Exploration of Specific Nanobodies As Immunological Reagents to Detect Milk Allergen of β-Lactoglobulin without Interference of Hydrolytic Peptides

Milk proteins are widely used for food supplementation, despite the potential risk of food allergy, especially against β-lactoglobulin (BLG), which makes BLG surveillance critical. Possible interaction of detecting antibodies with BLG-derived peptides will result in unprecise inspection. Thus, in this study, it was proposed to generate nanobodies (Nbs) and validate the immunological detection of intact BLG rather than hydrolytic peptides. Nbs were successfully retrieved and characterized with high stability and target specificity. A competitive enzyme-linked immunosorbent assay (cELISA) was developed with a linear range from 39 to 10,000 ng/mL and a detection limit (LOD) of 4.55 ng/mL, with a recovery of 86.30%-95.09% revealed by analysis of spiked samples. Meanwhile, a sandwich ELISA (sELISA) was established with Nb82 and BLG polyclonal antibody (pAb-BLG) providing a linear range from 29.7 to 1250 ng/mL and an LOD of 13.82 ng/mL with a recovery of 87.82%-103.97%. The interaction of selected Nbs with BLG-derived peptides was investigated by Nb structure modeling and BLG docking. No binding on hydrolytic peptides was revealed, confirming the precision of Nb-mediated immunoassays. In summary, this study successfully identified BLG-specific Nbs for immunoassay development and guaranteed the monitoring of intact BLG without interference of hydrolytic peptides, providing experimental evidence that our Nbs recognize intact food allergen.