Single domain antibodies are specially suited for quantitative determination of gliadins under denaturing conditions.
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.
Doña
Vanina
V
Laboratorio de Investigación en el Sistema Inmune - LISIN, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
Urrutia
Mariela
M
Bayardo
Mariela
M
Alzogaray
Vanina
V
Goldbaum
Fernando Alberto
FA
Chirdo
Fernando G
FG
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002446:Celiac Disease / Q000097:blood*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005903:Gliadin / Q000008:administration & dosage / Q000097:blood* / Q000737:chemistry; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D000906:Antibodies; D005903:Gliadin