Generation of an Ovomucoid-Immune scFv Library for the Development of Novel Immunoassays in Hen’s Egg Detection

Author:

Rodríguez Santiago1ORCID,García-García Aina1ORCID,Garcia-Calvo Eduardo1ORCID,Esteban Vanesa2ORCID,Pastor-Vargas Carlos3ORCID,Díaz-Perales Araceli4ORCID,García Teresa1ORCID,Martín Rosario1

Affiliation:

1. Departamento de Nutrición y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (UCM), 28040 Madrid, Spain

2. Departamento de Alergia e Inmunología, IIS-Fundación Jiménez Díaz, Universidad Autónoma de Madrid (UAM), 28040 Madrid, Spain

3. Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain

4. Centro de Biotecnología Y Genómica de Plantas, Universidad Politécnica de Madrid-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (CBGP, UPM-INIA), 28223 Madrid, Spain

Abstract

Hen’s egg allergy is the second most common food allergy among infants and young children. The possible presence of undeclared eggs in foods poses a significant risk to sensitized individuals. Therefore, reliable egg allergen detection methods are needed to ensure compliance with food labeling and improve consumer protection. This work describes for the first time the application of phage display technology for the generation of a recombinant antibody aimed at the specific detection of hen’s ovomucoid. First, a single-chain variable fragment (scFv) library was constructed from mRNA isolated from the spleen of a rabbit immunized with ovomucoid. After rounds of biopanning, four binding clones were isolated and characterized. Based on the best ovomucoid-binding candidate SR-G1, an indirect phage enzyme-linked immunosorbent assay (phage-ELISA) was developed, reaching limits of detection and quantitation of 43 and 79 ng/mL of ovomucoid, respectively. The developed ELISA was applied to the analysis of a wide variety of food products, obtaining a good correlation with a commercial egg detection assay used as a reference. Finally, in silico modeling of the antigen-antibody complex revealed that the main interactions most likely occur between the scFv heavy chain and the ovomucoid domain-III, the most immunogenic region of this allergen.

Funder

Ministerio de Ciencia e Innovación

Consejería de Educación e Investigación de la Comunidad de Madrid

Publisher

MDPI AG

Subject

Plant Science,Health Professions (miscellaneous),Health (social science),Microbiology,Food Science

Reference50 articles.

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