Generation and application of a fluorescein-specific single chain antibody.
A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.
Potsdam University, Biotechnology, Institute of Biochemistry and Biology, Karl-Liebknecht-Str 24/25, D-14476, Golm, Germany. jschenk@uni-potsdam.de
Jörg A
JA
Schenk
Frank
F
Sellrie
Volker
V
Böttger
Astrid
A
Menning
Walter F M
WF
Stöcklein
Burkhard
B
Micheel
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli; D019793:Fluorescein / Q000032:analysis* / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D006801:Humans; D007124:Immunoenzyme Techniques; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D016650:Fluorescein-5-isothiocyanate; D019793:Fluorescein