Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.

Immunoassays are the most commonly used quantitative techniques to determine the gliadin content of food aimed at coeliac patients. Though the minimal amount of gliadins inducing the typical histopathological changes at the intestinal mucosa in coeliacs is still a matter of debate, current research is focussed on the development of methods having higher sensitivities. One of the main drawbacks in gliadin analysis is the low eYciency of the conventional extraction procedure using 60% ethanol. The use of reducing (2-mercaptoethanol) and denaturing (guanidinium chloride) agents has been recommended to improve the extraction eYciency. Owing to the well-known eVects of these agents on native conformation of proteins, and their widely reported interference on the antigen/antibody interaction in other systems, we assessed whether gliadin detection by immunoassays is aVected by the presence of those agents. Using two ELISA formats with a panel of polyclonal and monoclonal antibodies, we found that recognition by speciWc antibodies of partially or totally denatured gliadins is severely impaired. The magnitude of the interference Keywords Gliadin analysis • ELISA • Coeliac disease • Antigen denaturation Abbreviations 2-ME 2-Mercaptoethanol GuHCl Guanidinium chloride mAb Monoclonal antibody