Generation and characterization of a novel single-chain antibody fragment specific against human fibrin clots from phage display antibody library.
A novel single-chain fragment variable (scFv) antibody was developed directed against human fibrin clots by using the human single fold scFv libraries I+J (Tomlinson I+J). Three positively binding scFvs were evaluated by scFv-phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing. Then the positive scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC) with a yield of about 1 mg/l, the expression of soluble scFv was verified by Western blot analysis. The purified scFv could specifically recognize human fibrin clots and indicate no binding ability with human fibrinogen shown by ELISA. Furthermore, we will amplify the gene of the positive scFv by polymerase chain reaction (PCR) for future study of its role in diagnosis and therapy of thrombus-correlated diseases.
Yan
Jun Peng
JP
Key Laboratory of Virology, Ministry of Education, College of Life Science, Wuhan University, Hubei, PR China 430072.
Ko
Ju Ho
JH
Qi
Yi Peng
YP
D000595:Amino Acid Sequence; D001777:Blood Coagulation / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005337:Fibrin / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D005337:Fibrin