Usage of nanobody-beta-galactosidase fusion in immunoassays and its application in detecting a peanut allergen

Small-sized nanobodies (NBs) offer many advantages over traditional antibodies and antibody fragments. β-Galactosidase (β-gal) has been used as a detection enzyme in immunoassays when horseradish peroxidase (HRP) or alkaline phosphatase (ALP) could not be used. However, more research is needed to fully realize the benefits of using β-gal in immunoassays. This study fused a previously isolated NB specific to peanut allergen Ara h 3 (Nb16) with the tetrameric <i>Escherichia coli</i> β-gal. Kinetic signals generated using ONPG demonstrated the advantage of using β-gal in ELISA experiments. Peanut allergen Ara h 3 was successfully detected with the Nb16-β-gal, with a detection limit of 0.3 ppm, outperforming detection with the same NB and HRP. For detecting peanut proteins in baked foods, the detection limit was better than 1.56 ppm. Stable signals produced with S-Gal/X-Gal showed the benefits of using β-gal in immunoblots. The readily available, stable β-gal substrates and the ease of recombinant production of NB-β-gal chimeras are among the advantages of using β-gal over HRP and ALP as detection enzymes in immunoassays.