Serology assays to detect circulating antibodies directed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gained popularity for primarily two reasons. They were originally not regulated under the emergency use authorization (EUA) and could be easily implemented. Furthermore, specimens for serologic testing are easily obtained via phlebotomy and do not require a specialized collection device. However, given the time window that exists between initial infection and antibody production, serology assays cannot be used to diagnose acute SARS-CoV-2 infection. Nonetheless, these assays have continued to be used widely outside of research setting and are even offered as direct to consumer products by private for-profit clinical laboratories.

The concept of presumptive immunity is commonly used for infectious diseases with effective vaccines and assumes that individuals with antibodies are protected from the infection. The long-standing scientific rationale behind presumptive immunity is antibody-mediated viral neutralization – antibodies produced by the host lymphocytes bind to viral particles and inhibit viral entry into host cells, thereby preventing infection. Several small studies provided evidence suggesting the benefits of neutralizing antibodies to SARS-CoV-2 and protection from reinfection. Addetia et al described an outbreak of SARS-CoV-2 among a fishing boat crew and reported that three crewmembers who had developed neutralizing antibodies prior to departure did not develop re-infection, despite the high infection rate of the outbreak (1). A study from Netherlands found that high neutralizing titers (≥1:160) were predictive of non-infectivity (2). However, neutralizing assays are laborious to perform and often require specialized biosafety facilities typically available only at research laboratories. An attractive alternative was to predict the presence of neutralizing antibodies based on automated serology test results.

We evaluated the feasibility of this idea by simultaneously measuring total antibodies using three commercially available serology assays, as well as neutralizing titers, from 71 plasma specimens of patients with known SARS-CoV-2 infection (3). When the presence of neutralizing antibodies was defined using a low cutoff titer ≥1:32, we found a concordance of 0.61-0.69, with a negative percent agreement (NPA) 73-100%. However, when the cutoff neutralizing titer was increased to ≥1:128, concordance decreased to 0.31-0.50, with NPA 31-55%. A positive serology test result was only ~70% predictive of high neutralizing titers at ≥1:128. Although several subsequent studies have claimed that serology assays could predict for neutralizing antibodies (4,5), it is important to realize that the neutralizing titer cutoffs used in these studies are low at 1:20-1:50.

We also emphasized in our study that signal cutoffs for serology assays should be increased when the intended use is to predict for high neutralizing titer. This is consistent with an observational study from Mayo Clinic, which examined the efficacy of convalescent plasma (CCP) as a treatment for SARS-CoV-2 infection. CCP provides passive immunity through transfusion of plasma collected from individuals who had recovered from SARS-CoV-2 infection. The study reported that, among a subgroup of patients who received CCP during early disease, patients who received CCP units from donors with higher serology assay signal had better outcomes than those who received units from donors with lower serology assay signal (6). In the EUA granted to CCP, manufacturers are required to label CCP units as “high titer” vs. “low titer”. Only the Ortho IgG serology assay is approved for CCP labeling and an assay signal cutoff of 12 is used to define high vs. low titer units. This cutoff was reported to correspond to a neutralizing titer of 1:250, based on a neutralizing assay from the Broad Institute (7). While the FDA has stated that other assays may be considered for unit labeling in the future (8), how this can be achieved is unclear. Some questions are: If serology assays will now be calibrated against neutralizing assays, which neutralizing assay should be used as the reference procedure? If the neutralizing assay from Broad Institute will be the reference procedure, has its precision and repeatability been established? Given the laborious nature of neutralizing assays, will reference materials for high neutralizing titer be made available for calibration of serology assays?

In summary, commercially available SARS-CoV-2 serology assays are poor proxies for neutralizing antibodies. The assay cutoffs established by manufacturers are not optimal to predict for the presence of high titer neutralizing antibodies. While increasing assay cutoffs can help counter this issue, modifications to these assays (including their cutoffs) are not regulated under the EUA and modified assays will be categorized as lab-developed tests. Finally, while presence of circulating antibodies poorly predicts neutralizing antibodies, it is important to remember that SARS-CoV-2 immunity also involves cellular immunity, which is not captured by in vitro neutralizing assays.

Acknowledgement: The author would like to acknowledge Drs. Christopher Farnsworth and Neil Anderson for constructive feedback.


  1. Addetia A, Crawford KHD, Dingens A, Zhu H, Roychoudhury P, Huang M-L, et al. Neutralizing Antibodies Correlate with Protection from SARS-CoV-2 in Humans during a Fishery Vessel Outbreak with a High Attack Rate. Journal of Clinical Microbiology. Available from:
  2. Kampen JJA van, Vijver DAMC van de, Fraaij PLA, Haagmans BL, Lamers MM, Okba N, et al. Shedding of infectious virus in hospitalized patients with coronavirus disease-2019 (COVID-19): duration and key determinants. medRxiv. Cold Spring Harbor Laboratory Press; 2020;2020.06.08.20125310.
  3. Tang MS, Case JB, Franks CE, Chen RE, Anderson NW, Henderson JP, et al. Association between SARS-CoV-2 Neutralizing Antibodies and Commercial Serological Assays. Clin Chem. Available from:
  4. Suhandynata RT, Hoffman MA, Huang D, Tran JT, Kelner MJ, Reed SL, et al. Commercial Serology Assays Predict Neutralization Activity Against SARS-CoV-2. Clin Chem. Available from:
  5. GeurtsvanKessel CH, Okba NMA, Igloi Z, Bogers S, Embregts CWE, Laksono BM, et al. An evaluation of COVID-19 serological assays informs future diagnostics and exposure assessment. Nature Communications. 2020;11:3436.
  6. Joyner MJ, Senefeld JW, Klassen SA, Mills JR, Johnson PW, Theel ES, et al. Effect of Convalescent Plasma on Mortality among Hospitalized Patients with COVID-19: Initial Three-Month Experience. medRxiv. Cold Spring Harbor Laboratory Press; 2020;2020.08.12.20169359.
  7. Clinical Memorandum: Emergency Use Authorization for Convalescent Plasma. Available from: (accessed November 23, 2020).
  8. Toolkit for Nov 2020 COVID-19 Convalescent Plasma (CCP) Unfer Emergency Use Authorization (EUA). Available from: (accessed November 22, 2020).