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Legume proteins are widely used as food ingredients, but only some (soybean, lupin, and peanut) must be declared under consumer safety regulations to protect allergy sufferers. It is not yet mandatory to declare pea proteins as allergens even though they are predicted to be allergenic based on cross-reactivity in sensitized people. The processing of legume proteins can modify their allergenic properties and hence the need for specific and precise methods for the detection of all major legume allergens. There are many commercially available tests for known food allergens but not for ingredients that are yet to be classified as allergenic. We therefore generated sets of pea-specific antibodies targeting globulins to be used in a multiplex assay for the simultaneous detection of soybean, lupin, peanut, and pea proteins. We focused on the 7S globulin family, which is the least conserved among the four legumes, allowing the specific detection of proteins from each species. Having confirmed the specificity and sensitivity of the multiplex assay, we evaluated different processing steps for proteins rich in pea globulins to demonstrate the impact of food processing on antibody binding. Our sensitive multiplex assay provides a fast and reliable method for the specific detection of soybean, lupin, peanut, and pea allergens and is therefore ideal for food safety and authenticity testing applications.

Soybean allergens in food samples are currently detected in most cases using enzyme-linked immunosorbent assays (ELISAs) based on antibodies raised against bulk soybean proteins or specifically targeting soybean trypsin inhibitor, conglycinin, or glycinin. The various commercial ELISAs lack standardized reference material, and the results are often inaccurate because the antibodies cross-react with proteins from other legumes. Furthermore, the isolation of allergenic proteins involves laborious denaturing extraction conditions. To tackle these challenges, we have developed a novel sandwich ELISA based on monoclonal antibodies raised against the soybean 2S albumin Gly m 8 and a recombinant Gly m 8 reference protein with native-analogous characteristics. The antibodies do not cross-react with other legume proteins, and the extraordinary stability and solubility of Gly m 8 allows it to be extracted even from complex matrices after processing. The Gly m 8 ELISA therefore achieves greater specificity and reproducibility than current ELISA tests.