Screening of Nanobody Specific for Peanut Major Allergen Ara h 3 by Phage Display

Abstract

Peanut allergy is a major health problem worldwide. Detection of food allergens is a critical aspect of food safety. The VHH domain of single chain antibody from camelids, also known as nanobody (Nb), showed its advantages in the development of biosensors because of its high stability, small molecular size, and ease of production. However, no nanobody specific to peanut allergens has been developed. In this study, we constructed a library with random triplets (NNK) in its CDR regions of a camel nanobody backbone. We screened the library with peanut allergy Ara h 3 and obtained several candidate nanobodies. One of the promising nanobodies, Nb16 was further biochemical characterization by gel filtration, isothermal titration calorimetry (ITC), cocrystallization, and Western blot in terms of its interaction with Ara h 3. Nb16 specifically binds to peanut major allergen Ara h 3 with a dissociation constant of 400 nM. Furthermore, we obtained the Ara h 3-Nb16 complex crystals. Structure analysis shows the packing mode is completely different between the Ara h 3-Nb16 complex crystal and the native Ara h 3 crystal. Structural determination of Ara h 3-Nb16 will provide the necessary information to understand the allergenicity of this important peanut allergen. The nanobody Nb16 may have application in the development of biosensors for peanut allergen detection.

Nanobodies—Useful Tools for Allergy Treatment?

“…So far, only one allergen-specific nanobody is described in the literature. This nanobody is reported to be specific for the major peanut allergen, Ara h 3 and was isolated from a synthetic library of humanized nanobodies via phage display (95). The interaction between Ara h 3 and the Ara h 3-specific nanobody resulted in a dissociation constant of 400 nM representing medium affinity binding and was further investigated by the structural determination of formed co-crystals (95).…”

“…This nanobody is reported to be specific for the major peanut allergen, Ara h 3 and was isolated from a synthetic library of humanized nanobodies via phage display (95). The interaction between Ara h 3 and the Ara h 3-specific nanobody resulted in a dissociation constant of 400 nM representing medium affinity binding and was further investigated by the structural determination of formed co-crystals (95). The authors acknowledged that additional work is needed to improve the affinity of the isolated nanobody to make it an attractive tool for the development of biosensors for peanut allergen detection.…”

Nanoanticuerpos: desarrollo biotecnológico y aplicaciones

“…Los Nac también se han utilizado en inmunoensayos para detectar a patógenos transmitidos por alimentos (He et al, 2020;Hu et al, 2021), así como a los alérgenos del cacahuate (Chen et al, 2019) y de los cereales (García-García, Madrid, González, García & Martín, 2020). Además, se han creado inmunosensores para identificar a las microalgas tóxicas que afectan a la industria pesquera y turística (Oloketuyi et al, 2020;Mazzega, Beran, Cabrini & Marco, 2019).…”

Characterization and Genome Analysis of a Novel Escherichia coli Bacteriophage vB_EcoS_W011D

“…recombinant phage to control diseases caused by bacterial infection among animals (Stanley, 2018;Chen et al, 2019).…”