Development of Monoclonal Antibodies against Pea Globulins for Multiplex Assays Targeting Legume Proteins

Abstract

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.

Improved Sensitivity of Allergen Detection by Immunoaffinity LC-MS/MS Using Ovalbumin as a Case Study

“…Antibodies that recognize non-linear epitopes are occasionally found to bind with higher affinity than those recognizing linear epitopes [ 28 ]. ELISAs are generally sensitive and specific, but food processing often destroys native protein structures, preventing the detection of allergens with non-linear epitopes [ 29 , 30 , 31 ]. ELISAs are also used for the quantitative detection of food allergens [ 32 ], but the OD readout must be transformed into mg/mL or mg/kg using a conversion factor which is based on internal calibration [ 33 ].…”

Enzymatic Hydrolysis and Fermentation of Pea Protein Isolate and Its Effects on Antigenic Proteins, Functional Properties, and Sensory Profile

“…Modification of pea allergens by enzymatic treatment has been studied to a lesser extent. Pea protein isolate (PPI) treated with trypsin [ 14 ], Alcalase [ 15 ], flavourzyme, papain, and pepsin [ 16 ] have shown a reduced immunogenicity by means of ELISA methods. Frączek, Kostyra [ 14 ] found that a higher degree of hydrolysis resulted in a higher reduction in immunogenic potential.…”