Development of a quantitative immunoassay for gluten-free food analysis based on a new antigen-binding

DOÑA V, URRUTIA M, BAYARDO M, GOLDBAUM F, CHIRDOF

Verlag Wissenschaftliche Scripten, Alemania

Conferencia; 22nd Meeting Working Group on Prolamin Analysis and Toxicity; 2007

Coeliac disease is a permanent gastrointestinal disorder characterised by a permanent intolerance to a group of proteins (prolamins) present in wheat, and related cereals [1]. Histological changes in the intestinal mucosa as well as symptoms revert when patients follow a strict diet, free of toxic proteins. Gluten-free food certification requires both an efficient procedure for prolamin recovery from food samples and the use of powerful quantitative methods (commonly, immunoassays). Extraction with 60% ethanol has been the most common procedure used for prolamin recovery from food samples. However, due to the fact that prolamins are cross-linked through disulphide bonds to other prolamins and food matrix components, the efficacy of extraction involving 60% ethanol is low. To increase prolamins solubility, the use of reducing (2-mercaptoethanol) and denaturing agents (guanidine hydrochloride) has been proposed [2]. Previous work from our laboratory showed that these additives may affect antibody reactivity, causing interference in antigen antibody/ interaction and leading to quantitative errors [3]. Camelids produce, in addition to conventional antibodies, other molecules consisting of homodimeric heavy chains. In these antibodies, the antigen binding site is formed by one single domain named VHH. Under denaturing conditions, this structure is more stable than the antigen binding site in conventional antibodies [4]. In addition, recombinant VHH or VHH-phage from libraries are easily produced by genetic engineering techniques [5].