Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis

• Published in: Analytical and bioanalytical chemistry 2016-01, Vol.408 (3), p.895-903
• Main Authors: Qiu, Yu-Lou, He, Qing-Hua, Xu, Yang, Wang, Wei, Liu, Yuan-Yuan
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Antigens; Antigens - chemistry; Antigens - genetics; Antigens - immunology; Assaying; bacteriophages; Biochemistry; Biofilms; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography; deoxynivalenol; ELISA; engineering; enzyme-linked immunosorbent assay; Food Science; Fractionation; grains; Health aspects; Immunoassay; Immunoassay - instrumentation; Immunoassay - methods; Immunoassays; Laboratory Medicine; Mathematical analysis; Methods; Molecular Mimicry; Monitoring/Environmental Analysis; Monoclonal antibodies; Mutagenesis; mutants; Nanostructure; Observations; Peptides; Phage; Research Paper; Residues; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; Trichothecenes - chemistry; Trichothecenes - genetics; Trichothecenes - immunology
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-015-9181-5

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102–Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.