Camelization of a murine single-domain antibody against aflatoxin B1 and its antigen-binding analysis

Aflatoxin B-1 (AFB(1)), a highly toxic mycotoxin, always contaminated in a variety of agricultural products. Camelid variable domain of heavy chain antibody (VHH) is a noteworthy reagent in immunoassay, owing to its excellent characteristics. Immunization of camelid animals is a straightforward strategy to produce VHHs. In this study, to avoid the dependence on the large animals, the camelized, murine antibody (cVHs) against AFB(1) was prepared in vitro based on the identities between murine VH and camelid VHH and then to develop an immunoassay for AFB(1). A murine anti-AFB(1) VH fragment (VH-2E6) was selected for camelization through replacement of conserved hydrophobic residues in framework region 2 (FR2) (cVH-FR2), point mutation at position 103 in the FR4 region (cVH-103), and CDR3-grafted with a high AFB(1)-affinity VHH (cVH-Nb26). The cVH-Nb26 had a yield of 5 mg/L as refolded protein expressed from Escherichia coli and 10 mg/L expressed from Pichia pastoris. Compared with anti-AFB(1) single-chain fragment variable (scFv) 2E6, cVH-Nb26 performed more than 20-fold enhancement of AFB(1)-binding interactions. Although the AFB(1)-affinity of cVH-Nb26 cannot meet the application requirement in the present form, our study provides effective strategies for preparation of camelized antibody in vitro, which could be a promising immunoreagent for AFB(1) detection.