To develop early diagnostic reagents, effective vaccines, and even drugs against SARS-associated coronavirus (SARS-CoV), the human single fold single-chain antibody fragments, (scFv) libraries I + J (Tomlinson I + J) were used to identify novel scFvs, which can specifically bind to SARS-CoV. Interestingly, two scFvs (135 and 139) exhibited higher binding specificity to SARS-CoV with the OD450 value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of V-H gene (351 bp) and V-L gene (327 bp), so the two scFvs were uniformly named as SA59B and chosen for further analysis. SA59B scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The soluble 30 kDa SA59B scFv-antibody was verified in SDS-PAGE and Western-blot. The purified SA59B scFv-antibody was labeled with HRP by the glutaraldehyde method, and the concentration of HRP and SA59B scFv-antibody in the SA59B-HRP solution reached 2.4 and 2.28 mg/ml, respectively. Then, the binding ability of SA59B-HRP to SARS-CoV was evaluated by ELISA with S/N of 11.6, indicating higher binding specificity between them. Finally, both the SA59B sequence specificity and its application for diagnosis, prophylaxis or therapy of SARS were discussed. (c) 2005 Elsevier Inc. All rights reserved.