High-specificity quantification method for almond-by-products, based on differential proteomic analysis

Abstract

A highly specific competitive enzyme-linked immunosorbent assay (ELISA) protocol has been developed to identify and classify almond products based on differential proteomic analysis. We applied two-dimensional electrophoresis to compare the differences between almond and apricot kernels to search for almond-specific proteins. The amino acid of apricot Pru-1 was sequenced and aligned to almond Pru-1. One peptide, RQGRQQGRQQQEEGR, which exists in almond but not in apricot, was used as hapten to prepare monoclonal antibody against almond Pru-1. An optimized ELISA method was established using this antibody. The assay did not exhibit cross-reactivity with the tested apricot kernels and other edible plant seeds. The limit of detection (LOD) was 2.5–100μg/g based on different food samples. The recoveries of fortified samples at levels of twofold and eightfold LOD ranged from 82% to 96%. The coefficients of variation were less than 13.0%. Using 7M urea as extracting solution, the heat-treated protein loss ratios were 2%, 5% and 15% under pasteurization (65°C for 30min), baking (150°C for 30min) and autoclaved sterilization (120°C for 15min), respectively.