The S1 or RBD regions from the spike (S) protein offers better specificity. serology Introduction In December 2019, a novel coronavirus first emerged in Hubei province in China and the virus was identified as 2019-nCoV, later designated SARS-CoV-2 [1,2]. The associated disease is referred to as COVID-19. On 30 January 2020, the World Health Organization declared COVID-19 a Public Health Emergency of International Concern and declared a pandemic on 11 March 2020. As of 1 June 2020, the COVID-19 outbreak has led PKC (19-36) to 6,057,853 confirmed cases and 371,166 deaths globally [3]. The virus exhibited efficient human-to-human transmission and the epicentre has shifted from mainland China to Europe and USA, and then to South America [3]. Laboratory diagnostic assessments play a pivotal role for any infectious disease outbreak response, which is also true for COVID-19. Within days of the SARS-CoV-2 genome release into the public domain name, multiple PCR assessments were rapidly developed and implemented at the frontline for diagnosis of acute pneumonia patients in China and globally [4]. When the outbreak progressed further, it was evident that PCR assessments alone could not meet the other needs of the COVID-19 response, such as retrospective contact tracing, investigation of asymptomatic contamination rate and assessment of herd immunity [5]. There is an urgent need for serology or antibody assessments. Research laboratories and pharmaceutical companies are racing to produce assessments that can detect COVID-19 contamination with sufficient specificity and sensitivity [5]. In addition to SARS-CoV-2, there are six other coronaviruses (OC43, 229E, SARS-CoV, NL63, HKU1 and MERS-CoV) known to infect humans [6C10]. This presents a major challenge when aiming for test specificity. Although the possibility of antibody cross-reactivity among all these human coronaviruses (hCoVs) exists, SARS-CoV presents the highest chance of cross-reactivity with SARS-CoV-2 because of the close phylogenetic relationship and the high genome and protein sequences identity [11]. Determining the level of cross-reactivity between COVID-19 and SARS antibodies is especially important for countries like China and Singapore, which were affected by both outbreaks [12,13]. The current study focused on the development of serologic assessments which can reliably differentiate COVID-19 contamination from SARS contamination. The major viral antigens used most frequently in antibody assessments for coronavirus infections are two of the four major structural proteins, the nucleocapsid protein (N) and the spike protein (S) [10]. The CoV S protein is a large envelope protein, and is 1,273 aa long for SARS-CoV-2 [14]. The S protein is cleaved by host protease into two subunits, the N-terminal S1 PKC (19-36) EM9 subunit (aa 1-685) responsible for receptor binding PKC (19-36) and the C-terminal S2 subunit, responsible for membrane fusion. The receptor-binding domain (RBD) is located at the C-terminal region (aa 319-591) of the S1 subunit, and recombinant RBD alone has been shown to be sufficient to bind the cell entry receptor, angiotensin-converting enzyme 2 (ACE2) [15,16]. In this study we examined the performance of N, S1 and RBD proteins from SARS-CoV-2 and SARS-CoV in four different test platforms. Our results show that the RBD protein provides the best specificity, whereas the N protein of either virus is not suitable to detect virus-specific antibodies due to PKC (19-36) very high level of cross-reactivity. We demonstrated that capture ELISA can further enhance test specificity and the assay format can be easily adapted to studying isotype- (IgG, IgM, etc.) and subtype-specific antibody responses PKC (19-36) for more basic research needs. Unexpectedly, we discovered that the N-specific antibodies waned faster than the RBD-specific antibodies in convalescent sera obtained from SARS survivors, seventeen years after infection. The significance and the mechanism behind this observation warrants further investigation in the future. Materials and methods Cells and virus Human embryonic kidney (HEK293T) cells (ATCC# CRL-3216) were maintained in Dulbeccos modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum. The first SARS-CoV-2 isolate from Singapore, BetaCoV/Singapore/2/2020 (Accession ID EPI_ISL_406973), was used for virus neutralization.