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Lab-on-a-Chip-Based Devices for Rapid and Accurate Measurement of Nanomaterial Toxicity
Published in Suresh C. Pillai, Yvonne Lang, Toxicity of Nanomaterials, 2019
Mehenur Sarwar, Amirali Nilchian, Chen-zhong Li
On the other hand, a tradeoff for using bioreporter-based LoCs is their low response time. In cases of portable systems (in-field measurements), where a response time of minutes is required, the requirement of 30 minutes to a few hours for most engineered bioreceptors to produce sufficient reporter proteins for a readable signal becomes especially problematic. It is also worth mentioning that with these bacterial-based platforms, achieving detection limits of submicromolar levels, which are required for environmental standards, is difficult to attain.
Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX
Published in mAbs, 2021
Anne E.G. Lenferink, Paul C. McDonald, Christiane Cantin, Suzanne Grothé, Mylene Gosselin, Jason Baardsnes, Myriam Banville, Paul Lachance, Alma Robert, Yuneivy Cepero-Donates, Stevo Radinovic, Patrick Salois, Marie Parat, Hafida Oamari, Annie Dulude, Mehul Patel, Martin Lafrance, Andrea Acel, Nathalie Bousquet-Gagnon, Denis L’Abbé, Alex Pelletier, Félix Malenfant, Maria Jaramillo, Maureen O’Connor-Mccourt, Cunle Wu, Yves Durocher, Mélanie Duchesne, Christine Gadoury, Anne Marcil, Yves Fortin, Beatrice Paul-Roc, Maurizio Acchione, Shawn C. Chafe, Oksana Nemirovsky, Joseph Lau, Francois Bénard, Shoukat Dedhar
Clinically used cG250 antibody has been described to evoke an ADCC response.35 To evaluate the ADCC response of our antibodies, we used SK-RC-52 target cells (high CAIX) in conjunction with Promega’s human FcγRIIIa (for c11H9, c12H8, c2C7, c2D7; Figure 4(a) and murine FcγRIV (for m4A2, m9B6; Figure 4(b)) in a Jurkat-Luc bioreporter assay. We observed a difference in the ADCC response level between the chimeric and murine antibodies, which is likely a combination of the antibody affinity for hCAIX (e.g., m9B6 does not reach a plateau as was shown in the cell-binding studies) and the human and mouse Fcγ receptor for the respective human and mouse Fc portions of the antibodies. To our surprise, we observed that only antibodies that bind to the catalytic domain of CAIX (i.e., c2C7, c2D7, m4A2, m9B6), but not those that bind the PG-like domain (i.e., c11H9, c12H8), are unable to induce an ADCC response (Figure 4(a,b)). These data thus imply that the nature of an antibody-binding epitope (i.e., structured versus unstructured) may determine whether an ADCC response is induced.
Cell-based bioreporter assay coupled to HPLC micro-fractionation in the evaluation of antimicrobial properties of the basidiomycete fungus Pycnoporus cinnabarinus
Published in Pharmaceutical Biology, 2016
Päivi Järvinen, Susanna Nybond, Laurence Marcourt, Emerson Ferreira Queiroz, Jean-Luc Wolfender, Aila Mettälä, Matti Karp, Heikki Vuorela, Pia Vuorela, Annele Hatakka, Päivi Tammela
Coupling of HPLC micro-fractionation of a crude extract with a cell-based E. coli K-12 (pTetLux1) bioreporter assay was employed in this study to accelerate the process of identifying bioactive component(s) from extracts. The ethyl acetate crude extract of culture filtrates of P. cinnabarinus (strain FBCC103) was selected to represent an example of a crude extract, which had been identified to demonstrate interesting antimicrobial activity in the preliminary screening campaign on TLC bioautography (Table 1) and in the follow-up assays conducted on the six most interesting extracts (Table 2). The discovery of bioactive compounds from natural products can be challenging due to the fact that the active metabolite can be present in very small amounts. Using traditional broth dilution antibacterial assay based on turbidity measurement, only modest growth inhibition of E. coli (ATCC 25922) by the crude extract of P. cinnabarinus could be detected (14.4 ± 2.0%) (Table 2) due to the insensitivity of the assay. The inhibition of E. coli K-12 (pTetLux1) by the same crude extract was 98.1 ± 0.6%. In the broth microdilution method, the bacterial growth is detected by the turbidity of the microbial mass and needs to be quite high to achieve a good signal separation between the background and the maximum well in order to properly evaluate the activity of the samples. Therefore, within the first hours of bacterial growth, the signal window is not good enough to detect any activity. In most cases, the endpoint measurement is set after 24 h of growth. The natural product fractions might contain interesting active metabolites, but are present in such low concentrations that the bioactivity is missed using the standard methods. Thus, in this work, the aim was to overcome the issue by using a bioreporter-based assay. The crude extract showed a dose-dependent response in the E. coli K-12 (pTetLux1) bioreporter assay with an IC50 value of 0.10 mg/mL (Supporting information Figure S1). The extract was micro-fractionated by HPLC-UV in order to localise the compound responsible for the bioactivity. The micro-fractions were collected to a micro-plate, evaporated to dryness and the 120 min bioassay was subsequently carried out. Using this approach, it was possible to correlate the inhibitory activity to one peak (compound 1) in the chromatogram, displaying activity above the set hit limit of 26% (average-3SDs) (Figure 1). The hit limit in screening assays is usually set at 3SDs away from the mean of the sample signals to gain 99.73% confidence limit to the sample data (Zhang et al. 1999).