SINGLE-CHAIN F_v ANTIBODY-ALKALINE PHOSPHATASE FUSION PROTEINS PRODUCED BY ONE-STEP CLONING AS RAPID DETECTION TOOLS FOR ELISA

ABSTRACT

A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain F_v (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5′ and 3′ end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv–aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv–aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.

ACKNOWLEDGMENTS

We thank Prof. Arne Skerra (Technical University München, Center of Life and Food Sciences Weihenstephan, Germany) for providing the expression vector pASK75, and also Dr. Claudia Schmidt-Dannert (University Stuttgart, Institut für Technische Biochemie, Germany) for providing the E. coli alkaline phosphatase gene. This work was supported by the Deutsche Forschungsgemeinschaft (HO 383/35-1/2).

This paper is dedicated to Professor George Guilbault on the occasion of his 65th birthday.