Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect [4-arginine]microcystin in freshwater

Stanislav Melnik, Anna-Cathrine Neumann, Ryan Karongo, Sebastian Dirndorfer, Martin Stübler, Verena Ibl, Reinhard Niessner, Dietmar Knopp, Eva Stoger

Antibody MC10E7 is one of a small number of monoclonal antibodies that bind specifically to [Arg4]-microcystins, and it can be used to survey natural water sources and food samples for algal toxin contamination. However, the development of sensitive immunoassays in different test formats, particularly user-friendly tests for on-site analysis, requires a sensitive but also cost-effective antibody. The original version of MC10E7 was derived from a murine hybridoma, but we determined the sequence of the variable regions using the peptide mass-assisted cloning strategy and expressed a scFv (single-chain variable fragment) format of this antibody in yeast and a chimeric full-size version in leaves of Nicotiana tabacum and Nicotiana benthamiana to facilitate inexpensive and scalable production. The specific antigen-binding activity of the purified antibody was verified by surface plasmon resonance spectroscopy and ELISA, confirming the same binding specificity as its hybridoma-derived counterpart. The plant-derived antibody was used to design a lateral flow immunoassay (dipstick) for the sensitive detection of [Arg4]-microcystins at concentrations of 100-300 ng/L in freshwater samples collected at different sites. Plant-based production will likely reduce the cost of the antibody, currently the most expensive component of the dipstick immunoassay, and will allow the development of further antibody-based analytical devices and water purification adsorbents for the efficient removal of toxic contaminants.

External organisation(s)
Universität für Bodenkultur Wien, Technische Universität München
Plant Biotechnology Journal
Publication date
Peer reviewed
Austrian Fields of Science 2012
106023 Molecular biology
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