Antigenicity of the Low Molecular Weight Proteins in Selected Tree Nuts, Oilseeds, Legumes and Cereals

The antigenic properties of the low molecular weight (LMW) proteins, polypeptides and peptides of several plant foods were evaluated. Trichloroacetic acid (0.6 M) was used to isolate the non-proteins nitrogen fraction of selected tree nuts, oilseeds, legumes and cereals. The antigenicity and cross-reactivity for LMW proteins, polypeptides and peptides were evaluated with polyclonal antibodies raised against almond, almond major protein (AMP), cashew major protein (CMP), peanut, pecan, pistachio and walnut glutelin and monoclonal antibodies raised against AMP (mAb 4C10 and 4F10) and cashew (Ana o 1-4B7 and Ana o 2-4H9). Three immunological assays were utilized to determine antigenicity and cross-reactivity, including Dot blotting, Western blotting and ELISA. The ELISA utilized in this study used rabbit anti almond as the primary antibody, and the almond standard curve had an IC50 value of 0.4837 ± 0.028 mg/ml. Significant antigenicity and cross-reactivity in the NPN fraction was found with Dot blot, Western blot and ELISA assays. Antigenic peptides with a molecular weight range of 7.69-31.02 kDa were identified. TCA extracted tree nut and oilseed samples were typically more cross reactive than legumes and cereals. Typically, cereals were not determined to be cross-reactive in more than one assay. Polyclonal antibodies that were raised against whole proteins recognized more antigenic LMW species than polyclonal antibodies that were more specific. Also, monoclonal antibodies did not recognize any cross-reactive species in the NPN fraction. This study serves as a preliminary tool for LMW antigen identification and can guide the direction of future research in this area. Establishing clinical relevance in humans will be important before these LMW antigens can be considered allergens. If identified antigens are determined to be allergens, research focused on reducing their allergenicity can be approached.