Detection of β-lactoglobulin under different thermal-processing conditions by immunoassay based on nanobody and monoclonal antibody

The problems of inaccurate detection values of thermal-processed β-lactoglobulin (β-LG) content seriously affect the screening of allergens. A monoclonal antibody (mAb) against β-LG was successfully prepared and a highly sensitive sandwich ELISA (sELISA) was constructed with specific nanobody (Nb) as the capture antibody with detection limit of 0.24 ng/mL. Based on this sELISA, the ability of Nb and mAb to recognize β-LG and β-LG interacting with milk components was explored. Combined with protein structure analysis to elaborate the mechanism of shielding β-LG antigen epitopes during thermal-processing, thus enabling the differentiation between pasteurized and ultra-high temperature sterilized milk, the detection of milk content in milk-containing beverages, and the highly sensitive detection and analysis of β-LG allergens in dairy-free products. The method provides methodological support for identifying the quality of dairy products and reducing the risk of β-LG contamination in dairy-free products.