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

Oswaldo Cruz Foundation, Research Center Rene Rachou, Av. Augusto de Lima 1715, Belo Horizonte, MG 30190-002, Brazil.
Vaccine (Impact Factor: 3.62). 07/2009; 27(33):4537-42. DOI: 10.1016/j.vaccine.2009.05.045
Source: PubMed


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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    • "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]. "
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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.
    Full-text · Article · Dec 2013 · BMC Veterinary Research
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    • "Therefore, if ELISA were capable of predicting accurately the results of the more informative, but technically more challenging TNA, then ELISA could be selected for further assay development. As a first step in deciding if one assay should be favored over the other, we studied the degree of agreement between the antibody estimate obtained by ELISA and those obtained by TNA in the same sample [12]. The anti-PA antibody response in mice immunized once with varying doses of either AVA or rPAV were measure by ELISA and TNA. "
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    ABSTRACT: Only one anthrax vaccine (Anthrax Vaccine Adsorbed, AVA) is licensed in the US to date, although new vaccines are under development. Logistic difficulties with the potency testing of AVA, plus humane considerations, have prompted the development of an alternative test method that can be used as a potency test not only for AVA, but also for newer vaccines. A potency test is not limited to measuring the concentration and quality of the antigen in the final formulation at the time of vaccine release, but should also detect changes in these characteristics during the dating period of the product, to ensure that the vaccine has retained its potency. We have developed a mouse immunogenicity test with potential for use as a potency test for anthrax vaccines. This model is based on the measurement of antibodies induced by a fixed dose of antigen. The test consists of two stages: a) the induction of antibodies in mice with one pre-selected test dose of vaccine; and b) the measurement of the response. We have established the test dose for AVA and experimental vaccines based on the use of anthrax Protective Antigen (PA). Two types of assays, an ELISA and a toxin neutralization assay (TNA) have been employed to measure antibodies to PA. TNA may be more useful in predicting vaccine efficacy, since it measures the neutralizing activity of sera against the cytotoxic effect of the toxin formed by PA when associated with Lethal Factor. However, anti-PA ELISA is less demanding from a technical point of view. Therefore, if ELISA were capable of detecting accurately changes in antigen quantity and quality, then it could be selected as the antibody-measuring test. Studies in our laboratory suggest that even though ELISA and TNA results are correlated, they may not be strictly interchangeable for quantification of anti-PA antibodies after a single immunization of mice with a test dose of anthrax vaccine. We have also found that TNA is better suited than ELISA to detect changes in immunogenicity caused by vaccine exposure to high temperature for very short periods (two minutes). These results open the possibility that an immunogenicity test in which TNA is used to quantify the anti-PA antibody responses can be used to measure vaccine potency of anthrax vaccines at the end of the manufacturing process and periodically after the finished product has been placed in storage, instead of an active protection test that requires lethal challenge. (C) 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of Integrated Laboratory Systems, Inc.
    Full-text · Article · Dec 2011 · Procedia in Vaccinology
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    ABSTRACT: This paper is intended to present an approach to decision making in the operation of electrical power systems that will use a simple genetic algorithm as a teacher for the process of supervised learning of a feedforward, backpropagation artificial neural network. The fitness function used in the genetic algorithm is based on a load flow program and used to determine the optimal condition of the critical switches of the system. Reward and penalty functions are applied to it in order to emphasize environmental, economic, security, robustness, public policy and other considerations as they are predetermined by the philosophy of operation of the electric utility. These considerations (policies) become a part of the training set and operation of the neural network. The fitness function used by the genetic algorithm in order to rank the possible solutions is based on a load flow program. The binary nature of the genetic algorithm is particularly appropriate for the operation of switches. The result of the methodology is the equivalent of an online implicit load flow program used to redesign the configuration of the power system in real-time by opening and closing critical switches that are placed along the power system. Experiments leading towards the development of this methodology using real data from the Peninsular Control Area (The Yucatan Peninsula) of the National Mexican Interconnected Power Grid are also presented
    No preview · Conference Paper · Sep 1996
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