A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays.

Vadim R Viviani,Paulo Lee Ho,Jaqueline Rodrigues Silva

doi:10.3389/fbioe.2021.755045

Vadim R Viviani, Paulo Lee Ho + Show 1 more

Open Access

https://doi.org/10.3389/fbioe.2021.755045

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Abstract

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1–250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5–250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

Highlights

Bioluminescence, the emission of visible light by living organisms has been extensively used for bioanalytical purposes during the past decades, including their use as reagents for ATP and enzymatic assays, and reporter genes for bioimaging biological and pathological processes and biosensors (Viviani and Ohmiya 2006; Roda et al, 2009)
In order to develop efficient bioluminescence based immunoassays, here we report the construction and uses of a novel bioluminescent fusion protein based on the ZZ portion of protein A and a brighter luciferase arising from Amydetes vivianii firefly, which emits a more blue-shifted emission than other firefly luciferases (Viviani et al, 2011; Pelentir et al, 2019)
We constructed a fusion protein using the cDNA of the ZZ portion of protein A at the N-terminus of a brighter luciferase (Figure 1), originally cloned from Amydetes vivianii firefly from campus of Sorocaba of Federal Univ. of São Carlos, which emits one of the most blue-shifted emissions among firefly luciferases (Viviani et al, 2011)

Summary

Introduction

Bioluminescence, the emission of visible light by living organisms has been extensively used for bioanalytical purposes during the past decades, including their use as reagents for ATP and enzymatic assays, and reporter genes for bioimaging biological and pathological processes and biosensors (Viviani and Ohmiya 2006; Roda et al, 2009). Sensitive and fast detection and diagnostic methods such as immunoassays are especially demanded. Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. Radioactive methods involving I125 labelled Protein A to detect antigenic proteins have been used for Western Blots and Immunoassays (Kessler, 1975, 1981; McConahey and Dixon, 1980). The radioactive methods were replaced by safer colorimetric, fluorescent or chemiluminescent methods.

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