Depletion of algal toxin-contaminated water using selective biofilters based on plant-produced antibodies (plantibodies)
Zusammenfassung der Projektergebnisse
It was the aim of this project, to produce a highly affine and specific antibody (plantibody) mAb MC10E7 against algal toxins in plants, followed by its characterization and investigations on its ex vivo applicability for selective, efficient and cost-effective depletion of spiked model water samples. The work was based on a murine anti-MC-LR antibody (mAb MC10E7) generated in a former research project at the TUM (Munich). The project was split into the genetic/biotechnological part which was performed at the project partner BOKU (Vienna, Austria) and the characterization of the generated plant-based antibody and production/evaluation of biofilter material done at TUM. The first testing of plantibody functionality which was performed with scFv-fragments prepared in the methylotrophic yeast Pichia pastoris failed. It revealed that the murine hybridoma cell line contained a second light chain transcript with an unrelated but otherwise normal VL sequence. Following, peptide mass fingerprinting of the functional hybridomaderived antibody using MALDI-TOF-MS was performed and expression vectors with functional antibody transcript sequences could be constructed. The scFv fragments prepared showed an about 16-fold lower antigen-binding activity compared to the full length murine IgG. Therefore, a full-size chimeric version for human IgG1 was constructed and expressed in Nicotiana species using small-scale transient expression. Highest yields of ~460 mg per kg FW (329 mg of purified antibody) were achieved in N. benthamiana leaves. The purified plantibodies were characterized in detail for their binding properties using an indirect competitive ELISA on microplates (MTPs) and Surface Plasmon Resonance (SPR) spectroscopy. The monoclonal mouse antibody was used as reference. For the ELISA, a special MC-LR-BSA conjugate was synthesized and coupling rate determined. The microcystin-binding properties of murine and plant-based antibody were in very good agreement, i.e., binding rate constants (ka, kd) and dissociation constant (KD in the picomolar range) and cross-reactivity pattern (only binding of MCs with L-Arg residue in position 4) were comparable. Purified plantibodies were entrapped in a porous sol-gel-derived glass matrix (SGG) using a two-step microencapsulation procedure in silica-based material. Following, immunoaffinity supports were tested regard non-specific binding of MC-LR, binding capacity, regeneration capability and antibody leaching performed in comparison with the murine antibody as the control. Next, different depletion modes were tested using surface water samples spiked with high MC-LR concentrations (100 µg L-1). The depletion efficiency was clearly dependent on the incubation time of biofilter and sample. Almost 100% toxin removal and relatively fast depletion (~5 hours) was obtained with suspension mode, i.e., by suspending the ground and sieved (mesh size 250 µm) functionalized SGG material in the sample. In addition, in terms of cost-effectiveness for future application, only roughly purified fractions were tested and quite the same efficiency and binding capacity were observed. The repeated use of biofilter material after regeneration yielded significant lower depletion efficiency owing to the deterioration of the antibody activity by the organic eluent. First time, a lateral-flow test (immunological dipstick) using a plant-based antibody was manually prepared and assembled and initial proof-of-principle testing performed using fresh water samples and revealed its highly suitability for the on-site analysis.
Projektbezogene Publikationen (Auswahl)
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(2018) Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect 4-argininemicrocystin in freshwater. Plant biotechnology journal 16 (1) 27–38
Melnik, Stanislav; Neumann, Anna-Cathrine; Karongo, Ryan; Dirndorfer, Sebastian; Stübler, Martin; Ibl, Verena; Niessner, Reinhard; Knopp, Dietmar; Stoger, Eva
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Depletion of algalcontaminated water using selective biofilters based on plant-produced antibodies (plantibodies). ANAKON 2015, Graz, 23.-26.03.2015
A.-C. Neumann, V. Ibl, R. Niessner, E. Stöger, D. Knopp
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Herstellung und Charakterisierung von Plantibodies gegen Cyanotoxine zur selektiven Immunfiltration. 9. Deutsches BioSensor Symposium, München, 11.-13.03.2015
A.-C. Neumann, V. Ibl, R. Niessner, E. Stöger, D. Knopp
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Antibodies as highly affine and specific reagents for bioanalytical applications – plants as emerging antibody source. The 1st International Conference on Applied Sciences (ICAS-1), 20.-21.07.2016, Ho Chi-Minh City, Vietnam
D. Knopp and E. Stöger
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Approach to depletion of cyanotoxin-MC-LR from surface water using plantibody-doped porous filter. 18th International Symposium on Advances in Extraction Technologies & 22 nd International Symposium on Separation Sciences (ExTECH), 03.-06.07.2016, Torun, Poland
D. Knopp, A.-C. Neumann, S. Melnik, R. Niessner, E. Stöger
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Determination of microcystin-LR in surface water by a magnetic bead-based colorimetric immunoassay using antibody-conjugated gold nanoparticles. Anal. Methods 8 (2016) 57-63
Neumann, A.-C., Wang, X., Niessner, R., Knopp, D.
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Plant molecular farming: Much more than medicines. Annu. Rev. Anal. Chem. 9 (2016) 271-294
Tschofen, M., Knopp, D., Hood, E., Stoeger, E.
