In vitro selection of anti-gliadin single-domain antibodies from a naïve library for cDNA-display mediated immuno-PCR

• Published in: Analytical biochemistry 2020-01, Vol.589, p.113490, Article 113490
• Main Authors: Jayathilake, Chathuni, Kumachi, Shigefumi, Arai, Hidenao, Motohashi, Maiko, Terai, Takuya, Murakami, Akikazu, Nemoto, Naoto
• Format: Article
• Language: English
• Subjects: cDNA display; cDNA display mediated immuno-PCR (cD-IPCR); Gliadin; Gluten toxicity; In vitro selection; Single-domain antibody
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2019.113490

Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. In this study, cDNA display method was used to select specific single-domain antibodies against toxic gliadin from an alpaca-derived naïve VHH library. The cDNA display method is a promising in vitro display technique, which uniquely converts an unstable mRNA-protein fusion molecule to a stable mRNA/cDNA-protein fusion molecule using a well-designed puromycin linker. Three candidate VHHs were selected and the affinities of the VHHs were observed by pulldown assay and indirect ELISA method. In addition, a novel cDNA display mediated immuno-PCR method (cD-IPCR) was successfully applied to detect gliadin in food. We believe this work demonstrates the potential application of the cDNA display method in selecting binders against toxic and heterogeneous targets such as gliadin with an immunization-free preparation manner. [Display omitted] •The cDNA display method was first applied to screen binders against gliadin protein from a naïve alpaca VHH library.•The binding of selected VHHs with gliadin were analyzed by affinity assays such as pulldown assay and ELISA. .•The affinity data were also confirmed by cDNA-display mediated immune-PCR (cD-IPCR).•Specificity analysis of anti-gliadin VHH antibodies was demonstrated for detection of gliadin in food.