Identification of single-chain antibody fragments specific against SARS-associated coronavirus from phage-displayed antibody library.
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 (B5 and B9) exhibited higher binding specificity to SARS-CoV with the OD(450) value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of V(H) gene (351bp) and V(L) gene (327bp), 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 30kDa 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 2.28mg/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.
Liu
Zheng-Xue
ZX
Key Laboratory of Virology, Ministry of Education, College of Life Science, Wuhan University, Wuhan 430072, PR China.
Yi
Guo-Hua
GH
Qi
Yi-Peng
YP
Liu
Ying-Le
YL
Yan
Jun-Peng
JP
Qian
Juan
J
Du
En-Qi
EQ
Ling
Wei-Fang
WF
D000595:Amino Acid Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000032:analysis / Q000737:chemistry* / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D017421:Sequence Analysis / Q000379:methods*
D007128:Immunoglobulin Fragments; D019151:Peptide Library
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