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

Abstract

Milk proteins are widely used for food supplementation, despite the potential risk of food allergy, especially against βlactoglobulin (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.

Trimeric Bet v 1-specific nanobodies cause strong suppression of IgE binding

“…While allergen-specific nanobodies as tools for food surveillance to identify traces of nut, lupine, and milk allergen contaminants have already been explored, the application of allergen-specific nanobodies as biologics for allergy prevention or treatment is a rather new endeavour ( 31 – 33 , 36 , 55 – 57 ). Passive administration of allergen-specific monoclonal antibodies has lately been proven to provide a fast and efficient treatment option for allergies ( 21 , 23 ).…”

Development of a Biotinylated Nanobody-Based Gold Nanoparticle Immunochromatographic Assay for the Detection of Procymidone in Crops

“…The protein G affinity chromatography column was used for purification of the isolated cells . Subsequently, the total RNA was extracted and immediately reverse-transcribed to cDNA using a reverse transcription kit . Nested PCR was performed to amplify the VHH genes, and the specific primers are shown in Table S1.…”

“…17 Subsequently, the total RNA was extracted and immediately reverse-transcribed to cDNA using a reverse transcription kit. 18 Nested PCR was performed to amplify the VHH genes, and the specific primers are shown in Table S1. The target band was recovered via two rounds of amplification, and the vector containing the target genes was obtained by digesting the plasmid vector pcomb3xSS by SfiI and linking by T4 DNA ligase.…”