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

Yaozhong Hu,Yi Wang,Ying Sun,Shuo Wang,Feier Yang,Huan Lv,Sihao Wu,Xuemeng Ji,Chuan Zhang,Yang Lu,Serge Muyldermans,Jing Lin,Bowei Zhang

doi:10.3390/foods10102428

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.

Highlights

Lupine belongs to the Leguminosae family in taxonomy together with other legumes including peanut, soybean and pea [1]
Crude lupine protein extract was prepared with an acceptable efficiency
The results potentially demonstrated the recognition between the selected Nbs and a lupine protein, and confirmed the successful selection of specific Nbs

Summary

Introduction

Lupine belongs to the Leguminosae family in taxonomy together with other legumes including peanut, soybean and pea [1]. Lupine related allergies are on the rise to attract the global concern since the reported cases of allergic reactions, either against lupine allergens directly or from cross-reactivities to other legumes, especially peanut [3,4,5]. The storage proteins, including the allergen of Lup an 1, have been identified as the main allergen causing allergic reactions [7,8]. The surveillance for lupine allergens in foods and the availability of reliable detection methods seems essential and feasible. These analytical techniques should guarantee avoiding intake of lupine allergens and should ensure the compliance with food labeling regulations, the disclosure of adulterations, and consumer protection

Methods

Results

Conclusion