Quantification of major 2S allergen protein of yellow mustard using anti-Sin a 1 epitope antibody

Abstract

The major allergenic protein in yellow mustard (YM, Sinapis alba L.) is the 2S napin Sin a 1. The level of Sin a 1 in YM seeds was quantified using anti-epitope antibody (AE-Ab) generated against the epitope sequence QGPHVISRIYQTAT as the capture antibody in a non-competitive enzyme linked immunosorbent assay (NCI-ELISA), and Sin a 1 containing napin purified from YM (var Andante) as the reference standard. The AE-Ab showed high specificity towards Sin a 1 epitope-containing napin long chain and showed no cross reactivity with other proteins of YM or other Brassicaceae proteins of similar genetic homology. The assay quantified Sin a 1 with a limit of detection and quantitation (LOD and LOQ) of 3.08 ppm and 3.23 ppm, respectively with acceptable recoveries and precision. The Sin a 1 content in YM varieties produced in 2006, 2010, 2011 was in the range of 2.65–4.68 g (AC Base); 3.81–5.98 g (AC Pennant) and 3.11–4.92 g (Andante) per 100 g of seeds. Sin a 1 composed more than 50% of napin protein fraction of YM seed. A trend of increased Sin a 1 level with the increased contents of total seed protein and napin was observed from this data. • Complete extraction of yellow mustard napin can be achieved at pH 3. • Sin a 1 anti-epitope antibody showed specific binding with napin large chain. • Sin a 1 allergen in yellow mustard was in the range of 2.65–5.98 g/100 g seeds. • Napin and Sin a 1 levels showed a positive relationship with total protein content of the seed.