Quantitative determination of antigens under denaturing conditions using specific recombinant VHH fragments from llama

DOÑA VV; BAYARDO M; IAROSSI B; URRUTIA M; GOLDBAUM F; CHIRDO FG

Cordoba

Congreso; VII Latin American Congress of Immunology; 2005

Asociación latinoamericana de Inmunologia

Gliadins, a group of wheat proteins, are toxic for coeliac patients and must be excluded from their diet. To certify food products aimed for coeliacs, gliadins must be quantified using high detectability ELISA. To improve gliadins recovery from food samples, ethanol added with denaturing agents, 2 mercaptoethanol (2ME) and guanidine chloride (G), is used for extraction. Since conventional antibodies do not resist such denaturing condition, we tested the use of antigen-specific VHH fragments of heavy chain antibodies from Llama (Lama glama). VHHs are resistant to denaturing conditions and good candidates to obtain functional antigen-binding recombinant fragments. The aim of this study was to analyse the stability of recombinant VHH fragments and to develop quantitative assays able to detect antigens under denaturing conditions. We produced a phage display library of VHH fragments from Llama immunised with gliadins. Positive clones were selected after three rounds of antigen specific panning. Four out of twelve clones, tested by indirect ELISA, showed above 90% of reactivity in the presence of 15% ethanol and above 50% in the presence of 0.5M G and 0.5% 2ME. In competitive ELISA, all the four clones, produced quantitative assays with detection limit ranging between 20 - 40 ng gliadin/ml. Tests using these clones are able to detect gliadin even in buffers containing 0.1% 2ME and 0.01M G, but gliadins become undetectable when antigen denaturation occur prior to ELISA. In conclusion, we selected antigen specific VHH fragments, which are resistant to denaturing conditions and produce high detectatibility quantitative assays.