key: cord-1045555-z6g0sfxo authors: Cerutti, Helena; Ricci, Veronica; Tesi, Giulia; Soldatini, Claudia; Castria, Marinunzia; Vaccaro, Marco Natale; Tornesi, Stefania; Toppi, Simona; Verdiani, Silvana; Brogi, Alessandra title: Large scale production and characterization of SARS‐CoV‐2 whole antigen for serological test development date: 2021-02-19 journal: J Clin Lab Anal DOI: 10.1002/jcla.23735 sha: d08f7f35b310c6a4092a7ec7a31186ff6e6879f0 doc_id: 1045555 cord_uid: z6g0sfxo BACKGROUND: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID‐19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS‐CoV‐2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory. METHODS: An enzyme‐linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings. RESULTS: Preliminary data from 10 sera (5 patients with COVID‐19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS‐CoV‐2‐positive individuals. CONCLUSION: This ELISA appears to be a specific and reliable method for detecting COVID‐19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus. On December 31 st 2019, China notified the World Health Organization (WHO) of a novel coronavirus outbreak which was first observed a few weeks earlier in a patient presenting with severe respiratory disease in Wuhan. 1 In January 2020, the virus was identified as a novel coronavirus with more than 70% similarity with the severe acute respiratory syndrome coronavirus (SARS-CoV), and it was officially named by the WHO as 2019 novel coronavirus (nCoV) or SARS-CoV-2. 1 Currently, SARS-CoV-2 has attributed to a worldwide pandemic resulting in over 40 million infections and 1 million deaths, with numbers constantly growing. 2 Coronaviruses are enveloped, non-segmented, positive-sense single-stranded RNA viruses with a diameter of 60-140 nm, including the spike, with genome sizes ranging from 26 to 32 kilobases (the largest known viral RNA genome). 3, 4 Coronaviruses have four structural proteins essential for virion assembly and viral transmission: the viral membrane containing the transmembrane (M) glycoprotein; the spike (S) glycoprotein; and the envelope (E) protein, surrounding a disordered or flexible, probably helical, nucleocapsid (N) protein. 4, 5 Projections composed of S glycoprotein trimers protrude from the surface of the virus with an approximate length of 20 nm. The main difference of this new coronavirus compared to SARS-CoV (which also belongs to the beta genera), appears to be localized in the S glycoprotein. 6 The symptoms of coronavirus disease 2019 (COVID-19) appear after an incubation period ranging between 2 and 11 days and most commonly include fever, cough, fatigue, and dyspnea, often associated with lymphopenia. 7 Considering the evolution of the worldwide pandemic, an improved insight into the development of virus-specific antibodies after SARS-CoV-2 infection is of high importance. To date, it is still unclear whether patients previously diagnosed with COVID-19 develop immunity, and if this immunity remains. Therefore, serological investigations play an essential role to not only better understand the host response to the virus and provide more accurate estimations of the spread within the population, but they also have an impact on vaccine and therapy development. In addition, as we approach the colder seasons, a differential diagnosis which differentiates SARS-CoV-2 infections from other viral infections (coronavirus or not) that cause respiratory syndromes, is fundamental. 7, 8 Due to the paucity of evidence regarding immunity and serology of SARS-CoV-2, well-validated serological assays are urgently required. Thus, the aim of this study is to use inactivated SARS-CoV-2 viral cultures as an antigen source to characterize the whole viral antigen and produce serologic tests. Vero E6 cells were seeded into 24-well plates 72/24 h before infection with tenfold serial diluted SARS-CoV-2 and incubating for a further 1 h at 37°C. After the absorption phase, 1 ml of DMEM supplemented with 5% FBS was added and the plates were incu- A virus inactivation protocol utilizing beta-propiolactone (BPL), a commonly used reagent for viral inactivation in vaccine preparations, [9] [10] [11] [12] was adopted and optimized. After thawing, virus batches were inactivated with BPL (Natalex, Warsaw, Poland) and 0.1% [v/v] of BPL was added in 3 consecutive additions to ensure inactivation. For each cycle of BPL treatment, viral suspension was incubated at room temperature (RT) for 3 h, shaken, and then incubated for a further 2 h at 37°C. Viral inactivation was verified by CPE assay, the absence of CPE and TCID 50 /ml <1 × 10 1.5 confirmed inactivation. In cases of incomplete inactivation, protocol stated that treatment with 0.1% BPL should be repeated and checked; however, although it was evident that after the first addition the virus was completely inactivated, due to safety concerns, BPL was added three times (Table S1 ). As an assay positive control, a live virus with a 10 4 -10 5 TCID 50 /ml, was added to Vero E6 cells, and the CPE after 48/72 h, and the TCID 50 /ml titer were measured. Each batch of inactivated virus was concentrated and purified by centrifugation. Batches were centrifuged at 10,000 g to remove cell debris and concentrated by ultracentrifugation at 100,000 g. As inactivated viruses are also present in debris pellets, the pellets were ultrasonically broken-down and collected in a phosphate-buffered saline solution (PBS, pH 7.2-7.8). Batches were stored at −80°C. Protein levels in the virus stock were quantified using the Bradford assay, as previously described. 13 The viral protein and a twofold serial dilution of a protein standard (bovine serum albumin, Sigma-Aldrich) in PBS were mixed with Coomassie Plus (Bradford) solution (Thermo Fisher Scientific, Waltham, MA, US) and incubated at RT for 10 min. Absorbance was measured at 595 nm and used to calculate protein concentration (Spectrophotometer, Amersham, BioSciences, Buckinghamshire, United Kingdom). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli protocol, 14 using a 10% (w/v) Pre-Cast gel (Bio-Rad, Hercules, CA, US). Samples were prepared in a Laemmli buffer with or without βmercaptoethanol, for reducing and non-reducing conditions, respectively. Protein detection was performed by the silver stain system, using Pierce™ Silver Stain Kit (Thermo Fisher Scientific). Proteins were then electroblotted onto a nitrocellulose membrane with 0.2 µm pores (Bio-Rad). The nitrocellulose was then blocked with 5% milk powder dissolved in 0.1% tris-buffered saline solution (TBS, Bio-Rad). 15 Similarly, the analysis with commercial MAB specifically targeting Subunit 1 of the S and the N proteins demonstrated the same differences between reducing and non-reducing electrophoresis conditions as those observed with the serum samples, as shown in Figure 3A ,B, respectively. Similarly, the sera (Table 1 ) and commercial MABs ( Table 2) 16 However, the need for reliable serological assays is rapidly growing. It is well acknowledged that serological assays play a fundamental role in providing epidemiological analysis, and enhance the understanding of immunity profiles from recovered patients, which allows for development and implementation of serologic therapies (e.g. convalescent plasma) and vaccines. 8 Furthermore, several studies support the role of serological testing for more reliable detection of a positive infection, 17 TA B L E 2 ELISA results of commercial recombinant monoclonal antibodies, anti-S1 spike subunit, and anti-nucleocapsid efficient strategy for qualitative and differential detection of the specific anti-SARS-CoV-2 antibodies (IgG, IgM, and IgA). The authors thank "Laboratory of Virology at National Institute for The data that support the findings of this study are available from the corresponding author, [HC], upon reasonable request. 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