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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
Viviani, Vadim R. [1, 2] ; Silva, Jaqueline Rodrigues [1, 2] ; Ho, Paulo Lee [3]
Total Authors: 3
Affiliation:
[1] Fed Univ Sao Carlos UFSCar, Grad Program Biotechnol & Environm Monitoring, Sorocaba - Brazil
[2] Fed Univ Sao Carlos UFSCar, Dept Phys Chem & Math, Sorocaba - Brazil
[3] Inst Butantan, Ctr BioInd, Nucleo Prod Vacinas Bacterianas, Sao Paulo - Brazil
Total Affiliations: 3
Document type: Journal article
Source: FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY; v. 9, OCT 18 2021.
Web of Science Citations: 0
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 (t(1/2) > 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.</p> (AU)

FAPESP's process: 10/05426-8 - Arthropod bioluminescence: biological diversity in Brazilian biomes, biochemical origin, structural/functional evolution of luciferases, molecular differentiation of lanterns, biotechnological, environmental and educational applications
Grantee:Vadim Viviani
Support Opportunities: Research Projects - Thematic Grants