Construction of sandwich ELISA method for quantitative detection of α-lactalbumin in hypoallergenic formula based on a novel monoclonal antibody prepared by the recombinant protein.

α-lactalbumin is a major allergen severely impends the food safety of milk allergic people. In this study, a biotin-streptavidin-amplified sandwich enzyme linked immunosorbent assay (sELISA) was developed by using the anti-recombinant α-lactalbumin monoclonal antibody (rALA-mAb) 3B20 as capture antibody and biotinylated anti-ALA polyclonal antibody (Bio-ALA-pAb) as detection antibody for specific quantification of ALA proteins in hypoallergenic formula. The assay has provided a tremendous wide linear detection range of 61.04 ng/mL–62.50 μg/mL compared with previous research, which spanned three orders of magnitude, with an improved limit of quantitation (LOQ) to 1.61 ng/mL. Recoveries of 6 kind of ALA spiked food matrixes were in an ideal range. The accuracy and precision of this method are 2.78% and 10.05% (intra-plate) and 10.23% (inter-plate) with an acceptable level of variation, respectively. The biotin-streptavidin-amplified sELISA saves more antibody in comparison to the previous classic ELISA. Furthermore, the established method was tested in the detection of ALA in the hydrolyzed hypoallergenic formulas of infants, the results were in great agreement with electrophoresis method. Compared with other commercial kits, this rALA-mAb based assay can be effectively used for qualitative and quantitative detection of ALA in highly processed food. As a potential analytical tool, the newly developed method is suitable for detecting ALA residues undeclared in dairy products samples available in the market, thereby will help to reduce the incidents of milk allergies.