Colloidal gold-based immunochromatographic assay for β-lactoglobulin variants a and B in dairy products and insights into antibody recognition mechanisms

Food fraud practices, such as milk adulteration, exacerbate allergy risks by introducing undeclared allergens, primarily β-lactoglobulin (β-Lg). Therefore, the effective detection of β-Lg in food is crucial for ensuring consumer safety. In this study, we screened hybridoma cell lines 11F10 and 4G1, which recognized β-Lg variants A (β-Lg A) and B (β-Lg B). Genes of these hybridoma cell lines were extracted, amplified, and cloned into expression vectors to produce recombinant antibodies (rAbs). Using rAbs 11F10 and 4G1, colloidal gold-based immunochromatographic strips were developed for detecting β-Lg A and B in dairy products. The strips exhibited a visual limit of detection of 200 μg/kg in cheese and amino acid formulas, and 200 ng/mL in raw milk. The results of molecular docking indicated that the critical amino acid residues for 11F10 were Asp57, Tyr32, and Arg49, whereas those for 4G1 included Tyr101, Tyr106, and Tyr49.