A novel brighter bioluminescent fusion protein based on ZZ domain and amydetes vivianii firefly luciferase for immunoassays


Butantan affiliation
Publication type
Article
Language
English
Access rights
Open access
Terms of use
CC BY
Appears in Collections:
Metrics
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.
Reference
Viviani VR., Silva JR, Ho PL. A novel brighter bioluminescent fusion protein based on ZZ domain and amydetes vivianii firefly luciferase for immunoassays. Front Bioeng Biotechnol. 2021 Oct;9:755045. doi:10.3389/fbioe.2021.755045.
Link to cite this reference
https://repositorio.butantan.gov.br/handle/butantan/4043
Issue Date
2021


Files in This Item:

fbioe-09-755045.pdf
Description:
Size: 1.55 MB
Format: Adobe PDF
View/Open
Show full item record

This item is licensed under a Creative Commons License Creative Commons