Generation and characterization of a novel single-chain antibody fragment specific against human fibrin clots from phage display antibody library

• Published in: Thrombosis research 2004, Vol.114 (3), p.205-211
• Main Authors: Yan, Jun Peng, Ko, Ju Ho, Qi, Yi Peng
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibody-targeted thrombolysis; Associated diseases and complications; Biological and medical sciences; Blood and lymphatic vessels; Blood Coagulation - immunology; Cardiology. Vascular system; Cloning, Molecular - methods; Diabetes. Impaired glucose tolerance; Diseases of the peripheral vessels. Diseases of the vena cava. Miscellaneous; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Enzyme-Linked Immunosorbent Assay; Fibrin - chemistry; Fibrin - immunology; Fibrin clots; Humans; Immunoglobulin Fragments - chemistry; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - immunology; Immunoglobulin Fragments - isolation & purification; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Immunoglobulin Variable Region - isolation & purification; Medical sciences; Molecular Sequence Data; Peptide Library; Phage display; Protein Binding; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Reproducibility of Results; Sarcoidosis. Granulomatous diseases of unproved etiology. Connective tissue diseases. Elastic tissue diseases. Vasculitis; Sensitivity and Specificity; Single-chain fragment variable antibody
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/j.thromres.2004.06.013

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.