Development of a novel duplex lateral flow test for simultaneous detection of casein and β-lactoglobulin in food

•A multiplex LFIA test was developed for simultaneous detection of β-lactoglobulin and casein.•Detection of β-lactoglobulin and casein was displayed in independent lines.•Limit of detection of 0.5 ppm for β-lactoglobulin and 2 ppm for caseins.•The new test was evaluated against incurred food processed matrices. Milk by-products such as whey and caseinate are widely used as ingredients or processing aids in food industry. However, since they could cause allergic reactions they are included in Allergen Control Plans. β-Lactoglobulin is the major whey protein and caseins are main proteins in milk. Selection of a unique target to analyze the presence of milk in foods could be insufficient when the source of milk proteins is unknown. A new test based on lateral flow immunocromatography that combines the simultaneous and independent detection of both proteins (β-lactoglobulin and casein) in one rapid test was developed. The assay was validated according to AOAC guidelines being able to detect β-lactoglobulin (0.5 ppm), casein (2 ppm), whey and powder milk (1–5 ppm). No cross-reactivity was found with a panel of 38 food commodities. The method is a rapid and suitable tool to identify milk proteins in processed food, ingredients, and rinsing water.