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

Abstract

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.

Versatile Application of Nanobodies for Food Allergen Detection and Allergy Immunotherapy

“…Taken together, Nbs process prominent characteristics such as the high stability against high temperature and pressure or extensive applicability under chemical solvents for the surveillant of trace food allergens. 22,44 General Strategies for the Selection of Nbs against Food Allergens. Generally, antigen-specific Nbs are selected on the basis of phage display technology to obtain the positive clones with protein-based phenotype and phagemid included genotype simultaneously.…”

“…44 Meanwhile, the researcher also validated the application of the unbiased strategy through selecting specific Nbs against peanut and lupine allergens. 22 Chen et al screened specific Nbs against the peanut allergen of Ara h 3 through a precise strategy by preparing the purified Ara h 3 first. 41 It could be more straightforward by using homogeneous allergens for the selection of allergen-specific Nbs.…”

“…44 In another study, specific Nbs against lupine allergen of Lup an 1 were selected, and a sandwich ELISA was developed providing a linear range of 0.036−4.4 μg/mL with a detection limit of 1.15 ng/mL, which is relatively more sensitive than the mAb-based immunoassay. 22 The increasing research outputs are expected with the increasing interest to develop Nbs against different food allergens, which will provide the promising tools to not only develop detecting methods against food allergens but also serve as research reagents for structure determination and epitope mapping of food allergens. Nb-Mediated Immunotherapy of Food Allergies.…”

“…Many other countries also specify the indication of food allergens through labeling, though the regulatory provisions have dissimilarities and variations. , Herein, reliable and precise detecting techniques are of great importance for food manufacturers to clear the presence of intentionally supplemented or unintentionally contaminated allergens to ensure compliance with such legislation and avoid economical loss since the unexpected allergen is obscured. , The frequently applied detecting strategies against food allergens mainly include protein-based immunoassay of enzyme-linked immunosorbent assay (ELISA) and DNA-based real-time polymerase chain reaction (RT-PCR) . DNA-based assay can provide high precision but is relatively inefficient or inconvenience for in situ application and cannot be applied for the surveillance of certain allergens. , The immunoassay based on allergen-specific antibodies is detecting approaches with extensive application, and varied techniques with the characteristics of rapid, multiplex for food allergen detection have been developed. − Although the immunoassay is commercially available for the detection of certain allergens, the limitation remains to restrict the application and detection of certain allergens including narrow detection limits, insufficient specificity and sensitivity, significant cross-reaction of conventional antibodies, and insufficient reproducibility. These drawbacks dominantly rely on the employed conventional monoclonal antibodies (mAbs), and the alternative candidates for the development of immunoassay with comparable advantages can be explored and investigated.…”

Quantum-Dot-Bead-Based Fluorescence-Linked Immunosorbent Assay for Sensitive Detection of Cry2A Toxin in Cereals Using Nanobodies

“…Nanobodies, which were first discovered by Hamers-Casterman in 1993, have attracted much attention in recent years [ 23 , 24 , 25 , 26 ]. They are a class of single-domain antibodies derived from heavy-chain antibodies of camelid or related species.…”