RESUMEN
The Working Group on Prolamin Analysis and Toxicity (WGPAT) organized a collaborative study to confirm whether the two R5 antibody-based ELISA test kits are able to detect gliadin in the lower mg/kg (ppm) level. Twenty laboratories investigated 12 blind-coded samples, spiked and naturally contaminated, to show the possibility of determining traces of gliadin in heat-treated or nonheat-treated foods by ELISA. It was shown that very small amounts of gliadin (below 100 ppm) could be detected by ELISA with a reproducibility RSD(R) (37%) and a repeatability RSD, (27%) common for ELISA under these conditions. The recovery of gliadin from the spiked samples was between 84 and 109%, based on the results of all laboratories, including those with poor performance. No false positives were found by the method (P < or =0.05), but one negative sample was contaminated during the bakery process. It is recommended that the method be accepted by AOAC as Official First Action.
Asunto(s)
Aminoácidos/análisis , Anticuerpos Monoclonales/química , Enfermedad Celíaca/metabolismo , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Análisis de los Alimentos/métodos , Gliadina/análisis , Glútenes/análisis , Hordeum/metabolismo , Técnicas para Inmunoenzimas/métodos , Prolaminas/análisis , Secale/metabolismo , Triticum/metabolismo , Algoritmos , Ensayo de Inmunoadsorción Enzimática/métodos , Contaminación de Alimentos , Juego de Reactivos para Diagnóstico , Reproducibilidad de los ResultadosRESUMEN
The second generation of a competitive ELISA for prolamin quantification based on the R5 antibody was studied for method performance and suitability to detect partially hydrolyzed prolamins in food. To be able to convert signal intensities to gluten concentrations, as required by the Codex Alimentarius Standard, a new calibrator consisting of a peptic-tryptic digest of wheat, rye, and barley prolamins was used for the first time. LOD and LOQ of the assay were 1.36 and 5.0 mg prolamin/kg food, respectively. Analysis of beer samples and a hydrolyzed wheat product showed that the assay provided significantly higher prolamin concentrations, compared to the sandwich ELISA based on the same antibody, which is only suitable for the detection of intact prolamins. Spiking experiments with defined concentrations of partially hydrolyzed prolamins gave recoveries ranging from 92 to 136%.