Purification and analysis of water based on plant-made antibodies

Although the use of genetically modified plants for bioremediation, or the in situ cleaning of contaminated sites, has been known for quite some time, little attention has so far been paid to the production of antibodies in plants and their ex vivo application in selective depletion. Therefore, highly affine and specific antibodies against algal toxins using microcystin as an example will be produced in plants at low cost within this research project. The basis is a monoclonal antibody (Mab 10E7, species: mouse) generated in a former research project. The sequence of the variable domains will be determined, optimised for plants and subcloned into suitable plant transformation vectors, which already contain constant antibody sequences, . In addition, a scFv fragment containing different tag sequences and fusion proteins will be constructed. Leaf-based (tobacco) as well as seed-based (barley) systems will be used. Affinity-purified plantibodies will be characterised in detail for their binding properties using microtitre plate- ELISA and surface plasmon resonance (SPR). The monoclonal mouse antibody will be used as reference. To assure cost-efficiency for future applications, roughly purified fractions (sequential pH and temperature treatment followed by filtration) will be tested for the upscaling. Following immobilisation of the plantibody fractions on suitable substrates, for instance membranes, porous polymer monoliths or in porous glasses, their application for depletion will be defined using model water samples spiked with microcystins.

Eutrophication of water bodies caused by an excessive influx of nutrients like nitrogen and phosphorus resulting from urbanization and industrialization and also higher ambient temperatures can promote cyanobacterial (blue-green algae) blooms, which is a worldwide environmental problem. Many of the cyanobacterial species are able to produce potent cyanotoxins, for example hepatotoxins such as nodularin and microcystins (MCs), and therefore present a serious health risk to wildlife, domestic animals and humans. Among the MC variants, microcystin-leucine-arginine (MC-LR) is the most frequent and most toxic microcystin congener. The World Health Organization recommends that MC-LR levels should be limited to 1 g/L in drinking water, the primary source of exposure. In addition, cyanotoxins in recreational bathing waters may constitute an acute human health and safety hazard owing to inhalation and dermal contact. 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 only available from a murine hybridoma clone, but we determined the sequence of the variable regions using a peptide mass-assisted cloning strategy and expressed recombinant versions of the antibody in yeast and in leaves of Nicotiana tabacum and N. benthamiana, which are more stable and economically attractive than hybridoma lines. 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 100300 ng L-1 in fresh water samples collected at different sites. Plant-based production reduces the cost of the antibody, formerly the most expensive component of the dipstick immunoassay, and therefore has a significant impact on the overall cost. Our work confirms that plants are suitable for the rapid and cost-effective production of sufficient amounts of functional recombinant MC10E7 for downstream applications such as antibody-based sensitive test systems and water purification adsorbents for the efficient removal of toxic contaminants.