Study of variation in Ara h1 and Ara h3 expression in Australian and Indonesian peanut genotypes, based on the antibody-based phenotyping assays

Trace amounts of peanut protein trigger allergenic reactions in hypersensitive individuals. Many peanut cultivars are grown across the world; however, the information about allergen contents which is potentially valuable for peanut breeding programs is limited. The aim of this study is to investigate the variability of allergenic contents of 157 peanut genotypes from Australia and Indonesia, analysed by the developed antibody-based phenotypying assays specific to Ara h1 and Ara h3, and semi-qualitatively verified by SDS-PAGE.An ELISA specific to total peanut protein has been developed to investigate trace amounts of peanut protein in food and food products. The limit of detection of this assay was 1.4 μg L-1. This assay was highly specific to peanut, only showing very low cross reactivity for pine nuts, cashew, Lupinus angustifolius, and green bean. The recovery from the twelve food products spiked with peanut protein was within the accepted range of 79.7% - 122.3%. Ara h1 and Ara h3 from four peanut genotypes of different market types were successfully purified by employing two sequential steps of purification involving anion exchange chromatography and size exclusion chromatography. Polyclonal antibodies were successfully raised against the purified Ara h proteins. A phenotyping assay specific to Ara h1 utilized an indirect double antibody sandwich format using sheep and rabbit antibodies as capture and detection antibodies. This assay could detect as little as 12.3 μg L-1 of Ara h1. A direct sandwich ELISA specific to Ara h3 was developed based on the rabbit-rabbit antibodies as capture and detection antibodies. The limit of detection of this assay was 1.4 μg L-1. Quantification of the 157 Australian and Indonesian peanuts genotypes using ELISA and SDS-PAGE showed that Jepara and MLG 7721(Tegal) genotypes consisted of low Ara h1, Ara h2, Ara h3 and Ara h6. This finding is valuable for impending peanut breeding programs to develop hypoallergenic cultivars