A Mass Spectrometry ImmunoAssay (MSIA) specifically designed for the detection of egg allergens in wines is described. This approach is based on an immunoaffinity enrichment procedure combined with targeted MS/MS detection of selected egg peptide markers. Polyclonal antibodies raised against native ovalbumin, chosen as the target protein tracing for egg powder, were immobilized onto low backpressure monolithic MSIA customized disposable tips. Ovalbumin-free wine samples were fortified with standard protein at different concentration levels in the low µg/mL range. A simple protocol was devised consisting in a 1:4 dilution of the wine sample with a basic solution for pH adjustment, followed by a semi-automated purification/enrichment step on customized MSIA disposable tips fitted on a multichannel electronic pipette. Among the main figures of merit LOD and LOQ values as low as 0.01 and 0.03 ?g/mL, respectively, of 18 % should be noticed. Noteworthy, the developed assay outperformed current MS-based methods for the detection of allergenic protein in wine matrices, thanks to the immunoaffinity enrichment. In addition compared to other immunoassays, the present approach boasts the unquestionable advantage to provide an unambiguous identification of the target protein, by simultaneous detection of three unique peptide markers each giving three specific MS/MS transitions.
DEVELOPMENT OF A MASS SPECTROMETRY IMMUNOASSAY FOR UNAMBIGUOUS DETECTION OF EGG ALLERGEN TRACES IN WINES
ROSA PILOLLI;LINDA MONACI
2016
Abstract
A Mass Spectrometry ImmunoAssay (MSIA) specifically designed for the detection of egg allergens in wines is described. This approach is based on an immunoaffinity enrichment procedure combined with targeted MS/MS detection of selected egg peptide markers. Polyclonal antibodies raised against native ovalbumin, chosen as the target protein tracing for egg powder, were immobilized onto low backpressure monolithic MSIA customized disposable tips. Ovalbumin-free wine samples were fortified with standard protein at different concentration levels in the low µg/mL range. A simple protocol was devised consisting in a 1:4 dilution of the wine sample with a basic solution for pH adjustment, followed by a semi-automated purification/enrichment step on customized MSIA disposable tips fitted on a multichannel electronic pipette. Among the main figures of merit LOD and LOQ values as low as 0.01 and 0.03 ?g/mL, respectively, of 18 % should be noticed. Noteworthy, the developed assay outperformed current MS-based methods for the detection of allergenic protein in wine matrices, thanks to the immunoaffinity enrichment. In addition compared to other immunoassays, the present approach boasts the unquestionable advantage to provide an unambiguous identification of the target protein, by simultaneous detection of three unique peptide markers each giving three specific MS/MS transitions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
