(1993)produced a fusion protein based on the protein A fused to N-terminal deletedPhotinus pyralisfirefly luciferase. successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1250 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 (5250 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. Keywords:luciferase, immunoassays, Amydetes vivianii, SARS-CoV-2, western blot, ZZ-domain == 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). In times of pandemics, sensitive and fast detection and diagnostic methods such as immunoassays AAF-CMK 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. In the past, radioactive methods involving I125labelled Protein A to detect antigenic proteins have been used for Western Blots and Immunoassays (Kessler, 1975,1981;McConahey and Dixon, 1980). Later, the radioactive methods were replaced by safer colorimetric, fluorescent or chemiluminescent methods. The chemiluminescence methods are in general more sensitive and specific. They usually rely on the conjugation of HRP to a secondary antibody which recognizes IgG, which upon mixing AAF-CMK with the chemiluminescent substrate solution, consisting of luminol or a derivative and H2O2, emits a blue chemiluminescence (Nesbitt and Horton, 1992). The need of novel sensitive chemiluminescence immunoassays is especially important in times of emerging viruses and pandemics of SARS-CoV-2. As an example of the wide use of chemiluminescence immunoassays for SARS-CoV-2, a recent report shows that among the current 54 commercially available antibody-based assays for SARS-CoV-2, 13 of them are luminescent and 10 are chemiluminescent. The chemiluminescent tests display between 66 and 75% sensitivity for IgM, and 75100% for IgG (Kojouri, 2020). Methods and reagents for automated chemiluminescence enzyme immunoassays for SARS-CoV-2 nucleocapsid protein and spike proteins are also being developed. Bioluminescence based immunoassays consisting on the fusion of luciferases or photoproteins to antigen or an antibodies were also proposed, but in general, they were not so popular like the chemiluminescent ones. Luciferase from bacteria (Vibrio harveyi) was fused to protein A showing its applicability in bioluminescent immunoassays (Lindbladh et al., 1991). The photoprotein obelin fromObelia longissima(Cnidaria) was attached to ZZ-domain of protein A demonstrating the possibility of its application in immunoassays (Frank et al., 1995). Obelin was also conjugated to anti-thyroid hormones (human thyrotropin and thyroxine), and the sensitivity of these bioluminescent immunoassays were similar to those using TSPAN33 radioisotopes (Frank et al., 2004).Kobatake et al. (1993)produced a fusion protein based on the protein A fused to N-terminal deletedPhotinus pyralisfirefly luciferase. However, despite being antigenically active, this construct displayed weaker luminescent activity than wild-type firefly luciferase. Later, the authors fused protein A to the full-length firefly luciferase, obtaining a more active construct, with high affinity for IgG, detecting up to 5 pg of tumor marker a-fetoprotein (AFP) (Zhang et al., 2000). More recently, the nanoluciferase fromOplophorus gracilirostris(deep-sea shrimp) was fused to a nanobody against aflatoxin B1, a potentially carcinogenic mycotoxin produced by fungi in cereals, resulting in an attractive, simple, and rapid analytical tool for quantification of the pollutants in commercial foods (Ren et al., 2019). An immunoassay for antibodies against SARS-CoV-2 proteins based on the fusion of viral S e N protein fragments with NanoLuc luciferase was also developed. This method was specific to quantify the levels of SARS-CoV-2 antibodies in patients (Haljasmgi et al., 2020). A quantitative bioluminescence immunoassay for immunohistochemistry based onCypridinaluciferase conjugated secondary antibody and its luciferin, was also developed and successfully used to detect the tumor marker carcinoembryonic antigen (Wang et al., 2020). Bioluminescent sensors to detect multiple antibodies based on microfluidics and BRET were also proposed (Kosuke et al., 2020). Although nowadays most immunoassays use IgG based secondary antibodies, protein AAF-CMK A still remains a useful and cheaper alternative, especially for affinity purification of antibodies (Huang et al., 2006). Protein A was first isolated fromStaphylococcus aureus, and due to its high affinity for the portion Fc of immunoglobulins, has been extensively used in immunoassays. Furthermore, the small ZZ portion of.