RESUMO
A bioinformatics approach to developing antibodies to specific proteins has been evaluated for the production of antibodies to heat-processed specified risk tissues from ruminants (brain and eye tissue). The approach involved the identification of proteins specific to ruminant tissues by interrogation of the annotation fields within the Swissprot database. These protein sequences were then interrogated for peptide sequences that were unique to the protein. Peptides were selected that met these criteria as close as possible and that were also theoretically resistant to either pepsin or trypsin. The selected peptides were synthesised and used as immunogens to raise monoclonal antibodies. Antibodies specific for the synthetic peptides were raised to half of the selected peptides. These antibodies have each been incorporated into a competitive enzyme-linked immunosorbent assay (ELISA) and shown to be able to detect the heat-processed parent protein after digestion with either pepsin or trypsin. One antibody, specific for alpha crystallin peptide (from bovine eye tissue), was able to detect the peptide in canned meat products spiked with 10% eye tissue. These results, although preliminary in nature, show that bioinformatics in conjunction with enzyme digestion can be used to develop ELISA for proteins in high-temperature processed foods and demonstrate that the approach is worth further study.
Assuntos
Biologia Computacional , Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Conservação de Alimentos , Produtos da Carne/análise , Carne/análise , Proteínas/análise , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Bovinos , Encefalopatia Espongiforme Bovina/prevenção & controle , Encefalopatia Espongiforme Bovina/transmissão , Feminino , Contaminação de Alimentos/prevenção & controle , Cabras , Camundongos , Camundongos Endogâmicos BALB C , Pepsina A/metabolismo , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Proteínas/imunologia , Reprodutibilidade dos Testes , Fatores de Risco , Sensibilidade e Especificidade , Ovinos , Software , Suínos , Temperatura , Tripsina/metabolismoRESUMO
A commercial aflatoxin detection ELISA kit, "RIDASCREEN(®) FAST Aflatoxin", was validated with corn samples naturally contaminated with aflatoxin and non-contaminated reference corn samples according to the Japanese Notification Method ShokuAnHatsu 0816-7. The trueness, intra-laboratory repeatability, intermediate precision, limit of detection and limit of quantitation were found to be 91%, 10%, 6.4%, 0.6µg/kg and 2µg/kg, respectively, and the performance of the kit was recognized as complying with all criteria in the Supplement Table of the Notification. These data suggest that this kit is useful as a simplified device to screen out negative corn samples contaminated with less than 4µg/kg.