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Recombinant proteins co-expressed and co-purified in the presence of antibody fragments
Ario De Marco, 2021, independent scientific component part or a chapter in a monograph

Keywords: protein complexes, affinity purification, nanobodies
Published in RUNG: 17.12.2020; Views: 2018; Downloads: 0
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Sandra Oloketuyi, 2020, doctoral dissertation

Abstract: Early detection and monitoring of toxic microalgae remains a challenge that will be necessary to overcome for understanding harmful algal bloom events and their consequent ecological and environmental impacts. Although there are diagnostic techniques based on microscopy, fluorescence in situ hybridization (FISH), DNA microarrays, real-time PCR, sandwich hybridization assays employed for the detection of harmful algal species, such techniques are not suitable for field monitoring and especially for the quantification of Alexandrium minutum due to abundance of cryptic species that cannot be differentiated based on morphology or genomic traits. These approaches are also time-consuming and laborious, hence there is need for alternative simple, rapid and cost-effective methods. In this study, our aim was to develop different nanobody-based capture strategies for the detection of A. minutum. Nanobodies were successfully displayed on the surface of Escherichia coli to facilitate detection of A. minutum. The method is simple and cost effective as no antibody purification step is necessary. As an alternative, A. minutum was quantified by exploiting the peroxidase activity of a G-quadruplex DNAzyme covalently bound to nanobodies and by means of a label free electrochemical immunosensor which exploit nanobodies bound via an irreversible SpyTag-SpyCatcher system as the specific immunocapture element. Such immunosensor was prepared by immobilizing the SpyTagged anti-A. minutum nanobody (SpyTagged C1) by means of a selfassembled monolayer (SAMs) of L- cysteine (L-Cys) displayed onto a gold nanoparticle-modified glassy carbon electrode. Electrochemical Impedance Spectroscopy (EIS) was performed to measure the electrical response of the circuit as the function of A. minutum immunocaptured cells. The results of EIS studies confirmed that the sensing chip detected A. minutum selectively, exhibited a wide dynamic range spanning from 103 to 109 cell L-1 and a limit of detection of 3.1× 103 cell L-1. The immunosensor data can be recorded and then analyzed with a portable potentiostat. Such device can be interfaced with a smart phone, a condition suitable for the rapid A. minutum quantification in situ.
Keywords: Alexandrium minutum, nanobodies, bioreagents, immunosensor, toxic microalgae
Published in RUNG: 17.06.2020; Views: 3917; Downloads: 230
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Recombinant expression of nanobodies and nanobody-derived immunoreagents
Ario De Marco, 2020, review article

Keywords: nanobodies, prokaryotic expression, eukaryotic expression, inclusion bodies
Published in RUNG: 14.04.2020; Views: 2304; Downloads: 0
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Opportunities offered by recombinant antibody technology: our 15-year experience with nanobodies
Ario De Marco, invited lecture at foreign university

Keywords: nanobodies, diagnostics, in vitro selection
Published in RUNG: 28.02.2020; Views: 2591; Downloads: 0
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Nanobodies isolated from pre-immune libraries as tunable biotechnological tools
Ario De Marco, invited lecture at foreign university

Keywords: nanobodies, panning, modeling
Published in RUNG: 27.02.2020; Views: 2341; Downloads: 0
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Pre-immune libraries of recombinant nanobodies: outcome from 15-year experience
Ario De Marco, invited lecture at foreign university

Keywords: Nanobodies, in vitro panning, protein functionalization
Published in RUNG: 14.02.2020; Views: 2643; Downloads: 0
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A consensus protocol for the in silico optimisation of antibody fragments
Miguel Soler, Barbara Medagli, Marta S. Semrau, Paola Storici, Gregor Bajc, Ario De Marco, Alessandro Laio, Sara Fortuna, 2019, original scientific article

Keywords: nanobodies, in silico design, Her2
Published in RUNG: 21.11.2019; Views: 3086; Downloads: 0
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