Immunological assays for SARS-CoV-2: an analysis of available commercial tests to measure antigen and antibodies

The rapid spread of SARS-CoV-2 coronavirus infection has led to the development of molecular and serologic tests in a short period of time. While tests such as RT-PCR have applications in the immediate diagnosis revealing the presence of the virus, serological tests can be used to determine previous exposure to the virus and complement acute diagnosis. Antibody production can occur as early as 5 days post-infection. Both IgM and IgG specific anti-SARS-COV-2 antibodies can be a useful tool to test faster and larger groups of individuals. The objective of this study was to carry out a review of the different serological tests offered to detect antigen or antibodies against SARS-CoV-2. This information should be useful for decision takers in different countries to choose a test according to their needs. Based on web pages that listed serological assays, we found 226 coming from 20 countries, the majority are indirect tests for specific antibodies detection (n 180) and use immunochromatography methods (n 110) with samples coming from blood-derived products (n 105). Measuring IgM/IgG at the same time (n 112) and a procedure time of <20 min (n 83) are the most common. The overall average sensitivity was 91.8% and specificity was 97%. Most of the tests are currently for in vitro diagnosis (IVD). This information gathered could change day by day due to the expedite process of production and emergency of authorization use.


Introduction
Nidoviruses are positive-sense single-stranded RNA viruses that infect a large number of vertebrates. Within these is the family of coronaviruses which has four groups, including betacoronavirus, that caused epidemic outbreaks in recent decades (1). Although coronaviruses were described as causing common respiratory symptoms in the 1960's (2), they may be responsible for between 7 to 15% of uncomplicated upper respiratory infections (3). SARS (severe acute respiratory syndrome) in 2002 from China was the first report of a coronavirus outbreak with a mortality around 10%. The virus was probably transmitted to humans by a mammal (Civet cat) but probably originally derived in bats. The second outbreak was MERS (Middle East respiratory syndrome), originating in Saudi Arabia, transmitted by camels, but also originally derived in bats; with a mortality close to 40% (1). Now, we have a third epidemic, the coronavirus (CoV) SARS-CoV-2, which produces COVID-19 (coronavirus disease 2019) which began in the Wuhan province in China but has now turned into a pandemic. The sequence of the virus genome isolated from patients is similar to a bat virus (4). In China, the infection produced mild respiratory symptoms in about 80% of those infected, however, 5% were admitted to the Intensive Care Unit (ICU) of which 2.3% received mechanical ventilation and a mortality of 1.3% (5). The rapid case growth around the world means that in a short time the health systems with scarce resources, such as ICU teams, could saturate rapidly (6). The current standard assay for COVID-19 diagnosis is the detection of viral RNA using RT-PCR in nasopharyngeal swabs (7). Rapid and simple immunoassay tests have been developed to detect antigen or IgM and IgG antibodies (separately or simultaneously) against the SARS-CoV-2 virus in human blood even within 15 minutes. Antibody response can be detected as early as 5 days post-infection (8) and the antibody-secreting cells peak around day 7-8 post-infection (9,10). One of the first peer reviewed studies of this kind of assays showed a test with a sensitivity of 88.66% and a specificity of 90.63% in 397 patients with SARS-CoV-2 confirmed by PCR (11). There are currently more than 200 immunoassays for SARS-CoV-2 to detect antigens or specific antibodies (12). The goal of this study is to carry out a comprehensive review of the wide offer of serological kits to detect SARS-CoV-2 antigen or antibodies, in order to help institutions and policymakers define the best option for a possible massive testing.
There is an urgent need for rapid serological tests for SARS-CoV-2 that will be a useful tool for public health in the upcoming days. 3

Methods
Two approaches were used for the literature search, web searches for pages listing serology tests for SARS-COv-2 and Pubmed ( https://www.ncbi.nlm.nih.gov/pubmed/ ) for peer reviewed literature. Descriptive information from each test was obtained from technical data sheets (TDS) or in their respective company web page. Variables obtained were: country of origin, type of immuno-assay, procedure time, sample type, fixed antigen and antibodies isotype for indirect assays, sensibility, specificity, current regulatory status and published studies. We used not reported (N/R) to specify when information about a variable was not found; and N/A when a variable does not apply. A Pubmed search was conducted for articles describing studies of serology with human samples for SARS-CoV-2. Keywords used were: human + serology + either nCoV, SARS-CoV-2 and Covid-19 or human + antibodies + either nCoV, SARS-CoV-2. We examined the articles, looking for ones that mentioned the use of commercial antigen or antibody detection kits. Data was obtained until April 5th 2020. A total of 18.6% of the assays reported internal validation defined here as the data either found in the TDS or provided by the manufacturer web pages. The number of 6 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
On the other hand, the specificity of the assays ranged between 90.3% and 100%,  the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Discussion
Although the current standard for SARS-CoV-2 is the amplification of viral RNA by RT-PCR, this technique requires special equipment and trained individuals (7). Also, detection of the virus is dependent on the sample origin and time of sampling (15,16). Detection of anti-virus specific SARS-CoV 2 antibodies could help to determine the exposure of a large population to the virus (5,(8)(9)(10). In infected individuals, antibody detection by ELISA using nucleocapsid protein as antigen was identified at Initial reports of the new CoV causing acute respiratory distress syndrome came up in Wuhan, China. Since then, several assays have been developed in order to improve the diagnosis, most of them from China (4,19). As expected most of the available tests detect antibodies using blood-derived samples. Although RT-PCR is considered the most sensitive detection method in respiratory fluid samples, it increases the risk of contamination of healthcare workers (20). Blood-derived samples are easier to obtain, and compared to RT-PCR, serological tests are faster, require less training and no equipment, so they can be used in almost any setting 8 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.10.20061150 doi: medRxiv preprint (11). The most common method behind is lateral flow immunochromatography (21) since these tests have a long shelf-life, do not require refrigeration and easily distinguishable visual results exclude the need for additional equipment compared with other methods like ELISA (22). Antigens used for detection are very important; the genome of SARS-CoV-2 codifies for several structural proteins, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins (23). The most common antigens used for indirect assays are the recombinant spike and nucleocapsid proteins (16,17). The S protein contains the domain for attachment to the human host cells (24), and the nucleocapsid protein is one of the major structural components involved in many processes of the virus including viral replication, transcription, and assembly (18). Interestingly, there is a 90.5% homology among nucleocapsid proteins of SARS-Cov-1 y SARS-CoV-2 (17), and SARS-CoV-2 showed an homology of about 85% with a coronavirus isolated from bats (4,16).
Most tests assessed levels of IgG and IgM simultaneously. The humoral response to some SARS-associated coronavirus shows a simultaneous increase in the level of all antibodies (25). Also, the dual detection of IgG-IgM improves the sensibility in comparison with individual IgG or IgM antibody assay (11), suggesting a possible improvement in the infection detection. In terms of efficiency, a shorter procedure time means a greater evaluation capacity of a population.
Test specificity and sensitivity are key for determining the role of these assays in diagnosis and public health programs (26). Unfortunately, only a minority of the tests present this information, maybe due to short time for development. However, most of them present a sensitivity and specificity well over 90%, but with a low number of 9 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04. 10.20061150 doi: medRxiv preprint infected individuals. Patients with RT-PCR confirmed virus have a median seroconversion rate of 93.1% for IgG and IgM (9) in a time dependent manner (9,11), and 15 days after disease onset seroconversion was 100% for both antibody isotypes (9). Meanwhile, the detection rate of molecular based methods decreased to as low as 45% in that same time (9,15,27). The latter shows that the specificity and sensitivity of each immunoassay are variable depending on the time of onset, with more positive results given in a later time of disease onset (17). Additionally, the different samples that can be used for serological diagnosis offer more consistent results, no significant differences were found in blood, serum or plasma samples (11). This is opposed to the great variability from samples used in viral RNA detection (sputum, naso/oropharyngeal swabs and bronchoalveolar lavage) (15,27,28). Both RT-PCR with antibody assays have advantages, however the combination of both can provide more accuracy to the initial diagnosis of SARS-CoV-2 infection (11,20). Moreover, current label requirements showed that most of the offered assays (50%) are intended for in-vitro diagnosis and this application represents the usefulness in a clinical environment. At this point of the pandemic, it would be difficult to suggest which tests are the best for clinical application. However, the information here presented sheds light into the large number of assays available, and the number increases day by day. This has to be used carefully, we suggest that researchers and policymakers focus on the ones with the most information available, such as a rigorous internal validation data, a well defined TDS, and are intended to be used for in-vitro diagnosis. More research is needed, especially studies that compare between different tests which provide more accurate information than studies with single assays (29). Yet, the initial data looks promising 10 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04. 10.20061150 doi: medRxiv preprint and immunoassays could help screen larger populations in less time, increasing the detection rate and increasing the testing capacity, one of the cornerstones needed to decrease the SARS-Cov2 spread . Recent studies show that convalescent patients have high levels of SARS-CoV2 neutralizing antibodies (NAbs), which increased with patient age (30). Interestingly, transfusion of convalescent plasma obtained from COVID-19 cases, improved clinical outcomes of patients with severe disease (31); thus suggesting that the antibodies produced by COVID-19 patients during the infection have a posterior protective effect. Large serological studies to detect virus-specific antibodies will be needed to determine the infected asymptomatic population and also could help to suspend social isolation in seropositive individuals.
11 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.10.20061150 doi: medRxiv preprint  the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.