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. 2016 Feb 18:7:27.
doi: 10.3389/fphar.2016.00027. eCollection 2016.

NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening

Nicolas Boute  1 , Peter Lowe  1 , Sven Berger  1 , Martine Malissard  2 , Alain Robert  1 , Michael Tesar  1
Affiliations

NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening

Nicolas Boute et al. Front Pharmacol. .

Abstract

Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a "swiss army knife solution" for today and future high throughput antibody drug screenings.

Keywords: NanoLuc®; Western blot; affinity determination; bioluminescence resonance energy transfer (BRET); expression analysis; homogeneous assay; library screening; luciferase; phage display.

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Figures

FIGURE 1
FIGURE 1
Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).
FIGURE 2
FIGURE 2
(A) Antibody libraries were created by cloning of assembled VH and VL genes after restriction digest with Nco I and Mlu I into phagemid vector pPL101. Cloned scFv were preceded by the pelB signal sequence and VH and VL genes were joined by a linker sequence (L1). The scFv was finally fused to the “short”(Δ) M13 gene III (corresponding to amino acids 251–406). After panning, the enriched phagemid population was digested by Nco I and Mlu I and subcloned into the expression vectors pPL302 and pPL303. Deduced amino acid sequences are shown for the most relevant fusion sites including linker sequences L1(QLKSSGSGSESKST), L2(GGGGSMEARLPAWQSGTVGKAT), L3(GGGSCYKDDDDKGGGGS), as well as for the anti-gIII (ATDYGAAIDGF) and the Strep-tagTM epitope (WSHPQFEK). (B) For the pIgG Nluc vector the NanoLuc gene was fused downstream to the heavy chain IgG1 CH3 gene domain including the linker sequence L4. Deduced amino acid sequences at the fusion site are shown for the linker L4 (GKGGGGS) and three N- and C-terminal amino acids of NanoLuc (MVF…[Nluc]…ILA). Genes for the heavy chain variable region, the constant regions and the signal peptide are depicted as VH, CH1-3 and S.
FIGURE 3
FIGURE 3
Comparison of luminescence intensity of purified Nluc (circles) and two representatives of the four Nluc fused antibodies (squares and diamonds) read without filter (filled) or with a 530 nm filter (empty). Antibody concentrations are corrected in equivalent Nluc as each antibody is linked to two Nluc. Each experimental point represents the means of three independent experiments made in triplicates, bars correspond to SD.
FIGURE 4
FIGURE 4
Evaluation of homogenous BRET screening assay for detection of scFv-Nluc binders on a directly (A) or indirectly labeled target (B). BRET signal measurement of increasing amounts of specific (squares) or aspecific (diamonds) Nluc fused scFv incubated either with a Alexa-fluor488 covalently labeled target (A) or an indirectly labeled target-Fc fusion with an (Fab’)2 anti-human Fc coupled to phycoerythrin (B). BRET signal of specific scFv-Nluc incubated with an (Fab’)2 anti-human Fc coupled to phycoerythrin in absence of target is represented with empty square. Experimental points are means of triplicates of three independent experiments, bars are SD.
FIGURE 5
FIGURE 5
Screening of 92 scFv-Nluc derived from library L3 panned against target 3 using either ELISA on recombinant protein (A), on cells (B) or the BRET assays with direct (C) or indirect (D) labeling of the target. For all graphs, the x-axis corresponds to the luminescence of a sample of scFv extract before experiment indicating the relative quantity of each scFv. For cellular ELISA, results are the difference between the signal with CHO cells stably expressing target 3 and the signal of untransfected CHO. The dotted lines correspond to the selected threshold for positive clones.
FIGURE 6
FIGURE 6
Binding competition ELISA on recombinant target 2 between a Nluc-fused anti-target 2 mAb (1 nmol/L) and specific competitors [mAb (squares) and scFv (circles) or irrelevant antibodies mAb (diamonds) and scFv (triangles)]. Each experimental point is the mean of three independent experiments performed in triplicates, bars correspond to SEM.
FIGURE 7
FIGURE 7
Western blot of targets 2 and 3 revealed with naked anti target 2 mAb and an anti-human kappa light chain-HRP secondary antibody (A), the Nluc fused to anti-target 2 antibody (B), the scFv-Nluc version of the anti-target 2 antibody (C) and an anti-target 3 scFv-Nluc (D). Membrane A labeling was revealed with ECL blotting substrate, while membranes (B–D) labeling was revealed with furimazine diluted 1000 fold in PBS, 0.1% BSA.
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