Single domain antibodies are specially suited for quantitative determination of gliadins in denaturing conditions

DOÑA, VANINA; URRUTIA M; BAYARDO M; ALZOGARAY V; GOLDBAUM F; CHIRDO F

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2010 vol. 58 p. 918 - 926

0021-8561

Food intended for celiac patients’ consumption must be analyzed for the presence of toxic prolaminsusing high detectability tests. Though 60% ethanol is the most commonly used solvent for prolaminsextraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increaseprotein recovery. However, ethanol and denaturing agents interfere with antigen recognition whenconventional antibodies are used. In the present work, a new method for gliadins quantification isshown. The method is based on the selection of llama single domain antibody fragments able tooperate under denaturing conditions. Six out of 28 VHH-phages obtained retained their bindingcapacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additionalcysteines that could be responsible for the higher stability. One of the clones (named VHH26) wasfully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA usingVHH26 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 ofproteins or any other immunogenic compound, in the presence of denaturing agents, when specificrecognition units with high stability are required.