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

Abstract

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.

Functional Hybrid Micro/Nanoentities Promote Agro-Food Safety Inspection

“…In addition, we must focus on the development of biosensors to detect macadamia nut allergens more quickly and accurately. Further research should be undertaken to intensively investigate the exact binding epitope and the structural intrinsic features of the macadamia antigen …”

“…Further research should be undertaken to intensively investigate the exact binding epitope and the structural intrinsic features of the macadamia antigen. 139 Nbs need further in-depth research and application in food safety detection. Although they overcomes the shortcomings of small-molecule functional antibodies, they still have shortcomings, such as low affinity and poor stability.…”

Selection of Specific Nanobodies against Lupine Allergen Lup an 1 for Immunoassay Development

“…Enrichment of Nb displayed phages against lupine protein extract was obtained by consecutive rounds of bio-panning based on a well-established protocol [16]. In general, a representative fraction of immune library was inoculated into 2 × TY medium to grow until exponential phase.…”

“…Camelidae species possess a unique functional antibody format known as heavy chain-only antibody (HCAb) with an antigen binding fragment that is restricted to one single variable domain of the heavy chain, referred to as VHH or nanobody (Nb) [15]. A straightforward and successful method has been developed to select specifically the Nbs against allergens [16]. More importantly, the unique characteristics of Nbs facilitate the recognition of their target antigens with relatively high affinity and specificity, with the potential to identify the unique epitopes that are difficult to be targeted by conventional antibodies [17,18].…”

Intrabody Targeting HIF-1α Mediates Transcriptional Downregulation of Target Genes Related to Solid Tumors

“…Specific Nbs against rPasB were retrieved from the immune library and the synthetic library after panning and screening processes [45]. Generally, one aliquot of the library stock was cultured in 300 mL 2 × TY medium supplemented with 1% (w/v) glucose and 100 µg/mL ampicillin until an OD 600nm of 0.6-0.8 was reached.…”