202-21].

The gold standard for assessing the presence of neutralizing antibodies is the plaque reduction neutralization test (PRNT), but this approach does not scale well [@doi:10.1038/s41467-020-17892-0].
An early high-throughput neutralizing antibody assay designed against SARS-CoV-2 used a fluorescently labeled reporter virus that was incubated with different dilutions of patient serum [@doi:10.1038/s41467-020-17892-0].
The cells used for incubation would turn green if antibodies were not present.
Essentially, this assay evaluates whether the virus is able to infect the cell in the presence of the serum.
The specificity of this assay was 100%, and the correlation between the results of this assay and of PRNT was 0.85 with the results suggesting that the sensitivity of the high-throughput approach was higher than that of PRNT [@doi:10.1038/s41467-020-17892-0].
While this approach was performed on a plate and using cells, other methods have been developed using methods such as bead arrays [@doi:10.1016/j.nbt.2021.10.002].

#### Duration of Immune Indicators

While the antibodies produced by the adaptive immune system in response to SARS-CoV-2 viral challenge, these indicators of seroconversion are unlikely to remain in circulation permanently.
Previously, a two-year longitudinal study following convalesced SARS patients with a mean age of 29 found that IgG antibodies were detectable in all 56 patients surveyed for at least 16 months, and remained detectable in all but 4 (11.8%) of patients through the full two-year study period [@doi:10.1086/500469].
These results suggest that immunity to SARS-CoV-1 is sustained for at least a year.
Circulating antibody titers to other coronaviruses have been reported to decline significantly after 1 year [@doi:10.1017/s0950268800048019].
Evidence to date suggests that sustained immunity to the SARS-CoV-2 virus remains for a shorter period of time, but at least 6 to 8 months after infection [@doi:10.3201/eid2703.204543; @doi:10.1126/science.abf4063; @pmid:33443036; @doi:10.1016/j.medj.2021.02.001].
However, this does not mean that all serological evidence of infection dissipates but rather that the immune response becomes insufficient to neutralize the virus.

In order to study the persistence of SARS-CoV-2 antibodies, one study assessed sustained immunity using 254 blood samples from 188 COVID-19 positive patients [@doi:10.1126/science.abf4063].
The samples were collected at various time points between 6 and 240 days post-symptom onset, meaning some patients were assessed longitudinally.
Of the samples, 43 were collected at least 6 months after symptom onset.
After 1 month, 98% of patients were seropositive for IgG to the spike protein, S.
Moreover, S IgG titers were stable and heterogeneous among patients over a period of 6 to 8 months post-symptom onset, with 90% of subjects seropositive at 6 months.
Similarly, at 6 to 8 months 88% of patients were seropositive for RBD IgG, and 90% were seropositive for SARS-CoV-2 neutralizing antibodies.
Another study examined 119 samples from 88 donors who had recovered from mild to severe cases of COVID-19 [@doi:10.1016/j.medj.2021.02.001].
A relatively stable level of IgG and plasma neutralizing antibodies was identified up to 6 months post diagnosis.
Significantly lower but considerable levels of anti-SARS-CoV-2 IgG antibodies were still present in 80% of samples obtained 6-8 months post symptom-onset.
Titers of IgM and IgG antibodies against the RBD were found to decrease from 1.3 to 6.2 months post infection in a study of 87 individuals [@doi:10.1038/s41586-021-03207-w].
However, the decline of IgA (15%) activity was less pronounced than that of IgM (53%) or IgG (32%).
It was noted that higher levels of anti-RBD IgG and anti-N total antibodies were detected in individuals that reported persistent post-acute symptoms at both study visits.
Moreover, plasma neutralizing activity decreased five-fold between 1.3 and 6.2 months in an assay of HIV-1 virus pseudotyped with SARS-CoV-2 S protein, and this neutralizing activity was directly correlated with IgG anti-RBD titers [@doi:10.1038/s41586-021-03207-w].
These findings are in accordance with other studies that show that the majority of seroconverters have detectable, albeit decreasing, levels of neutralizing antibodies at least 3-6 months post infection [@doi:10.1126/science.abd7728; @doi:10.1038/s41564-020-00813-8; @doi:10.1038/s41467-021-21444-5].
Determining the potency of anti-RBD antibodies early in the course of an infection may be important moving forward, as their neutralizing potency may be prognostic for disease severity and survival [@doi:10.1016/j.cell.2020.12.015].
The duration of immunity might also vary with age [@doi:10.1038/s41590-021-01089-8] or ABO blood type [@doi:10.1080/21505594.2021.2019959].
Autopsies of lymph nodes and spleens from severe acute COVID-19 patients showed a loss of T follicular helper cells and germinal centers that may explain some of the impaired development of antibody responses [@doi:10.1016/j.cell.2020.08.025].
Therefore, serological testing may be time-limited in its ability to detect prior infection.

Other immune indicators of prior infection have also been evaluated to see how they persist over time.
SARS-CoV-2 memory CD8^+^ T cells were slightly decreased (50%) 6 months post-symptom onset.
In this same subset of COVID-19 patients, 93% of subjects had detectable levels of SARS-CoV-2 memory CD4^+^ T cells, of which 42% had more than 1% SARS-CoV-2-specific CD4^+^ T cells.
At 6 months, 92% of patients were positive for SARS-CoV-2 memory CD4^+^ T cells.
Indeed, the abundance of S-specific memory CD4^+^ T cells over time was similar to that of SARS-CoV-2-specific CD4^+^ T cells overall [@doi:10.1126/science.abf4063].
T cell immunity to SARS-CoV-2 at 6 to 8 months following symptom onset has also been confirmed by other studies [@doi:10.1101/2020.11.01.362319; @doi:10.1101/2020.12.08.416636; @doi:10.1016/j.medj.2021.02.001].
In another study, T cell reactivity to SARS-CoV-2 epitopes was also detected in some individuals never been exposed to SARS-CoV-2.
This finding suggests the potential for cross-reactive T cell recognition between SARS-CoV-2 and pre-existing circulating HCoV that are responsible for the “common cold” [@doi:10.1016/j.cell.2020.05.015], but further research is required.
Therefore, whether T-cells will over a more stable measure through which to assess prior infection remains unknown.
Notably, commercial entities have tried to develop tests specifically for T cells, some of which have been authorized by the United States Food and Drug Administration (FDA) [@doi:10.1038/s41587-021-00920-9; @url:https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-adaptive-biotechnologies-t-detect-covid-test] to identify people with adaptive T cell immune responses to SARS-CoV-2, either from a previous or ongoing infection. 

#### Applications of Serological Tests

In addition to the limitations posed by the fact that antibodies are not permanent indicators of prior infection, serological immunoassays carry a number of limitations that influence their utility in different situations.
Importantly, false positives can occur due to cross-reactivity with other antibodies according to the clinical condition of the patient [@url:https://www.fda.gov/media/136625/download], and due to the long incubation times and delayed immune responses of infected patients, serological immunoassays are insufficiently sensitive for a diagnosis in the early stages of an infection.
Therefore, such tests must be used in combination with RNA detection tests if intended for diagnostic purposes [@doi:10.1056/NEJMp2025631].
False positives are particularly harmful if they are erroneously interpreted to mean that a population is more likely to have acquired immunity to a disease [@doi:10/gg559s].
Similarly, while serological tests may be of interest to individuals who wish to confirm they were infected with SARS-CoV-2 in the past, their potential for false positives means that they are not currently recommended for this use.
However, in the wake of vaccines becoming widely available, accurate serological tests that could be administered at point of care investigated in the hope