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

Aflatoxin B₁ (AFB₁), 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 AFB₁ was prepared in vitro based on the identities between murine VH and camelid VHH and then to develop an immunoassay for AFB₁. A murine anti-AFB₁ 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 AFB₁-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-AFB₁ single-chain fragment variable (scFv) 2E6, cVH-Nb26 performed more than 20-fold enhancement of AFB₁-binding interactions. Although the AFB₁-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 AFB₁ detection.