The COVIDAR group developed a quantitative serologic test based on ELISA for SARS-CoV-2 infection

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Researchers have developed and applied a robust, versatile antibody test to assist health authorities in managing the coronavirus disease 2019 (COVID-19) pandemic, according to a study published January 14 in the open-access journal PLOS Pathogens by Andrea Gamarnik of the Fundación Instituto Leloir-CONICET in Argentina, and colleagues.

As of November 22, there have been more than 57 million COVID-19 cases worldwide since the start of the pandemic, resulting in more than one million deaths globally. Surveillance and testing play an important role in controlling viral spread. In the new study, Gamarnik and colleagues developed and applied a robust, versatile test to detect antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – the virus that causes COVID-19.

Specifically, the enzyme-linked immunosorbent assay (ELISA) detects immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies against the whole spike protein and its receptor binding domain. More than half a million tests have been freely distributed to health institutions in Argentina.

Antibodies were detected in at least 34% of patients within seven days, and in 95% of patients within 45 days of symptom onset. Overall, antibody responses in asymptomatic cases varied widely but were generally lower than those of symptomatic patients.

The researchers also developed standardized protocols that were used to assess IgG antibody levels to select suitable blood samples from donors who have recovered from COVID-19 for therapeutic use and clinical trials across the country. Using this protocol, approximately 80% of donor blood samples were potentially suitable for therapies.

According to the authors, the study offers a powerful tool for detecting asymptomatic cases and finding better control measures for the pandemic.

The authors add, “The COVIDAR program has built bridges among basic virology researchers, clinical investigators, health care workers, non-profit organizations and policy makers in Argentina for coping with the pandemic. Hopefully, this transformative lesson will help establish collaborative models and priorities for improving public health.”


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 [10–12]. 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 [13–17].

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 [18–21].

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].

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.

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.

reference link: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009161


More information: Ojeda DS, Gonzalez Lopez Ledesma MM, Pallares HM, Costa Navarro GS, Sanchez L, Perazzi B, et al. (2021) Emergency response for evaluating SARS-CoV-2 immune status, seroprevalence and convalescent plasma in Argentina. PLoS Pathog 17(1): e1009161.
doi.org/10.1371/journal.ppat.1009161

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