Production and characterization of a bacterial single-chain Fv fragment specific to human truncated midkine.
The production (and characterization) of a monoclonal antibody against human truncated midkine (tMK), and the detection of tMK in G401 cells, a Wilms' tumor cell line, as well as in Wilms' tumor patient specimens, have been reported (Paul et al., Cancer Lett. 163 (2001) 245-251). Here we report the molecular cloning and expression of this monoclonal antibody as a single-chain Fv fragment (scFv) in Escherichia coli. The scFv protein, purified by immobilized metal affinity chromatography, showed a specific affinity to recombinant tMK and native tMK in G401 cells as detected by enzyme-linked immunosorbent assay and immunofluorescence microscopy, respectively. The binding of this protein to recombinant tMK was competitive with the parental monoclonal antibody. These results suggest that this scFv can also be used for Wilms' tumor detection.
Dansithong
W
W
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, 376-8515, Gunma, Japan.
Paul
S
S
Mitsumoto
T
T
Saruhashi
S
S
Shinozawa
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001419:Bacteria / Q000276:immunology; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology*; D002846:Chromatography, Affinity; D016207:Cytokines; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000302:isolation & purification*; D008856:Microscopy, Fluorescence; D000078792:Midkine; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D016207:Cytokines; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D000078792:Midkine