Emergency response for evaluating SARS-CoV-2 immune status, seroprevalence and convalescent plasma in Argentina

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used for clinical trials and therapies across the country. Using this protocol, about 80% of convalescent donor plasmas were potentially suitable for therapies. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs.


Introduction
The Americas have been profoundly impacted and have become the epicenter of Coronavirus Disease 2019 (COVID- 19), as of October 13th more than 18 million infections and more than 550,000 deaths have been reported on the continent. Surveillance and testing are fundamental in controlling viral spread and understanding pandemic evolution. Detection of viral RNA by qPCR is the gold standard for diagnosis of acute infections. Measurement of humoral responses has been used as a complement to nucleic acid testing, for diagnosis of suspected cases with negative qPCR results, and for detection of acute or past infections in asymptomatic cases [1]. However, serology became an essential tool for the management of the pandemic, including seroprevalence assessment of immunity in the population, measurement of neutralizing antibody titers in convalescent patients and antibody response upon vaccination [2,3].
Antibody mediated immunity is thought to protect individuals from SARS-CoV-2 infection by interfering with viral entry and/or viral replication. Antibody responses appear within the first week of symptoms onset in about 30 to 40% of infections and, in most cases, simultaneous or close seroconversion for IgM and IgG were observed [1,4]. Antibodies have been detected in more than 90% of infections after the third week of symptoms onset [5]. However, the duration and degree to which recovery from COVID-19 disease, or asymptomatic infection, confers prolonged immunity from reinfection is still unclear, even among individuals with high antibody titers [6,7]. We are still learning about the dynamic nature of antibody response linked to severe, mild, and asymptomatic COVID-19 manifestations and, as the pandemic progresses, algorithms and strategies to implement serologic testing in different epidemiological settings are under evaluation. For understanding this complex process, it is essential to have highly specific and sensitive serologic assays [8,9]. Based on the urgent need to attain reliable tests that measure antibodies to SARS-CoV-2, we redirected resources of a basic molecular virology laboratory, as part of a national task force for the emergency, for the development and production of an affordable and robust serologic assay for Argentina and neighboring countries. The COVIDAR serologic test was generated early after the first COVID-19 case in Argentina, and over half million tests have been already produced and distributed for free in the country.
An important application of serology measurement is the assessment of humoral responses to vaccines and identification of plasma from convalescent donors for possible therapies [10][11][12]. During the initial stages of the COVID-19 epidemic in China, convalescent plasma therapy was used compassionately and has since been implemented in the United States and many other countries [13][14][15][16][17]. Randomized clinical trials to evaluate the usefulness of convalescent plasma in different stages of the disease are ongoing (https://clinicaltrials.gov). Success of this intervention likely increases with the antibody titer of the donor as recently shown [15,16]. Thus, it is important to screen potential convalescent donors to select plasma with the highest antibody titers. This screening can be accomplished by measuring the amount of antibodies by titration and defining the virus-neutralizing activity of the plasma. However, the lack of standardization and correlation between antibody levels and neutralizing capacity of antibodies complicates implementation and evaluation of plasma therapy protocols for COVID-19. Studies using SARS-CoV-2 in plaque reduction assays or pseudotyped particle-based systems indicate that plasma derived from convalescent patients has potent neutralizing activity related to IgG molecules recognizing the spike protein, suggesting that IgG antibodies against spike have high potential to fulfill neutralizing functions in vivo [18][19][20][21]. We developed standardized protocols for antibody quantification using COVIDAR IgG and provided data showing a positive correlation with neutralizing antibody measurements. This information allowed the implementation of protocols in hospitals throughout the country assessing plasma donors for compassionate use (https://www.groupcpc-19.com/) and for randomized multicenter clinical trials [22,23].

Results
Due to the urgent need for tools to cope with the pandemic, the Argentine Ministry of Science redirected scientific capacities to generate COVID-19 control means. In this context, the COVIDAR group developed a quantitative serologic test based on ELISA for SARS-CoV-2 infection, which gained emergency approval by the National Administration for Drugs, Food and Medical Devices (ANMAT) by May 4 th . The assay uses two viral proteins, a trimer stabilized spike protein and the receptor binding domain (RBD), expressed in human cells. Combining these proteins in one assay provided higher sensitivity and reproducibility than using each protein in independent plates for IgG and IgM testing [24].
For this study, four different cohorts of patient samples were used. First, a full data set of 535 acute or convalescent COVID-19 qPCR positive serum samples; second a data base of antibody measurements and specific information of suspected COVID-19 with 3500 entries; third, 1074 serum samples from longitudinal studies of a cohort of 93 patients; and fourth, 561 COVID-19 convalescent plasma from potential donors.
IgG and IgM responses were initially evaluated in 535 qPCR positive patients ( Fig 1A). Samples were grouped according to days of symptoms onset (SO). In the early phase, within 7 days of SO, the IgG sensitivity was 35% (Fig 1A). The sensitivity for IgG detection increased from 72% to 74% between 2 and 3 weeks, and seroconversion increased up to 90.4% after 3 weeks. In addition, within 100 samples with >45 days of SO, 5 did not show presence of IgG, indicating that 5% of the patients displayed undetectable levels of IgG or they were non- responders up to 45 days. Regarding IgM, within 7 days of SO, sensitivity was 46%. IgM levels were highest between 15 and 21 days, and although IgM positivity increased after 3 weeks, the antibody level observed by optic density (OD) at 450 nm clearly decreased (Fig 1A).
Assessment of 277 pre-pandemic samples, including acute infections of other pathogens (dengue virus, hepatitis C virus, human immunodeficiency virus and seasonal coronaviruses), were performed. The cut off for IgG and IgM were defined to maximize specificity, ensuring no false positives. Thus, the cut off was defined as the mean of negative control plus 10 or 5 standard deviations for IgG and IgM, respectively (Fig 1B). High specificity of COVIDAR IgG has been supported by baseline seroprevalence studies of heath care professionals (HCP). In this regard, seroprevalence in HCP at region VIII of Buenos Aires Province, performed in June 2020, showed 0.74% of IgG positivity [25].
A data set of IgG and IgM, including 1379 samples of acute and convalescent infections with complete medical records, was used to analyze distribution of age, gender and symptoms. No significant differences concerning gender were observed. The amount of patients with high IgG levels in the group of severe symptoms was larger than those in the moderate and mild groups. The distribution of IgG levels for patients with severe symptoms were significantly different from those with mild symptoms (Wilcoxon rank sum test p-value<0.0001). The median OD level at 450 nm for IgG was 2, 2.9 and 3.9 for mild, moderate and severe groups, respectively (scale up to 4). This trend of antibody levels with symptoms was also evident for IgM, median 0.8, 1.4 and 2.9 for mild, moderate and severe groups, respectively ( Fig 1C). The distribution of IgM levels for patients with severe vs mild symptoms was significantly different (Wilcoxon rank sum test p-value<0.0001). Antibody measurements also correlated with age, covering a range from 16 to more than 65 years showing the highest antibody levels associated with older COVID-19 patients (Fig 1D). Antibody level distribution significantly differed between patients that were < than 45 versus those that were > than 45 years old (Wilcoxon rank sum test p-value<0.0001).
Serological courses were also followed in a separate cohort of 93 hospitalized patients, most of which (90 patients) underwent seroconversion during the follow-up period (Fig 2A). From this, 1074 antibody determinations were performed. We represented the seroconversion time for IgM and IgG for each patient, as the first moment in which antibodies were detected. In most cases, synchronous seroconversion of IgG and IgM was observed (67 patients, 72%). IgM seroconversion was earlier than IgG in 21% of the patients, while IgG appeared earlier than IgM in 7% of the patients (Fig 2A). This longitudinal study indicates that in the first week of SO 34% of the patients were IgG positive and 38% IgM positive, in agreement with data provided in Fig 1, obtained with a different data set and analysis. In addition, considering patients that were followed for at least 45 days of SO, the seroconversion rate in this cohort was 96.7%.
For other infectious diseases, information regarding the duration of IgM is relevant for identification of possible active infections. In our cohort of COVID-19 subjects, longitudinal studies of antibody responses showed a considerable heterogeneity of IgM kinetics ( Fig 2B).

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Antibody kinetics and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections between 40 and 60 days of SO, IgM dynamics were also very diverse ( Fig 2C). IgM titrations indicate that, while in some patients the antibody levels abruptly or slowly dropped before 30 days, in others IgM levels remained high (titers above 1/12,000), even at 60 days of SO ( Fig  2C), suggesting that IgM detection should be used with caution in SARS-CoV-2 infections. Serology testing has been performed systematically in HCP and nursing homes using COV-IDAR in asymptomatic populations for surveillance. Data indicate that asymptomatic infections were detected by IgG and IgM testing, though at lower levels compared with symptomatic patients (Fig 3A). In addition, longitudinal studies of SARS-CoV-2 infections in the absence of symptoms also indicated simultaneous or close seroconversion of IgM and IgG ( Fig 3B).

Whole blood antibody testing by ELISA assay validated for large scale seroprevalence studies
Well-designed serosurveys are essential to determine how prevalent SARS-CoV-2 infection is in the general population, in selected subsections of the population (such as health care workers), or in specific risk groups. Highly specific and sensitive assays are desirable for accurate seroprevalence studies; however, it is also necessary to have methods that are suitable for simple and fast sample collection. ELISA assays use venipuncture, and require refrigerated storage of samples during transportation, making the assay incompatible with massive testing. To overcome this limitation, we adapted the COVIDAR IgG and IgM assays to work with whole blood samples obtained by finger prick. A self-contained Serokit was validated for storage and transportation of whole blood using a glycerin based preservation system [26]. Similar methods have been extensively used in serosurvey studies of Chagas disease conducted in rural areas of Argentina [27].
To validate the process, 68 paired serum and whole blood samples from confirmed SARS--CoV-2 qPCR positive patients were used. All serum specimens were kept at 4˚C, while whole blood samples collected from the same individuals were divided as follows. Fifty two whole blood samples were kept at room temperature and, to evaluate stability, 16 samples were placed at 37˚C for 7 days using the Serokit preservation system. IgG and IgM antibody reactivity from serum samples showed 100% agreement with those from the same individual's whole blood samples stored using Serokit, either at room temperature or at 37˚C (Fig 4).
The validation of finger prick capillary whole blood sample application with Serokit storage widened COVIDAR use to field serosurveys. Until now, seroprevalence studies in nine slums of Buenos Aires, and surveillance studies among HCP in 30 hospitals have been conducted [28][29][30].

Standardized protocols for IgG quantification that correlate with neutralizing activity
An important limitation to evaluate antibodies in COVID-19 convalescent plasma is the lack of standardized quantitative assays that can be easily performed in different institutions.

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Antibody kinetics and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections Previous analysis using serologic tests assessing IgG titers directed to RBD and/or spike have shown a positive correlation with antibody neutralization [7]. Due to the urgent need for tools, we validated an IgG titration protocol using COVIDAR and performed correlations with neutralizing antibody titers. Protocols and COVIDAR kits for IgG titration were freely distributed in private and public institutions in 20 out of 23 provinces of the country, in which more than 100,000 tests have already been performed.
Five hundred and sixty-one convalescent donor plasma samples were titered in our laboratory for standardization. A distribution analysis of the amount of donors and IgG levels indicate that more than 80% of the samples displayed spike specific end point IgG titers above 1/ 400. Hyper-immune plasmas with titers as high as 1/400,000 were observed (Fig 5A). A significant difference of IgG titers was observed between donors that experienced mild symptoms and those that experienced moderate or severe symptoms, median of 1/800 and 1/6400,

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Antibody kinetics and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections respectively (p = 0.0001) (Fig 5A). Full titration curves are shown in heat maps to illustrate this distribution (Fig 5B).
The data using the IgG titration protocol was correlated with virus-neutralizing activity using SARS-CoV-2 pseudotyped VSV particles (CoV2pp) [31]. Plasma from 176 potential donors were used to assess neutralizing activity. Neutralization curves were fit using a 4-parameter logistic regression model and used to determine 50 and 80% CoV2pp absolute infection inhibition concentration (AbsIC50 and AbsIC80, respectively) by measuring luciferase reporter activity encoded in the CoV2pp. AbsIC50 and AbsIC80 values were correlated
Convalescent plasma samples with low IgG titers between 1/50 to 1/200, displayed detectable neutralizing antibodies, with IC80 from the reciprocal of 16 to 132, highlighting that all COVID-19 convalescent individuals with positive anti-spike IgG showed neutralizing antibodies (Fig 5C). A positive correlation between spike binding IgG titers and CoV2pp infection neutralizing activity was observed with convalescent donor plasma titers above 1/200 (Fig 5C). Plasmas with high spike IgG titers invariably displayed high neutralization activity estimated by IC50 or IC80.
We conclude that using end point titrations for total IgG against spike with COVIDAR is suitable for large scale studies, especially in areas of limited resources, as surrogate of neutralization potency and guidance for convalescent donor plasma selection.

Discussion
The development and appropriate application of serologic assays to detect antibodies to SARS-CoV-2 are essential to determine the pandemic evolution and establish mitigation policies. Here, we developed and produced a widely available serologic tool to assist health authorities for pandemic management. Protocols and reagents were distributed to evaluate immune status of hospitalized patients, surveillance of at risk groups, seroprevalence studies in different populations and evaluation of plasma from convalescent donors for possible therapies. The biggest challenge still stands regarding how to deploy this tool in a strategic manner to bring communities out of the current suffering.
We provided a robust serologic test for health institutions throughout the country and generated highly needed information of humoral responses of acute and convalescent SARS-CoV-2 infections. Specific IgG and IgM responses were found to be highly heterogeneous among different individuals. We observed early seroconversion within the first week of SO in at least 34% of the patients (Figs 1 and 2). This raised the question of whether IgM and/or IgG seroconversion time indicates the end of infectiousness or whether early antibody appearance may overlap with active infection and possible transmission. Although this is an issue that requires further attention, periodic serology testing has been useful for guiding focused qPCR testing, asymptomatic case identification and contact tracing.
The fraction of asymptomatic but infectious cases is a critical epidemiological characteristic that modulates pandemic potential. Viral load and antibody responses in asymptomatic individuals are both important to understand transmission and pandemic extension. It is still unclear how many people carry SARS-CoV-2 infection asymptomatically. It has been shown that the ratio of symptomatic to asymptomatic cases changes in different settings. Ratios from 2:1 to near 1:8 symptomatic to asymptomatic cases have been reported [28,32,33]. The factors that influence this large variation are still unknown. One concern is that the low level of antibodies found in asymptomatic cases may lead to their underestimation [34]. Our studies show wide individual variations in the antibody response and kinetics in both symptomatic and asymptomatic cases. We found that asymptomatic individuals displayed lower overall antibody levels than those observed in COVID-19 symptomatic patients (Fig 3). A positive correlation between disease severity and levels of IgG and IgM in acute and convalescent stages was also observed (Fig 1C and 5A), in agreement with previous studies [6,35]. Another important issue is the duration of IgM antibodies. Our longitudinal study using 93 infected individuals showed that IgM levels wane in 60% of the cases within 30 days of SO (or first qPCR detection for asymptomatic cases). However, IgM levels were still detected after 30 or even 60 days in 40% of the cases, suggesting that IgM detection does not necessarily reflect a recent infection.

Antibody kinetics and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections
Another important question is whether all infected individuals mount a robust antibody response to SARS-CoV-2 infection. In this regard, we found antibodies in 90% of the qPCR positive cases after 3 weeks of SO and, considering patients that were followed for at least 45 days, the seroconversion rate reached 95%. Thus, at least 5% of infected individuals resolved the infection with undetectable levels of antibodies. This observation could be assigned to low antibody responses, below the detection limit, or to non-responder individuals. In this regard, previous studies have shown the relevant role of T cell responses in infection resolution [36,37].
Factors that affect the spread and dynamic of the virus in different geographic, demographic, and socioeconomic areas are still unclear. Reliable reagents to perform large and periodic serosurveys are urgently needed. Here, we provided a tool for using whole blood by finger pricking, followed by IgG and IgM tests, to facilitate large sampling together with a robust ELISA assay. This approach has been used in different neighborhoods and defined at-risk populations in Argentina with SARS-CoV-2 prevalence from less than 1 to more than 50% [28,29], supporting the convenience and utility of the application.
It is important to stress the relevance of widely available quantitative serologic assays. Passive antibody transfer is a treatment strategy that has been used for COVID-19, including plasma and purified immunoglobulins derived from COVID-19 convalescent donors. Although the efficacy of convalescent plasma treatment remains uncertain, recent reports indicate a clinical benefit associated with high-titer units administered early in the course of the infection [16]. In this regard, an important limitation has been to obtain harmonized antibody titers information used in different clinical trials and therapeutic applications. By titering more than 500 convalescent plasma samples, we showed a diversity of titers, which correlated with donor disease severity (Fig 5A). We provided a standardized protocol, widely distributed and used in Argentina for quantitative IgG measurements. A positive correlation between IgG quantitation, using COVIDAR, with neutralizing activity measured by a pseudotyped virus was shown (Fig 5C). This protocol has been useful to normalize quantifications at hospitals and heath institutions using plasma for compassionate therapies and for different clinical trials [22,23]. Although IgG titers correlated with neutralizing activities, the relevant open question is how they correlate with protection [38,39].
While the pandemic still progresses, scientists, public health workers, and policy makers are being challenged to create new strategies based on evidence and experiences in different parts of the world. A consensus has emerged that serological testing provides an essential tool in the pandemic response, nevertheless, serology testing strategies should be dynamic and adaptable to specific needs and resources available in different regions. Our work provides widely and freely available robust serology reagents, protocols, and data on humoral responses of SARS--CoV-2 infected individuals that hopefully helps finding better control measures for the current pandemic.

SARS-CoV-2 protein expression
The ectodomain, soluble version of the SARS-CoV-2 spike protein (residues 1−1208 of Gen-Bank: MN908947) including a T4 foldon trimerization domain, a GSAS substituted at the furin cleavage site (residues 682-685), and an octahistidine tag was cloned into a pCDNA mammalian expression vector. A SARS-CoV-2 version S-2P (spike with two proline substitutions at 986 and 987) was originally used. However, due to the difficulty of purifying the necessary amount of spike protein for producing the ELISA plates, substitutions that increased protein yields and stability were incorporated. In this regard, a variant with four additional

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Antibody kinetics and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections proline substitutions was generated, HexaPro variant, as recently described [40]. The sequence of the receptor binding domain (RBD, amino acid 319 to 541, RVQP. . ..CVNF) along with the signal peptide (amino acid 1-14, MFVF. . ..TSGS) plus a hexahistidine tag was obtained from the Krammer laboratory [41] and subcloned into a pCDNA human expression vector.
For protein expression, FreeStyle 293-F (293-F) cells or adherent HEK 293-T (293-T) cells were maintained at 37˚C with 8% CO 2 and 5% CO 2 , respectively. 293-F cells were grown and transfected in Expi293 expression medium and 293-T cells were grown in D-MEM high glucose supplemented with 10% Fetal Bovine Serum 100U/ml penicillin, 100ug/ml streptomycin and transfected in Opti-MEM reduced serum media. Plasmids encoding spike and RBD were transiently transfected using polyethyleneimine hydrochloride. Transfected 293-F suspension cultures were maintained at 32˚C and transfected 293-T cells were maintained at 37˚C. The cultures supernatants were harvested at five days after transfection by centrifugation of the culture at 4000 g for 20 minutes at 4˚C, new medium was added and a second harvest was performed after four days. For FreeStyle 293-F cells viability was monitored and kept above 75% at all times.

Protein purification
Culture supernatants were applied using a peristaltic pump to HisTrap excel columns (GE Healthcare) with a flow rate of 2-3 ml/min, using 1 ml of media for each 100 ml of supernatant. Columns were washed with 10 volumes of buffer A (NaH 2 PO 4 57,5mM, NaCl 300 mM, pH 7.8) and next with 60 volumes of buffer A with 10 mM imidazol. Then, columns were connected to an HPLC system (Kanuer) and washed at 2 ml/min with the last buffer until absorbance at 280 nm reached a value below 0.005. Proteins were eluted with a 12 minute linear gradient from 10 to 1 M imidazole in buffer A. Fractions of 1 ml were collected and analyzed by SDS-PAGE. Buffer of fractions containing protein was exchange to PBS with NAP-5 columns (GE Healthcare) and pooled. Typical yields were around 6 and 45 mg/l for spike and RBD, respectively, with a purity level of at least 98%.

Enzyme-linked immunosorbent assays development
The ELISA protocol was adapted from previously established protocols used for Chagas disease by Laboratorio Lemos S.R.L. [42], and for SARS-CoV-2 by the Krammer Laboratory [41]. Ninety-six well high binding plates (Jet Biofil, Guangzhou, China) were coated overnight at 4˚C with 75 μl per well of a solution containing 100 ng of a mixture of full-length trimeric spike and RBD proteins in carbonate buffer, pH 9.6. The next day the coating solution was removed by washing three times with phosphate-buffered saline (PBS), pH 7.4. Serum samples diluted in PBS-T containing 0.05% Tween and 0.8% casein were added to the wells (100 μl of a 1:100 dilution for IgM and 200 μl of a 1:50 dilution for IgG determination), and incubated for 1 h at 37˚C. For end point titrations, samples were serially diluted in IgG SARS-CoV-2 negative serum or fetal bovine serum (FBS), added to the plates and incubated for 1 h at 37˚C. Following a washing step with PBS-T, 100 μl of diluted horseradish peroxidase (HRP)-conjugated with goat anti-human IgM (Sigma), or with mouse anti-human IgG antibodies (BD pharmingen), was added to the wells and incubated for 30 min. at 37˚C. Subsequently, the plates were washed with PBS-T, and the peroxidase reaction was visualized by incubating the plates with 100 μl of TMB solution for 30 min at 37˚C. The reaction was stopped by adding 100 μl of 1M sulfuric acid, and optical densities (OD) were immediately measured at 450 nm. For whole blood analysis, 50 μl of blood were preserved in collecting tubes containing 90 μl of buffered glycerin from the Serokit system [26]. Stored samples were then diluted in PBS-T containing 0.8% casein for quantification of IgG or IgM levels.

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Antibody kinetics and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections

Serum and plasma samples
Human plasma and serum samples were obtained from a number of different sources following the subsequent inclusion criteria: people aged over 16  A pre-pandemic serum panel of samples selected based on the date of collection (July and September 2019) was provided by the Blood Bank of the Hospital de Clínicas José de San Martín. Positive and negative controls were donated from the CEMIC blood bank. Longitudinal studies were performed using samples from de-identified patients from Sanatorio de La Trinidad Mitre and Hospital de Clínicas José de San Martín (n = 118). Samples were taken every 2 to 4 days until the patient was discharged from the hospital. Additional samples were collected when patient assisted for controls. Samples were collected between 1 to 90, or more days, of symptoms onset.
Asymptomatic patients identified by contact tracing of COVID-19 positive patients were obtained from BBEI COVID-19 collection, Hospital de Clínicas José de San Martín, Sanatorio de La Trinidad Mitre and Swiss Medical. Paired whole blood and serum samples used for Serokit validation were obtained from the BBEI COVID19 collection.
Sample collection and processing were performed from April 2020 to October 2020.
Patient sera were heat inactivated at 56˚C for 30 minutes and serially diluted in DMEM high glucose with 10% FBS. Serum neutralizations were performed by first diluting the inactivated sample 8-folds and continuing with a 2-fold serial dilution. A pre-titrated amount of pseudotyped particles (diluted to give approximately 5x10 5 Relative Luminescent Units) was incubated with a 2-fold serial dilution of patient sera for 30 minutes at room temperature prior to infection. Approximately 20 hours post-infection cells were processed for detection of luciferase activity. Cells were lysed and transferred to white, F-bottom 750 Lumitrac plates (Greiner, 655074). Plates were read via the GloMax Navigator Microplate Luminometer (Promega, GM200) using the Renilla Luciferase Assay System Luciferase Assay System (Promega, E2820).
Absolute inhibitory concentrations (absIC) values were calculated for all patient sera samples by modeling a 4-parameter logistic (4PL) regression with GraphPad Prism 8. The 4PL model describes the sigmoid-shaped response pattern. For clarity, it is assumed that the

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Antibody kinetics and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections response can be expressed so that the slope increases as the concentration increase. Absolute inhibitory concentration (absIC) was calculated as the corresponding point between the 0% and 100% assay controls. Fifty % and 80% inhibition were defined by the controls for all the samples on the same plate. For example, the absIC50 or absIC80 would be the point at which the curve matches inhibition equal to exactly 50% or 80% of the 100% assay control relative to the assay minimum.