CHARACTERIZATION OF ZZ-AMYLUC FUSION PROTEIN AND ITS OPTIMIZATION FOR BIOLUMINESCENT IMMUNOASSAY PURPOSES

• Grant number: 22/14071-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): February 01, 2023
• Effective date (End): February 29, 2024
• Field of knowledge: Biological Sciences - Biophysics - Radiology and Photobiology
• Principal Investigator: Vadim Viviani
• Grantee: Maysa de Souza Cavalcante
• Host Institution: Centro de Ciências e Tecnologias para a Sustentabilidade (CCTS). Universidade Federal de São Carlos (UFSCAR). Sorocaba , SP, Brazil
• Associated research grant: 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, AP.TEM

Abstract

Immunoassays are widely used in biomedical analyzes based on antigenantibody interaction, in which they are capable of detecting antibodies against specific antigens. In general, these assays mainly use chemiluminescence as a detection method, due to its sensitivity. However, current chemiluminescence methods based on conjugated HRP show a rapid decay of luminescence. Thus, the new fusion protein, ZZ-AmyLuc, built from the luciferase of the firefly Amydetes vivianii fused to the ZZ portion of protein A (VIVIANI et al., 2021), proved to be a good alternative. This protein has a stronger blue-green glow, with greater bioluminescent activity, in addition to being very thermostable and presenting much more sustained emission, capable of lasting several minutes (t1/2 > 30 min). In addition, it was notably capable of detecting type G immunoglobulins (IgG), especially the anti-SARS-COV-2 nucleoprotein and its respective antigen. The detection could be observed in photodetection cameras (CCD) and even in common smartphone cameras, supporting its high efficiency in both immunoassays and Western Blotting techniques. However, better physicochemical characterization of ZZ-AmyLuc is needed, as well as the optimization of its stability and sensitivity for use in bioluminescent immunoassays, particularly for the detection of the anti-SARS-COV-2 nucleoprotein. For that, it will be determined the kinetic properties, to know the KMs for ATP and D-luciferin, the optimal pH of the bioluminescent activity and IgG binding, the analysis of protein thermostability at 4ºC and 37ºC, as well as the optimization of its sensitivity in immunoassays and stability tests.