Voltammetric Immunosensor to Track a Major Peanut Allergen (Ara h 1) in Food Products Employing Quantum Dot Labels

Tracking unreported allergens in commercial foods can avoid acute allergic reactions. A 2-step electrochemical immunosensor was developed for the analysis of the peanut allergen Ara h 1 in a 1-h assay (<15 min hands-on time). Bare screen-printed carbon electrodes (SPCE) were used as transducers and monoclonal capture and detection antibodies were applied in a sandwich-type immunoassay. The short assay time was achieved by previously combining the target analyte and the detection antibody. Core/shell CdSe@ZnS Quantum Dots were used as electroactive label for the detection of the immunological interaction by differential pulse anodic stripping voltammetry. A linear range between 25 and 1000 ng·mL−1 (LOD = 3.5 ng·mL−1), an adequate precision of the method (Vx0 ≈ 6%), and a sensitivity of 23.0 nA·mL·ng−1·cm−2 were achieved. The immunosensor was able to detect Ara h 1 in a spiked allergen-free product down to 0.05% (m/m) of peanut. Commercial organic farming cookies and cereal and protein bars were tested to track and quantify Ara h 1. The results were validated by comparison with an ELISA kit.

Peanut Component Ara h 1 and 2 Sensitization in Patients with Food Allergy in Kuwait

<b><i>Introduction:</i></b> There is limited knowledge on the sensitization patterns to peanut proteins and food allergy in the Middle East. The objective of this study is to analyze the relationship between sensitization patterns to peanut proteins and clinical symptoms in a group of patients with physician-diagnosed peanut allergy (PA) in Kuwait. <b><i>Methods:</i></b> PA patients were evaluated by the skin prick test (SPT), serum total IgE, peanut-specific IgE (sIgE), and sIgE against Ara h 1–3, 8, and 9, and clinical data were collected. <b><i>Results:</i></b> Sixty-nine patients were included. A positive correlation between peanut SPT and sIgE was detected for all 3 storage proteins (Ara h 1–3) in patients &#x3c;6 years old and for Ara h 1 and 2 in older patients. ROC analysis of positive correlations showed that oral food challenge should be considered for definite diagnosis of PA only if the level of Ara h 2 is &#x3c;22.25 KUA/L, with level of Ara h 2 ≥15.4 allowing the detection of systemic reactions with a sensitivity of 55.56%. Patients presenting with systemic reactions more frequently had positive Ara h 1 (88.9%) and Ara h 2 (83.3%), compared with 44.1% and 52.9% in those with local reaction (<i>p</i> = 0.0046 and <i>p</i> = 0.0378). The levels of Ara h 1 and 2 were also significantly higher in patients with systemic reactions compared to those with a local reaction, with those differences being especially relevant for Ara h 2 (15.9 vs. 0.4) (<i>p</i> = 0.0005). <b><i>Conclusions:</i></b> The pattern of sensitization to peanut proteins in the Middle East is similar to that of the Western world. Measurement of sIgE antibodies to Ara h 1, 2, and 3 is useful in the diagnosis of PA and in the investigation of reactions to raw and roasted peanuts.

Electrochemical Immunosensor for the Simultaneous Determination of Two Main Peanut Allergenic Proteins (Ara h 1 and Ara h 6) in Food Matrices

Efficiently detecting peanut traces in food products can prevent severe allergic reactions and serious health implications. This work presents the development of an electrochemical dual immunosensor for the simultaneous analysis of two major peanut allergens, Ara h 1 and Ara h 6, in food matrices. A sandwich immunoassay was performed on a dual working screen-printed carbon electrode using monoclonal antibodies. The antibody–antigen interaction was detected by linear sweep voltammetry through the oxidation of enzymatically deposited silver, which was formed by using detection antibodies labeled with alkaline phosphatase and a 3-indoxyl phosphate/silver nitrate mixture as the enzymatic substrate. The assay time was 2 h 20 min, with a hands-on time of 30 min, and precise results and low limits of detection were obtained (Ara h 1: 5.2 ng·mL−1; Ara h 6: 0.017 ng·mL−1). The selectivity of the method was confirmed through the analysis of other food allergens and ingredients (e.g., hazelnut, soybean and lupin). The dual sensor was successfully applied to the analysis of several food products and was able to quantify the presence of peanuts down to 0.05% (w/w). The accuracy of the results was confirmed through recovery studies and by comparison with an enzyme-linked immunosorbent assay. Tracking food allergens is of utmost importance and can be performed using the present biosensor in a suitable and practical way.

A Voltammetric Nanodiamond-Coated Screen-Printed Immunosensor for The Determination of a Peanut Allergen in Commercial Food Products

A voltammetric immunosensor was developed to quantify a major peanut allergen, Ara h 1, using screen-printed carbon electrodes (SPCE) as transducers. A sandwich-type immunoassay was performed on nanodiamond-coated SPCEs using an alkaline phosphatase-labeled detection antibody and a mixture containing an enzymatic substrate (3-indoxyl phosphate) and silver nitrate. The immunological interaction was detected through the (linear sweep) voltammetric stripping of the enzymatically deposited silver. The immunosensor’s applicability was evaluated by analyzing breakfast cereals, cookies, and energy and cereal bars. Ara h 1 was successfully tracked in these commercial food products.

Structure and IgE cross-reactivity among walnut and peanut vicilin leader sequences

Background: Vicilin seed storage proteins are translated with N-terminal leader sequences (LSs) that are cleaved to yield the mature protein. These LSs were thought to be unstructured and rapidly degraded. However, Ara h 1 and Jug r 2 LS (A1LS, J2LS) have been identified in seeds, and immunodominant IgE epitopes detected. Here, common sequences containing structured CxxxC-repeat motifs were identified as potential mediators of IgE cross-reactivity despite very low (17%) sequence identity. Method: Linear IgE epitopes were identified by peptide microarrays, in which overlapping 15-mer peptides on glass slides, were incubated with sera from peanut, walnut or dual allergic individuals. Similar epitopes were computationally predicted. Peanut A1LS and walnut J2LS fragments (J2.1, J2.2, J2.3) each with a CxxxC vicilin LS motif were identified, cloned, expressed, purified and their structures solved using solution-NMR to locate and assess epitopes on the structure. Results: A1LS and J2LSs reveal similar helix-turn-helix motifs connected by disulfide bonds between adjacent CxxxC repeats forming α-hairpin structures. Peanut-allergic IgE bound more frequently to the J2LSs, regardless of walnut allergic status or A1LS binding. IgE binding pattern to peptides from both J2LS and A1LS, along with structure and computational predictions, suggest that the structure and conserved amino acid properties of peptides determine cross-reactivity. The properties of LS IgE epitopes were closely related to epitopes in 2S albumins. Conclusion: The shared α-hairpin structure is a stable scaffold that contributes to cross-reactivity despite low sequence identity. Biophysical properties are a better predictor of distant cross-reactivity than traditional measures of evolutionary conservation.

Human monocyte-derived type 1 and 2 macrophages recognize Ara h 1, a major peanut allergen, by different mechanisms

Evidence has suggested that major peanut allergen Ara h 1 activates dendritic cells (DCs) via interaction with DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin), a C-type lectin receptor, and contributes to development of peanut allergy. Since macrophages, as well as DCs, play a crucial role in innate immunity, we investigated whether natural Ara h 1 (nAra h 1) activates two different subsets of macrophages, human monocyte derived macrophage type 1 (hMDM1: pro-inflammatory model) and type 2 (hMDM2: anti-inflammatory model). hMDM1 and hMDM2 predominantly produced pro-inflammatory cytokines (IL-6 and TNF-α) and an anti-inflammatory cytokine (IL-10) in response to nAra h 1, respectively. hMDM2 took up nAra h 1 and expressed DC-SIGN at higher levels than hMDM1. However, small interfering RNA knockdown of DC-SIGN did not suppress nAra h 1 uptake and nAra h 1-mediated cytokine production in hMDM2. Inhibitors of scavenger receptor class A type I (SR-AI) suppressed the response of hMDM2, but not of hMDM1, suggesting that SR-AI is a major receptor in hMDM2 for nAra h 1 recognition and internalization. nAra h 1 appears to exert stimulatory capacity on DC and macrophages via different receptors. This study advances our understanding how a major peanut allergen interacts with innate immunity.

IgE-Binding Epitopes of Pis v 1, Pis v 2 and Pis v 3, the Pistachio (Pistacia vera) Seed Allergens

Sequential IgE-binding epitopes were identified on the molecular surface of the Pis v 1 (2S albumin), Pis v 2 (11S globulin/legumin) and Pis v 3 (7S globulin/vicilin)—major allergens from pistachio (Pistacia vera) seeds—using the Spot technique. They essentially consist of hydrophilic and electropositively charged residues well exposed on the surface of the allergens. Most of the epitopic regions identified on Pis v 1 and Pis v 3 do not coincide with the putative N-glycosylation sites and thus are not considered as glycotopes. Surface analysis of these epitopic regions indicates a high degree of conformational similarity with the previously identified epitopic regions of the corresponding allergens Ana o 1 (vicilin), Ana o 2 (legumin) and Ana o 3 (2S albumin) from the cashew (Anacardium occidentale) nut. These results offer a molecular basis for the IgE-binding cross-reactivity often observed between pistachio and cashew nut. They support the recommendation for prescribing pistachio avoidance in cashew allergic patients. Other conformational similarities were identified with the corresponding allergens Ses i 1 (2S albumin), Ses i 3 (vicilin) and Ses i 6 (legumin) from sesame (Sesamum indicum), and Jug r 1 (2S albumin), Jug r 2 (vicilin) and Jug r 4 (legumin) from walnut (Juglans regia). Conversely, conformation of most of the epitopic regions of the pistachio allergens often differs from that of epitopes occurring on the molecular surface of the corresponding Ara h 1 (vicilin), Ara h 2 (2S albumin) and Ara h 3 (legumin) allergens from peanut (Arachis hypogaea).