title: Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol.
abstract: OBJECTIVE
  There is currently much call for a reliable enzyme-linked immunosorbent assay (ELISA) protocol for determining gluten in foods to serve as a basis for further Codex Alimentarius regulations. Given its ability to recognize the potential coeliac-toxic epitope QQPFP, which occurs repeatedly in alpha-, gamma- and omega-gliadins, hordeins and secalins, the monoclonal antibody R5 raised against a secalin extract may prove to be an essential tool for gluten analysis. This study was designed to develop a highly sensitive and specific sandwich ELISA to quantify low levels of wheat, barley and rye prolamins in foods for coeliacs.
  
  
  METHODS
  Simple sandwich ELISA based on the use of a single monoclonal antibody (R5) as both the coating and detection was developed. A quantitative cocktail gluten-extraction procedure for heat-processed foods was also tested.
  
  
  RESULTS
  R5-ELISA was able to identify gliadins, hordeins and secalins with assay sensitivities of 0.78, 0.39 and 0.39 ng/ml, respectively. The assay's detection limit was 1.5 ng gliadins/ml (1.56 ppm gliadins, 3.2 ppm gluten). The system proved insensitive to the non-coeliac-toxic cereals maize, rice and oats, and was non-cultivar-dependent. It was also able to detect gliadins and hordeins in unprocessed and heat-processed wheat- and barley-based products, and to estimate the gluten content of hydrolysed foods.
  
  
  CONCLUSIONS
  We present a new generation of a robust sandwich R5-ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten-detection limit of 3.2 ppm is lower than the existing threshold of 20-200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat-processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5-ELISA system as proposed by the Codex Alimentarius Commission.
authors.affiliation: Unidad de Gluten, Centro Nacional de Biotecnología, Madrid, Spain.
authors.forename: Israel
authors.initials: I
authors.lastname: Valdés
authors.forename: Enrique
authors.initials: E
authors.lastname: García
authors.forename: Mercedes
authors.initials: M
authors.lastname: Llorente
authors.forename: Enrique
authors.initials: E
authors.lastname: Méndez
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D002446:Celiac Disease / Q000178:diet therapy; D002523:Edible Grain / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005504:Food Analysis / Q000379:methods*; D005903:Gliadin / Q000032:analysis; D005983:Glutens / Q000032:analysis* / Q000276:immunology; D006801:Humans; D010940:Plant Proteins / Q000032:analysis; D055315:Prolamins; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D010940:Plant Proteins; D055315:Prolamins; D005983:Glutens; D005903:Gliadin