Estimating various antibody classes against the SARSCoV-2 virus can reflect the true burden of disease in the population

INTRODUCTION

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has infected millions of individuals, causing several deaths, and has greatly disrupted economies worldwide.

SARS-CoV-2 is an enveloped single-stranded ribonucleic acid (RNA) virus. The major structural proteins of SARSCoV-2 are the nucleocapsid protein (N), spike protein (S), membrane protein (M), and envelope protein (E). The S(spike) protein and the N(nucleocapsid) protein are known to be potential antigens for serodiagnosis. The S protein is composed of 2 subunits, the S1 subunit and the S2 subunit. The receptor binding domain (RBD) region of the S1 subunit is of specific importance, as antibodies are produced against this region.^[1]

The detection of viral RNA by real-time polymerase chain reaction (RT-PCR) is the gold standard diagnostic test.^[2] In addition to RT-PCR, estimation of antibodies to the SARSCoV-2 virus can also be used as a modality to assess recently resolved or past infections. The fundamental principle behind antibody-based immunodiagnostics is the detection of antibodies developed in response to viral infection (Immunoglobulin G [IgG]/Immunoglobulin A [IgA]/Immunoglobulin M [IgM]) and/or viral antigen.^[3] IgA, IgG, and IgM are the main antibodies produced in response to the SARS-CoV-2 virus.^[4] IgG and IgM levels rise and fall on different days after the onset of infection.

IgA antibody plays a significant role in contributing to the mucosal immunity of the respiratory and gastrointestinal tract against SARS-CoV-2. The RBD-specific IgA present in the respiratory mucosa is one of the indicators of host immune response, which appears earliest in the disease. Therefore, many studies consider it as an early diagnostic marker of infection and hence were used to determine seroprevalence.^[5]

Based on these dynamics, antibody testing can be applied clinically to diagnose a current or very recent infection, and epidemiologically used as a surveillance tool. For example, in some cases, individuals present with symptoms of COVID-19 but test negative with RT-PCR because either the virus has been cleared or viral shedding does not occur at the time of sampling, and/or technical errors leading to a false-negative result. If sufficient time has passed since the initial infection, the presence of total antibodies against SARS-CoV-2 antigens can be used to confirm a clinical case of COVID-19.

Serological assays such as enzyme-linked immunosorbent assay (ELISA), Lateral flow immunoassays, and chemiluminescence immunoassay (CLIA) are available for the detection of antibodies against SARS-CoV-2.^[6] They vary from each other by the type of antibodies detected, sensitivity, and specificity. Among these methods, the ELISA detecting IgA antibody was used in this study.

Nationwide serosurveys conducted in India estimated the exposure to the SARS-CoV-2 virus.^[7] In the majority of them, IgG antibodies to N and S regions have been estimated by different serological assays.^[8] These serosurveys helped in estimating the seroprevalence of COVID-19 antibodies, thereby providing information on the burden and epidemiology of the disease, for planning appropriate strategies to reduce transmission of the disease.^[9]

The detection of total antibodies (IgA+IgM+IgG) has two major advantages. One, it would be an accurate indicator to assess immunological response by the host against SARSCoV-2 infection, rather than detecting only IgG, which appears later in the course of illness. Second, this would help in understanding the seroconversion of an individual if infected.

Few studies have included IgA antibody detection as a role in neutralizing infection, but in India, no studies have detected total antibodies (IgG+IgM+IgA) for estimating the seroprevalence of COVID-19 disease. This study aims at determining the presence of total antibodies (IgG+IgM+IgA) in samples tested before for IgG antibody by immunoassay and comparing the results of seroprevalence by estimating total antibodies and only IgG antibodies.

MATERIALS AND METHODS

This is a laboratory-based retrospective comparative study conducted in the Department of Microbiology over 6 months.

In Mumbai, a serosurvey conducted in the second half of August 2020, IgG antibodies against the N and S1RBD region of SARS-CoV-2 were detected using chemiluminescent Immunoassay (CLIA). CLIA is a fully automated 2-step sandwich immunoassay using indirect chemiluminescent technology.

The Solid Phase holds a preformed complex of streptavidin-coated micro-particles and biotinylated SARS-CoV-2 recombinant antigens. The antigen-coated particles subsequently capture SARS-CoV-2-specific antibodies in the specimen. The tested samples, both positive and negative, were stored at −20° C in cryogenic vials.

Among the stored samples, around 504 samples tested for IgG antibody by CLIA were selected by a convenient sampling method, anonymized, and renumbered from 1 to 504 and tested by WANTAI assay (Commercial ELISA kit) for the presence of total antibodies (IgG+IgM+IgA) against SARS-CoV-2. This assay was provided to us by the World Health Organization India for testing serum samples for the presence of total antibodies.

WANTAI SARS-CoV-2 Antibody ELISA is a two-step incubation antigen sandwich enzyme immunoassay kit, which uses polystyrene microwell strips pre-coated with recombinant SARS-CoV-2 antigen. The antigen used in the assay is the RBD of the SARS-CoV-2 spike protein. The results obtained were recorded in chart format and analyzed.

RESULTS

All results are shown in graphical format to visualize the distribution of prevalence more clearly.

Comparison of seroprevalence in the total study population by estimating only IgG antibody by CLIA and total antibodies (IgG+IgM+IgA) using the WANTAI assay (n = 504). Out of the total sample size (n = 504) for this study, 14 samples tested positive for only IgG antibodies, with a resulting seroprevalence of 2.8%, whereas 104 samples tested positive for total antibodies (IgG+IgM+IgA) by the WANTAI method, and the resulting seroprevalence was 20.6%. Thus, the estimated seroprevalence increased by 17.8% by testing for total antibodies. Seroprevalence calculated by estimating only IgG antibody was lower, as the antibody levels peak at 2 weeks after symptom onset and are depicted in Figure 1.

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Figure 1:

Comparison of seroprevalence by estimating only immunoglobulin (Ig)G antibody by chemiluminescent immunoassay (CLIA) and total antibodies (IgG+IgM+IgA) using WANTAI assay.

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Seroprevalence of SARS-CoV-2 by estimation of only IgG and total antibodies in slum versus non-slum areas. Out of 504 samples analyzed using the WANTAI assay, 268 samples belonged to slum areas of Mumbai, and 236 samples belonged to non-slum areas. In slum areas, 13 samples tested positive for only IgG antibodies by CLIA, giving a seroprevalence of 4.8% whereas 89 samples were positive for total antibodies by WANTAI assay, giving a seroprevalence of 33.2%. In non-slum areas, only 1 sample was positive for only IgG antibody by CLIA, giving a seroprevalence of 0.5% whereas 15 samples tested positive for total antibodies by WANTAI assay, giving a seroprevalence of 6.3%. This result is depicted in Figure 2.

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Figure 2:

Seroprevalence in slum and non-slum areas using chemiluminescent immunoassay (CLIA) and WANTAI assay.

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Gender-wise distribution of seroprevalence by the estimation of only IgG by CLIA and total antibodies by WANTAI assay, as depicted in Figure 3. In males, 8 samples were positive for IgG antibody using CLIA, with a resulting seroprevalence of 2.48% and 63 samples tested positive for total antibodies, with a resulting seroprevalence of 19.56%. In females, 6 samples were positive for IgG antibody using CLIA, with a resulting seroprevalence of 3.2% and 41 samples tested positive for total antibodies, with a resulting seroprevalence of 22.52%.

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Figure 3:

Gender-wise distribution of seroprevalence by the estimation of only immunoglobulin G (IgG) by chemiluminescent immunoassay and total antibodies by WANTAI assay. CLIA: Chemiluminescent Immunoassay.

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DISCUSSION

The detection of specific antibody classes, such as IgG, IgA, and IgM, against SARS-CoV-2, using different methods, can provide crucial insights into an individual’s immune status against COVID-19 disease. In this study, CLIA and ELISA were the two methods used for antibody detection. They varied in certain parameters.

CLIA uses a chemical reaction that produces light (chemiluminescence) when the antibodies in a patient’s blood sample bind to specific antigens from the virus (SARSCoV-2 in this case), whereas the WANTAI assay is an ELISA precoated with recombinant SARS-CoV-2 antigen.

Both techniques require a trained workforce to facilitate ease of performance. CLIA requires additional equipment for processing, whereas the WANTAI assay can be performed in a pre-existing immunology laboratory.

CLIA allows continuous loading of samples, making it more efficient for processing multiple samples without interruption, whereas the WANTAI assay is better suited for high-throughput processing of large numbers of samples at once, for instance, in serosurveys.

Moreover, the turnaround time (TAT) of CLIA is 30 minutes for 1 sample, making it a rapid test, whereas the WANTAI assay has a TAT of 5 hours as it is tested in a batch of 96 samples.

Many studies have focused on the advantages of using total antibodies rather than estimating only IgG antibodies for calculating seroprevalence.

A study conducted by Zhao et.al.^[10] seroprevalence by estimating total antibodies was 100% and that by estimating only IgG antibodies was 79.8%. Moreover, the study also measured the rate of seroconversion in patients as the patients were followed up. This was one of the limitations of our study, as it was a retrospective analysis of leftover samples.

Nyagwange et al. ^[11] conducted a study comparing WANTAI ELISA with other immunoassays for the measurement of antibodies and concluded that the WANTAI method has more sensitivity and specificity than any other immunoassays, thus proving its advantage over other methods for the detection of antibodies.

Trabaud et al.^[12] evaluated eight different immunoassays for the estimation of antibodies, and one of them was the WANTAI method. The study concluded that the performance of the immunoassays had some differences in sensitivity and specificity, but WANTAI ELISA was highly sensitive, thus proving the advantage of this assay over the other methods.

This study highlights the importance of the WANTAI ELISA test in the detection of total antibodies, contributing to our understanding of the exact burden of disease in slums vs nonslums. Therefore, the seroprevalence calculated by estimating total antibodies was higher, with a comparatively higher prevalence in slum areas. As in slum areas, due to non-compliance with contact precautions, the rate of infectivity might be higher, and hence the antibodies prevalent in the sera of patients against the infection.

There was an increase in seroprevalence by the estimation of total antibodies in both males and females. In males, it was 19.56% and in females, it was 22.52%. No studies to date have commented on the characteristic distribution of antibodies against SARS-CoV-2.

Thus, the use of total antibodies in estimating seroprevalence should be recommended in addition to estimating only IgG antibodies, as total antibodies will give a real insight into true seroprevalence.

Therefore, total antibody testing has a significant role in the following settings:

In clinically diagnosed COVID-19 patients not confirmed by RNA PCR testing, the positive result of antibody testing contributes to the diagnosis of the disease, for an asymptomatic healthy close contact person in the quarantine period, and to monitor the progression of the disease in COVID-19 RT-PCR-positive patients. Furthermore, the detection of total antibodies holds promise as an important diagnostic tool in identifying potential animal hosts for SARS-CoV-2 because the double-sandwich ELISA method is free from species restriction.^[13]

CONCLUSIONS

This study strongly supports the use of total antibody testing (IgG+IgM+IgA) as a more effective method for serological surveys and understanding the true spread of the virus. It highlights the importance of detecting early-stage infections and obtaining accurate seroprevalence estimates for better epidemiological insights. As SARS-CoV-2 potentially becomes a more common pathogen in human populations, total antibody assays will likely play a crucial role in ongoing surveillance and immune monitoring, especially in future epidemics or endemic phases of the virus.

This approach can help public health authorities better track the virus’s spread, make informed decisions about interventions, and assess population-level immunity, ultimately contributing to more effective management of the disease.