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 μg/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.
The declaration of lupine supplements is mandatory to avoid lupine allergy for sensitive individuals. However, reliable detection methods against lupine allergen remain critical to prevent the unintended consumption of allergen contaminated food. In this study, we have immunized an alpaca with lupine protein extracts and retrieved nanobodies (Nbs). Nevertheless, the target antigen has been recognized as Lup an 1, which has been classified as β-conglutin, and confirmed to connect with lupine allergy. After selection of the best Nb-pair, a sandwich enzyme-linked immunosorbent assay (ELISA) has been developed providing a linear range of 0.036–4.4 μg/mL with detection limit of 1.15 ng/mL. This immunoassay was confirmed by detecting the samples with spiked allergen, and a recovery from 86.25% to 108.45% with coefficient of variation (CV) less than 4.0% has been determined. Generally, this study demonstrated the selection of Nbs against allergen with crude protein content to develop the immunoassay for lupine surveillance in foods.
Peanut is widely used for food supplementation with potential allergic reactions in infants and adults, which prompted the development of reliable and accurate detection of peanut allergens with emphasis on Ara h 1. In this study, a nanobody (Nb)-based micro-total electrochemical immunoassay (Nb-μTEI) was proposed to be generated. Generally, an alpaca was immunized with Ara h 1 to yield a Nb reservoir for selection of four specific Nbs. Nb-mediated immunocapturing allowed the identification of the target as Ara h 1. The Nb-based electrochemical immunoassay was developed by constructing a capturing electrode with cycles of signal enhancement. After construction of the capturing electrode, Nb152 with HA-tag was directly applied to connect immobilized anti-HA IgG for the capture of different concentrations of Ara h 1, which was labeled by biotinylated Nb152 to facilitate signal development with alkaline phosphatase conjugated streptavidin (SA-ALP). A linear range from 4.5 to 55 ng/mL was acquired with LOD and LOQ of 0.86 and 2.10 ng/mL, respectively, with an 11-fold increase of the sensitivity compared with the established sandwich ELISA. The dedicated immunoassay was verified by detecting the antigen spiked in food samples and demonstrated the successful conjugation of Nb with advanced detecting techniques.
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