Construction and purification of a covalently linked divalent tandem single-chain Fv antibody against placental alkaline phosphatase.
Multivalency is a recognized means to increase the functional affinity of single-chain antibody fragments (scFvs) for optimizing tumor uptake at radioimmunotargeting. A unique divalent tandem single-chain Fv antibody [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) H7, has now been generated. The antibody differs from other dimeric single-chain constructs in that a linker sequence (L) is introduced between the repeats of VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed as a His-tagged TrxA fusion protein in the Escherichia coli strain Origami B. Following cleavage with AcTev protease, the antibody was obtained in soluble and active form in E. coli and could be purified by Ni-NTA and cation-exchange chromatography. Purity and immunochemical properties were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), Western blot, and Biacore analyses. The [sc(Fv)2] displayed proper stability and could be purified to homogeneity. This antibody also maintained immunoreactivity at 42 degrees C with only slight decrease at 52 degrees C. The high affinity displayed by the original antibody H7, 6.7 x 10(9) M(-1), was only slightly decreased to 1.2 x 10(9) M(-1) as determined by Biacore. The generation of such a divalent single-chain Fv with a molecular weight around 60 kd would be of value for clinical applications such as radioimmunolocalization and radioimmunotherapy.
Sheikholvaezin
Ali
A
Department of Clinical Microbiology, Immunology, Umeå University, Umeå, Sweden.
Eriksson
David
D
Riklund
Katrine
K
Stigbrand
Torgny
T
D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli; D058851:GPI-Linked Proteins; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000302:isolation & purification*; D007527:Isoenzymes / Q000276:immunology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000302:isolation & purification; D020080:Tandem Repeat Sequences / Q000235:genetics
D000906:Antibodies; D058851:GPI-Linked Proteins; D007135:Immunoglobulin Variable Region; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental