Expression of antibody variable region-human alkaline phosphatase fusion proteins in mammalian cells

Antibody fragments and their fusion proteins are indispensable tools as immunoassay reagents in diagnostics and molecular/cellular biotechnology. However, bacterial expression of cloned antibody genes with correct tertiary structure is not always guaranteed because of the lack of proper folding machinery and/or post-translational modifications. In addition, frequently used bacterial alkaline phosphatase as a fusion partner generally shows lower specific activity than the mammalian enzyme, which hampers its wider use as a detection reagent. Here we tried to express the fusion proteins of antibody variable region(s) and secreted human placental alkaline phosphatase (SEAP) using mammalian cell culture. As a result, functional VH-SEAP and single-chain Fv-SEAP fusion proteins were successfully obtained from COS-1 cells, which was confirmed by ELISA and Western blotting. This system will be applicable to the rapid production of various antibody-enzyme fusions suitable for ELISA and open-sandwich ELISA that utilizes antigen-dependent VH/VL interaction for antigen quantitation.

Research Highlights
► Fusion proteins of antibody VH or scFv and secreted placental AP (SEAP) were made.
► VH-SEAP could detect antigen via immobilized VL fusion protein by AP activity.
► scFv-SEAP could detect immobilized antigen.
► The effect of linker length on AP activity was investigated.