NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening

Boute, Nicolas; Lowe, Peter N.; Berger, Sven; Malissard, Martine; Ahrends, Robert; Tesar, Michael

doi:10.3389/fphar.2016.00027

Cited by 55 publications

(45 citation statements)

References 35 publications

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“…With the need to introduce multiple DNAs into the same cell to achieve CRISPR/Cas9 editing, we sought to improve the transfection efficiency using the Amaxa nucleofection system 39 , 63 – 65 . We first compared electroporation versus nucleofection using the nanoluciferase reporter system 47 , 63 . Nanoluciferase (also termed NanoLuc) is a newly developed luciferase gene from a deep sea shrimp that produces stronger bioluminescence than the firefly luciferase (Promega).…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis

Janssen

Chen

Molgora

et al. 2018

Sci Rep

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The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.

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Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis

Janssen

Chen

Molgora

et al. 2018

Sci Rep

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“…While our GFP-VAMP2 cell line offers a possibility to develop a “high content” analysis assay using automated microscopy or sandwich ELISA-based detection as described for BoNT/A-cleaved SNAP25 ( Fernández-Salas et al, 2012 ), we hypothesized that a direct linkage of an enzyme to the VAMP molecule will allow faster and easier detection of BoNT/B activity if we can capture the proteolytic product on microplates. One-step capture immunoassay utilizing enzymatic reporter molecules offers the dual advantage of fewer washing and incubation steps coupled with superior sensitivity due to signal amplification ( Boute et al, 2016 ). Using viral transduction we generated two stable cell lines expressing either peroxidase- or luciferase-linked VAMP2 products, namely APEX2-VAMP2 and NanoLuc-VAMP2, respectively.…”

Section: Resultsmentioning

confidence: 99%

A Cell Line for Detection of Botulinum Neurotoxin Type B

et al. 2017

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Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.

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“…We used marine luciferases as the fusion partner due to their small size and favorable luminescence characteristics 20 . Other groups have fused marine luciferases to antibody fragments using E. coli or phage expression system, where optimal folding and glycosylation of antibody fragments is unpredictable 28,29 . Further, antibody-luciferase fusion proteins need to be purified from interfering bacterial contaminants prior to assessing expression levels or binding to mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors

Natarajan

Gopalakrishnan

Matta

et al. 2020

Sci Rep

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Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modifiedwith chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferasebased assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.

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NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening

Cited by 55 publications

References 35 publications

CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis

CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis

A Cell Line for Detection of Botulinum Neurotoxin Type B

A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors

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