title: Phage display generation of a novel humanized single-chain antibody against brain natriuretic peptide with potent neutralizing activity.
abstract: Cerebral salt wasting syndrome (CSW) is defined as a renal loss of sodium and water during intracranial disease leading to hyponatremia, which is the most frequent electrolyte disorder in critically neurological patients. Abnormal brain natriuretic peptide (BNP) secretion is implicated as the main offender. Development of antagonist against BNP is therefore of potential clinical relevance. In this study, synthetic human BNP peptide (hBNP) was used as bait and a humanized single chain fragment variable (scFv) phage antibody library as the source of antagonists. After three rounds of biopanning, hBNP-specific phage clones were greatly enriched. The scFv gene from the best phage clone was inserted into pET-22b and expressed in Escherichia coli BL21 (DE3) PlysS cells. After purification by nickel-affinity and refolding, this scFv antibody (Ab) was proven to recognize hBNP specifically and sensitively in ELISA and dot-blotting assay. Its binding constant to hBNP was 1.98×10(-8) M, measured by surface plasmon resonance. Thus, the humanized scFv Ab prepared with this approach has potential therapeutic value for neutralizing abnormally high level of BNP correlated well with CSW.
authors.affiliation: Department of Molecular Medicine, Institute for Advanced Studies in Multidisciplinary Science and Technology, East China Normal University, Shanghai, PR China. zygluck@gmail.com
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology*; D001435:Bacteriophages; D061505:Cell Surface Display Techniques; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D020097:Natriuretic Peptide, Brain / Q000276:immunology* / Q000378:metabolism; D019151:Peptide Library; D020539:Sequence Analysis, Protein; D057127:Single-Chain Antibodies / Q000276:immunology*; D020349:Surface Plasmon Resonance; D019282:Wasting Syndrome / Q000628:therapy; D014882:Water-Electrolyte Balance
chemical_list: D057134:Antibodies, Neutralizing; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies; D020097:Natriuretic Peptide, Brain