPA and rLF-HMA, respectively, by the majority of laboratories reporting TNA validation and immunogenicity studies (15,(18)(19)(20)(21)(22)(23)(24). Our results are in agreement with such a concept, in that increasing rLF-A concentration decreased the measured potency (and, most likely, sensitivity) of anti-LF antibody neutralizing activity (Fig.…”

Section: Discussionsupporting

confidence: 81%

“…After a 4-h incubation, the degree of cytotoxicity is measured via a redox viability dye, which allows for a 50% effective dilution (ED 50 ) potency value to be obtained from the serum titration curve (18,19). The specific rPA and rLF concentrations of 50 and 40 ng/ml, respectively, have been used in most reported TNA validation and immunogenicity studies of animal and human vaccinations (15,(18)(19)(20)(21)(22)(23)(24). Although potency among rPA production lots is consistent (our unpublished observation), the potency of rLF is known to vary substantially among lots.…”

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confidence: 99%

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Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

Catania

Baranji

et al. 2013

Clin. Vaccine Immunol.

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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.

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Section: Discussionsupporting

confidence: 81%

mentioning

confidence: 99%

Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

Catania

Baranji

et al. 2013

Clin. Vaccine Immunol.

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“…This approach has been used in the field of vaccine development albeit with different model species [30,31]. Correlation between ELISA-measured IgG levels and TNA derived neutralization titres have been shown previously in various laboratory species [24,32] but not in a ruminant species. Therefore the ELISA was also compared to the TNA.…”

Section: Discussionmentioning

confidence: 99%

“…The TNA is a technique developed to measure the ability of antibodies in sera of immunized animals to neutralize the PA and its contribution to LT cytotoxicity for certain sensitive cell lines [22-24]. This technique is species independent and has been standardized for use with multiple species [25-27].…”

Section: Introductionmentioning

confidence: 99%

Quantitative anti-PA IgG ELISA; assessment and comparability with the anthrax toxin neutralization assay in goats

et al. 2013

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BackgroundPresently, 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.ResultsThe 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).ConclusionThis 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.

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“…Given the rarity of anthrax in humans, it is unlikely that true vaccine efficacy and a direct correlate of protection based on levels of AbPA in humans can be determined. While lethal toxin neutralizing antibodies (TNA) may also be relevant, studies in humans 13 and mice 14 have generally indicated a strong correlation between AbPA and TNA levels. Levels of AbPA in response to priming doses of AVA vary greatly among individuals, with 100- to 1000-fold differences in peak AbPA response following at least two doses of AVA.…”

Section: Introductionmentioning

confidence: 99%

The role of HLA–DR–DQ haplotypes in variable antibody responses to Anthrax Vaccine Adsorbed

et al. 2011

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Host genetic variation, particularly within the human leukocyte antigen (HLA) loci, reportedly mediates heterogeneity in immune response to certain vaccines; however, no large study of genetic determinants of anthrax vaccine response has been described. We searched for associations between the IgG antibody to protective antigen (AbPA) response to Anthrax Vaccine Adsorbed (AVA) in humans and polymorphisms at HLA class I (HLA-A, -B, and -C) and class II (HLA-DRB1, -DQA1, -DQB1, -DPB1) loci. The study included 794 European-Americans and 200 African-Americans participating in a 43-month, double-blind, placebo-controlled, clinical trial of AVA (clinicaltrials.gov identifier NCT00119067). Among European-Americans, genes from tightly linked HLA-DRB1-DQA1-DQB1 haplotypes displayed significant overall associations with longitudinal variation in AbPA levels at 4, 8, 26, and 30 weeks from baseline in response to vaccination with 3 or 4 doses of AVA (global p=6.53×10−4). In particular, carriage of the DRB1-DQA1-DQB1 haplotypes *1501-*0102-*0602 (p=1.17×10−5), *0101-*0101-*0501 (p=0.009), and *0102-*0101-*0501 (p=0.006) was associated with significantlylower AbPA levels. In carriers of two copies of these haplotypes, lower AbPA levels persisted following subsequent vaccinations. No significant associations were observed amongst African-Americans or for any HLA class I allele/haplotype. Further studies will be required to replicate these findings and to explore the role of host genetic variation outside of the HLA region.

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Comparability of ELISA and toxin neutralization to measure immunogenicity of Protective Antigen in mice, as part of a potency test for anthrax vaccines

Cited by 13 publications

References 28 publications

Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

Quantitative anti-PA IgG ELISA; assessment and comparability with the anthrax toxin neutralization assay in goats

The role of HLA–DR–DQ haplotypes in variable antibody responses to Anthrax Vaccine Adsorbed

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