The production and characterization of an anti-clenbuterol single-chain Fv antibody (CBLscFv)-bacterial alkaline phosphatase (AP) fusion protein are described. The CBLscFv and the phoA gene of Escherichia coli strain K12 chromosomal DNA were cloned by PCR and sequentially inserted into the expression vector pBV220 to express the CBLscFv-AP fusion protein in E. coli strain BL21(DE3)pLysS. SDS-PAGE and western blot analyses revealed that the fusion protein showed a molecular weight of 73 kDa and bound with the antibacterial AP monoclonal antibody. Determination of enzymatic activity indicated that k(cat) and K(m) values of the fusion protein were 113.60 s(-1) and 29.82 microM, respectively. Competitive direct enzyme-linked immunosorbent assay based on the obtained fusion protein indicated that the average concentration required for 50% inhibition of binding (IC(50)) and the limit of detection for CBL were 4.74 +/- 0.003 (n = 3) and 0.54 +/- 0.004 (n = 3) microg/l, respectively, and the linear response range extended from 1.13 to 69.68 microg/l. Cross-reactivity studies showed that the fusion protein did not cross-react with CBL analogs. The present findings indicate that the production of the CBLscFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggest that it could be further used to develop a one-step ELISA for the specific detection of CBL.