Potential Usefulness of IgA for the Early Detection of SARS-CoV-2 Infection: Comparison With IgM

Since December 2019, severe acute respiratory coronavirus 2 (SARS-CoV-2) has emerged as a significant threat to global health, leading to a devasting number of deaths worldwide (Zhang et al. 2022). Viral RNA can be analyzed from nasal and pharyngeal swabs, bronchoalveolar lavage fluid, and blood plasma using real-time reverse-transcription polymerase chain reaction (RT-PCR) (Huang et al. 2020; Zhou et al. 2020). Currently, confirmation of a diagnosis of SARS-CoV-2 infection mainly depends on RNA testing.

The performance of RT-PCR in the real world is not always conclusive, with some suspected patients required to repeat testing multiple times before a confirmatory diagnosis can be made (Liu et al. 2020a). During the waiting period, the relevant treatment and quarantine management are delayed. Furthermore, RT-PCR requires expensive instruments and reagents, extensive sample processing, and trained personnel, limiting its widespread application (Esbin et al. 2020).

RNA detection has relatively low sensitivity for samples from hemodialysis patients with SARS-CoV-2 infection. Only 53% of confirmed patients were initially diagnosed with SARS-CoV-2 infection by positive RNA results, with the remaining 47% of patients later identified by serological testing rather than RT-PCR (Tang et al. 2020a).

A serological test aids diagnosis of false negative results from RT-PCR caused by a sampling time more than five days after symptom onset (Petherick 2020; Watson et al. 2020). Immunoassays detect antibody subtypes (IgA, IgM, or IgG) or composite antibodies (Pan-Ig or total antibodies). They can be used for either qualitative or quantitative measurements (Arkhipova-Jenkins et al. 2021). Sensitive and specific serological assays can be very useful for early detecting SARS-CoV-2 infection. During the humoral immune response to SARS-CoV-2, IgM and IgA antibodies are produced earlier than IgG isotypes, and IgA antibodies can sometimes be detected earlier than IgM (Infantino et al. 2021). The IgA detected by immunoassay is considered a more reliable early serological marker than IgM (Pieri et al. 2020). This should drive the inclusion of IgA antibody determination in diagnostic kits and may be particularly useful for patients with atypical symptoms, asymptomatic patients, and acute settings with repeated negative RNA testing results (Infantino et al. 2021). However, IgA is less utilized in routine serological settings, despite being a potential early serological biomarker that correlates with infection severity and neutralization capacity (Seow et al. 2020).

Here, we comprehensively review the differences in kinetics and assay performance between IgA and IgM antibodies against SARS-CoV-2 and explore the usefulness of IgA-based immunoassays for the early determination of SARS-CoV-2 infection.

Electronic databases (PubMed, Scopus, Google Scholar, and Embase) and pre-print servers (medRxiv and bioRxiv) were searched for the period from 1 January 2020 until 1 January 2024, with the following terms: kinetics of antibodies, SARS-CoV-2, serological test, early detection, IgA isotype, and antibody response. English-language original papers, meta-analyses, systematic reviews, and observational studies were included. All potentially eligible papers, including pre-peer-review pre-prints, were exported to Endnote X 8.2 (Clarivate™, USA) and screened for relevace. The author included all retrieved studies as they satisfied quality criteria.

The potential usefulness of IgA and IgM for the early detection of SARS-CoV-2 infection

The humoral immune response to SARS-CoV-2 infection often results in an early IgM response, followed by subsequent class switching to IgG. Some studies have shown that IgA responses precede IgM responses, although the implications of this pattern are not yet understood (Ma et al. 2020; Verkerke et al. 2021).

Several studies have evaluated the earliest day of seroconversion. As reported by Yu et al. (2020), the first seroconversion days for IgA and IgM were 2 and 5 days after initial symptom onset, respectively. Among 183 samples, the positive detection rate of antibodies was 98.9% for IgA and 93.4% for IgM. This study showed an earlier IgA immune response than an IgM response.

To explore the seroconversion of antibodies after SARS-CoV-2 infection, Ma et al. (2020) divided samples into six groups based on time points after symptom onset. The IgA detection kit they tested exhibited a higher positive rate (88.2%) than the IgM kit (76.4%) at 4–10 days post-symptom onset. The median seroconversion day was 4–6 days after symptom onset for IgA and IgM (Ma et al. 2020). This study provides valuable information about coronavirus disease 2019 (COVID-19) serological testing, especially about the utility of IgA antibodies for the early detection of SARS-CoV-2 infection.

Guo et al. (2020) observed that IgA and IgM antibodies were detectable at day 5 after symptom onset. At 1–7 days, the positive detection rates were 92.7% and 85.4% for IgA and IgM, respectively. Other studies analyzed the positive detection rates for IgA and IgM antibodies within 7 days to address whether IgA is suitable as an early detection marker for acute SARS-CoV-2 infection. Infantino et al. (2021) evaluated the effect of IgA antibody analysis on the detection window between serological testing and RT-PCR assay. At 5–7 days, among eight patients seronegative for IgM antibodies, all tested positive for IgA antibodies (100%). Another study had similar findings when monitoring 48 patients for the first 7 days post-symptom onset (Sterlin et al. 2021). The positive detection rates for IgA and IgM anti-RBD antibodies were 31.3% and 14.6%, respectively, while the positive detection rates for IgA and IgM anti-N antibodies were 23.0% and 2.1%, respectively. A study by Zervou et al. (2021) enrolled 82 patients with RT-PCR-confirmed infection. Serum samples were collected within 7 days of symptom onset and tested for antibodies. IgA and IgM were positive in 60.0% and 53.3% of samples, respectively (Zervou et al. 2021). These findings suggest that IgA antibodies bridge the chronological gap between effective serological and RT-PCR testing (Fig. 1). The positive detection rates of IgA and IgM antibodies against SARS-CoV-2 antigens at an early stage are shown in Table I.

Fig. 1.

IgA prevalence ranged from 75% to 89% (median, 83%) after symptom onset or RT-PCR positivity (Arkhipova-Jenkins et al. 2021). Serological testing has been reported to be significant and applicable for diagnosis, population surveillance, and vaccine response (Udugama et al. 2020). However, the serological immunoassay is not recommended for population surveillance in low prevalence settings due to possible more false-positive results (Peeling et al. 2020). IgA antibodies peaked within one month of SARS-CoV-2 infection and then declined but remained detectable until at least 100 days (Gudbjartsson et al. 2020). Regarding the IgA prevalence and kinetics, IgA response to SARS-CoV-2 infection occurs early and is potent for detecting SARS-CoV-2 infection in both serum and saliva specimens (Casian et al. 2021).

Several researchers have explored the performance of IgA and IgM immunoassays for determining SARS-CoV-2 antibodies. Montesinos et al. (2020) assessed the performance of five immunoassays for detecting SARS-CoV-2 antibodies. An enzyme-linked immunosorbent assay (ELISA) for IgA demonstrated 83.6%, 86.1%, 91.5%, and 74.7% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively. In contrast, whereas chemiluminescence immunoassay (CLIA) for IgM achieved 58.7%, 100%, 100%, and 58.1% sensitivity, specificity, PPV, and NPV, respectively. Strength of that study is that it verifies the better sensitivity of IgA than IgM for detecting SARS-CoV-2 at an early stage. Another study evaluated the performance of seven commercial SARS-CoV-2 serology assays on 171 serum samples (Herroelen et al. 2020). IgA ELISA showed 79.3%, 91.1%, 96.4%, and 59.3% overall sensitivity, specificity, PPV, and NPV, respectively, whereas the gold-standard lateral flow assay (LFA) for IgM showed 42.4%, 100%, 100%, and 36.4% overall sensitivity, specificity, PPV, and NPV, respectively. Unlike most IgM assays, the IgA assays displayed good sensitivity and acceptable specificity.

Tré-Hardy et al. (2021) sought to evaluate the performance of ELISAs for IgA, IgM, and total antibodies (IgA, IgM, and IgG). The sensitivity was 89.7%, 48.7%, and 91.2% for IgA, IgM, and total antibodies, respectively. The specificity was 98.7%, 98.7%, and 94.9% for IgA, IgM, and total antibodies, respectively (Tré-Hardy et al. 2021). This study suggests that including IgA may enhance serological sensitivity at an early phase of SARS-CoV-2 infection.

Nicol et al. (2020) compared ELISA’s overall sensitivity and specificity for IgA and lateral flow immunoassay (LFIA) for IgM. The IgA ELISA showed 86.7% sensitivity and 82.7% specificity, whereas the LFIA IgM achieved 81.8% sensitivity and 95.3% specificity. Few false positive results were observed for either IgA ELISA or IgM LFIA. False positive results suggest that interference factors should be taken into account for serological testing assays (Wang et al. 2020).

To compare the performance of different antibody isotypes for the diagnosis of acute COVID-19, an ELISA-based screening assay was developed to detect IgG, IgA, or IgM specific for the RBD of the SARS-CoV-2 S protein. Using receiver operating characteristic analysis, the area under the curve (AUC) for IgA was 0.97, and that for IgM was 0.95. The AUC results show that the analysis of IgA antibodies is slightly better for acute COVID-19 diagnosis than IgM antibodies (Verkerke et al. 2021). Therefore, compared to IgM, IgA antibodies seem to be suitable for the early detection of SARS-CoV-2 infection.

Longitudinal monitoring of humoral immunity and identification of vaccine response predictors are essential for optimizing vaccine management (Lippi et al. 2021). IgA antibodies play a crucial role since IgA values in serum closely reflect the development of humoral immunity. Anis et al. (2023) evaluated the importance of IgA antibodies in breakthrough infection individuals; they found a higher positivity rate for IgA to S1 (70% vs. 28% in healthy individuals), and IgA to NP was not found in the control group. In the breakthrough group, the positivity rate of IgA to NP decreased significantly, while IgA to S1 increased over time at 4–8 weeks. IgA to S1, and NP were both negative in 13 patients at initial testing. The findings of this study imply that serum IgA antibodies have a role in breakthrough infection and prevention of severe disease.

Mutations in SARS-CoV-2 can result in failure to detect antibodies with kits designed to recognize epitopes from earlier variants. Changes have primarily been observed in the S protein. Targeting the N protein may be beneficial as it may remain relatively unaffected in different variants compared with the S protein (Sharma et al. 2023). IgA to N protein is effective in detecting COVID-19 breakthrough infection.

Saliva is a crucial component of mucosal immunity; the secretory IgA (SIgA) in saliva attacks the infectious virus in the respiratory entrance, thereby preventing the virus from attaching to epithelial cells. Pisanic et al. (2021) postulated saliva could act as a noninvasive alternative to serological testing for monitoring SARS-CoV-2 infection. Therefore they developed a multiplex SARS-CoV-2 antibody immunoassay to analyze IgA, IgM and IgG in serum and saliva.

At the time point 1–7 days after symptom onset, the detection sensitivity was 50% for IgA and 0% for IgM in saliva, while it was 28% for IgA and 10% for IgM in serum. Obviously, this immunoassay displayed a relatively higher sensitivity for salivary IgA detection at an early stage.

Newly developed IgA biomarkers, such as dimeric IgA, can be detected in LFA IgA assays with 100% sensitivity and 98.2% specificity, with an estimated shorter half-life of 6.3 days. The advantages of dimeric IgA render it a superior biomarker for recent SARS-CoV-2 infection in point-of-care testing (POCT) (Drummer et al. 2021). A saliva IgA assay could be necessary for the evaluation of levels of protective immunity or the effects of vaccination when available in the future (Wang 2021).

The principles that seroconversion of IgA antibodies precede IgM remain unclear. Recently, a study revealed that S protein-based epitopes for detecting SARS-CoV-2 IgA antibodies cross-react with antibodies against other pathogens. Mapping revealed that 40 IgA epitopes were detected on the SARS-CoV-2 S protein. Evaluation of the specificity of each epitope by microarray demonstrated that at least 23 epitopes were cross-reactive with Dengue fever virus and other pathogens. Twenty-four epitopes showed cross-reactivity with other associated coronaviruses. Of 6 RBD-IgA epitopes, only SC/18 (a peptide with residues 490–504 of S1) of Omicron variants displayed good specificity (De-Simone et al. 2023). This suggests that utilization of the whole S protein or the S1/S2/RBD segment may give false positive results. Notably, in two studies with identical patient groups, detection time points, and detection assays, anti-S(RBD) antibodies had a higher positive detection rate than anti-NCP antibodies (31.3% vs. 23.0%) (Sterlin et al. 2021) and (93.8% vs. 71.9%) (Cacaci et al. 2021). The high positive detection rate of anti-S(RBD) IgA antibodies at an early stage of symptom onset does not rule out the possibility of cross-reactivity with S(RBD)-based assays because anti-NCP antibodies are expected to be produced earlier than those against S1 and S2 (Burbelo et al. 2020). Jääskeläinen et al. (2020) evaluated an S1-based IgA ELISA and found only 73% specificity. A serum sample from one patient with human coronavirus OC₄₃ infection showed crossreactivity in the assay (Jääskeläinen et al. 2020).

IgA seems to be an ideal early serological biomarker that correlates with infection severity and neutralization capacity. The role of IgA in controlling SARS-CoV-2 is becoming increasingly apparent. Many serological immunoassays have been developed to detect SARS-CoV-2 specific IgA with target antigens derived from the S and/or N protein. It is important to recognize the pitfalls of the early detection of SARS-CoV-2 IgA antibodies against S antigens. Either using assays for antibodies targeting the N antigen, a combination of the N and S antigens, or a specific non-cross-reactive IgA peptide epitope may strengthen the accuracy of serological assays (Jalkanen et al. 2021). Evaluation of a serological assay should include specificity analyses against related viruses and possible interfering substances (Lipsitch et al. 2020; Lv et al. 2020). If the issue of specificity is addressed with the appropriate antigen, the early detection capacity and high accuracy of IgA antibody determination may greatly aid COVID-19 diagnosis.

The availability of tests with good performance will lead to a more accurate diagnosis of SARS-CoV-2 infection. The analysis of IgA antibodies with IgM/IgG antibodies significantly enhances diagnostic accuracy for COVID-19 (Cervia et al. 2021). Zhao et al. (2020) collected serial plasma samples from 173 patients and determined total antibody, IgM, and IgG levels against SARS-CoV-2, and performed the virus detection with RT-PCR at different time points. At 1–7 days postsymptom onset, total antibodies, IgM, and IgG showed 38.3%, 28.7%, and 19.1% sensitivity, respectively. Total antibodies are expected to demonstrate higher sensitivity than IgM or IgG alone because total antibodies include an analysis of IgA, IgM, and IgG rather than an individual isotype (Tang et al. 2020b). Interestingly, the sensitivity of RT-PCR at 1–7 days post-symptom onset was 66.7%; combining RT-PCR with total antibody analysis improved the diagnostic sensitivity from 66.7% to 78.7%. To our knowledge, serological assays are generally not relied on for diagnosing acute viral respiratory tract infections, as it takes time for the body to produce detectable levels of antibodies (Theel et al. 2020).

One lesson from the SARS-CoV was that IgM and IgA antibody analysis did not provide earlier evidence of infection than IgG antibody testing (Hsueh et al. 2004). To our knowledge, serological assays are generally not relied on for diagnosing acute viral respiratory tract infections, as it takes time for the body to produce detectable levels of antibodies. At the time of publication, total antibody testing has been widely developed, and includes assessment of IgA with IgM and IgG. Seroconversion of total antibodies against SARS-CoV-2 was observed to occur as early as the day of symptom onset (Tariq et al. 2021). One study using the Roche Elecsys® SARS-CoV-2 total antibody and Abbott Alinity SARS-CoV-2 IgG (Abbott Laboratories, USA) assays reached a similar conclusion (Harley and Gunsolus 2020). Of 13 patients 3–7 days after symptom onset, eight (61.54%), 11 (84.62%), and seven (53.85%) were reactive to the Abbott IgG, Roche total antibody, and Abbott IgM assays, respectively. These results indicate the robust early detection capacity of total antibodies for the diagnosis of SARS-CoV-2 infections. Therefore, integration of IgA with IgM and IgG (Nuccetelli et al. 2020) or with RT-PCR could be useful for the early detection of SARS-CoV-2 infection (Li et al. 2020; Zhao et al. 2020).

A limitation of this review is the small number of studies. Some immunoassays have not yet been validated and there is variability in the accuracy of the tests employed. Data from previous studies indicated that IgA assays have good sensitivity and acceptable specificity compared with IgM assays. IgA antibodies appear to precede IgM in the immune response to SARS-CoV-2. Analyses of the discrepancies in the performances of various serological testing kits indicate that they are related to the inherent characteristics of a serological assay, the target antigen used, sampling time, and the populations enrolled, which suggests that accurate diagnosis depends on the tests used. High sensitivity (> 95%) is critical for diagnosing individual patients. High specificity (> 98%) is preferred for seroprevalence studies. To date, the performance of IgA immunoassays could be more satisfactory. Developing new recombinant antigens and using ultrasensitive quantum dot materials, although this process still has a long way to go, can improve the performance of IgA immunoassays. The development of an LFIA IgA assay is expected to prompt POCT kits for the early screening of SARS-CoV-2 infection based on its accessibility, simple procedures, and lower detection time.