Development of a β-Lactoglobulin Sensor Based on SPR for Milk Allergens Detection
A sensitive and label-free surface plasmon resonance (SPR) based sensor was developed in this work for the detection of milk allergens. β-lactoglobulin (BLG) protein was used as the biomarker for cow milk detection. This is to be used directly in final rinse samples of cleaning in-place (CIP) systems of food manufacturers. The affinity assay was optimised and characterised before a standard curve was performed in pure buffer conditions, giving a detection limit of 0.164 µg mL−1 as a direct binding assay. The detection limit can be further enhanced through the use of a sandwich assay and amplification with nanomaterials. However, this was not required here, as the detection limit achieved exceeded the required allergen detection levels of 2 µg mL−1 for β-lactoglobulin. The binding affinities of the polyclonal antibody for BLG, expressed by the dissociation constant (KD), were equal to 2.59 × 10−9 M. The developed SPR-based sensor offers several advantages in terms of label-free detection, real-time measurements, potential on-line system and superior sensitivity when compared to ELISA-based techniques. The method is novel for this application and could be applied to wider food allergen risk management decision(s) in food manufacturing.
An SPR based sensor for allergens detection.
A simple, sensitive and label-free optical sensor method was developed for allergens analysis using α-casein as the biomarker for cow's milk detection, to be used directly in final rinse samples of cleaning in place systems (CIP) of food manufacturers. A Surface Plasmon Resonance (SPR) sensor chip consisting of four sensing arrays enabling the measurement of samples and control binding events simultaneously on the sensor surface was employed in this work. SPR offers several advantages in terms of label free detection, real time measurements and superior sensitivity when compared to ELISA based techniques. The gold sensor chip was used to immobilise α-casein-polyclonal antibody using EDC/NHS coupling procedure. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions giving a detection limit of 58ngmL-1 as a direct binding assay. The assay sensitivity can be further improved by using sandwich assay format and amplified with nanoparticles. However, at this stage this is not required as the detection limit achieved exceeded the required allergens detection levels of 2µgmL-1 for α-S1-casein. The sensor demonstrated good selectivity towards the α-casein as the target analyte and adequate recoveries from CIP final rinse wash samples. The sensor would be useful tool for monitoring allergen levels after cleaning procedures, providing additional data that may better inform upon wider food allergen risk management decision(s) that are made by food manufacturer. In particular, this sensor could potentially help validate or optimise cleaning practices for a given food manufacturing process.
Processing can alter the properties of peanut extract preparations.
As peanut allergy is an increasing public health risk, affecting over 1% of the United States and United Kingdom school children, it is important that methods and reagents for accurate diagnosis of food allergy and detection of allergenic foods are reliable and consistent. Given that most current experimental, diagnostic, and detection tests rely on the presence of soluble allergens in food extracts, we investigated the effects of thermal processing on the solubility and IgE binding of the major peanut allergens, Ara h 1 and Ara h 2. The soluble and insoluble fractions of peanuts that were boiled, fried, and roasted were subjected to electrophoresis and Western blot analysis using anti-Ara h 1 and anti-Ara h 2 antibodies and serum IgE from peanut allergic individuals. Overall protein solubility is reduced with processing and IgE binding increases in the insoluble fractions, due mostly to the increase in the amount of insoluble proteins, with increased time of heating in all processes tested. Therefore, it can be concluded that thermal processing of peanuts alters solubility, and the differences in protein solubility within various extract preparations may contribute to inconsistent skin prick test and immunoassay results, particularly when nonstandardized reagents are used.