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Vaccimonitor

Print version ISSN 1025-028XOn-line version ISSN 1025-0298

Abstract

PINO, Yadira et al. Validation of an ELISA assay to determine antibodies against LPS of Vibrio Cholerae. Vaccimonitor [online]. 2003, vol.12, n.1, pp.11-17. ISSN 1025-028X.

In order to evaluate the immunological response to any vaccine candidate, it is necessary to have standardized and validated laboratory techniques. For this reason the standardization of an indirect ELISA, used for determining serum antibodies against Ogawa LPS to cholera in sera of humans inoculated orally, was carried out; as well as the evaluation of the typical validation parameters for this assay. The optimal conditions for the coating concentration, blocking conditions and conjugate dilution were evaluated to select the best results for the positive control, the negative control and the blank. A 25 μg/mL concentration of Ogawa LPS in PBS left overnight for coating, 0,5% skimmed milk in PBS during 30 min at room temperature for blocking and anti-IgA-HRP diluted 1:5000 as conjugate were found to be the optimal variants for the assay. With respect to the technique validation, the parameters evaluated were precision, exactness, detection limit, specificity and robustness. A validation protocol allowing their evaluation was carried out. In all cases the assay was considered adequate, if the variation coefficient was less than 20%. For all the parameters evaluated this requisite was fulfilled, so that the results were considered to be reliable and reproducible.

Keywords : Cholera; ELISA; standardization; validation.

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