Unbiased Immunization Strategy Yielding Specific Nanobodies against Macadamia Allergen of Vicilin-like Protein for Immunoassay Development

Macadamia nut contains important food allergens that potentially cause allergic reactions with severe adverse effects in infants and adults. Reliable and accurate detection of macadamia is critical to avoid allergic reactions. However, knowledge on macadamia allergen is scarce and a reliable detection method has not been reported, yet. In this study, an unbiased immunization and selection strategy was employed to select nanobodies (Nbs) recognizing specifically macadamia allergen, as well as to establish a detection method to unveil a macadamia protein contamination. An alpaca was immunized with a crude protein extract of macadamia followed by construction of a Nb library from its lymphocytes. The panning and screening of this immune Nb repertoire resulted in the selection of six target-specific Nbs. Nb-mediated immuno-capturing combined with mass spectrometry allowed us to identify the target as the macadamia vicilin-like antimicrobial peptides 2-3 (MiAMP2), a novel food allergenic protein abbreviated as Mac i 1. Later on, an immunoassay of a heterologous sandwich ELISA method based on the selected Nb-pairs was established, providing a linear response in the range of 0.442-2,800 mu g/mL and with a limit of detection of 27.1 ng/mL. The dedicated immunoassay has been verified by detecting the antigen spiked in food samples. Our study provided evidence for the successful application of the unprejudiced strategy to retrieve Nbs against a priori undefined macadamia allergen. These target-specific Nbs were used to design a highly reliable and effective immunoassay.