While qualitative reverse transcription polymerase chain reactions (RT-PCR) are used to diagnose COVID-19, serologic tests for antibodies specific to the SARS-CoV-2 virus are used to detect past infection. Most serologic assays are qualitative and semi-quantitative but quantitative serologic assays are also being developed. Quantitative assays that determine antibody concentrations allow longitudinal monitoring of adaptive or humoral immunity in response to infection or vaccination.
Qualitative assays provide a simple “yes” or “no” answer by measuring the presence or absence of a substance or target. The results are not expressed in numerical values, but as descriptive terms such as “positive,” or “reactive,” and “negative,” or “non-reactive.” (1) An example of a qualitative test is a general pregnancy test which determines the presence or absence of human chorionic gonadotrophin in the patient’s urine but does not quantify the amount present.
Most SARS-CoV-2 antibody tests report a qualitative result, denotating a “yes” or “no” answer to whether antibodies (immunoglobulins G, A, and/or M) that bind viral antigen are present or absent in a sample. Such qualitative assays use a single calibrator to establish a cutoff value and provide a simple “positive” or “negative” result. Although such assays do not tell us the level or concentration of antibodies present in the sample, they help us understand whether the patient has developed humoral (B-cell) immune response against the virus.
Researchers are still trying to understand whether these antibodies to the virus could protect people from getting re-infected with the virus and how long this immunity lasts. At this time, getting a “positive” result on the serological COVID-19 test does not mean one should avoid the guidance for masking and social distancing provided by the CDC.
Semi-quantitative assays measure approximate concentrations. Neither qualitative nor semi-quantitative tests provide a precise measurement of the analyte, but the latter can provide an estimate of how much substance is present, which is often represented numerically. Semi-quantitative results are reported in relative or arbitrary units based on a calibration curve that graphically scores reference material prepared by the manufacturer of the assay. (2)
Quantitative assays report precise concentrations of an unknown analyte and employ a six-point calibration. Calibration fits data on a curve by analyzing samples containing analytes (calibrators) of known concentrations. Subsequently, the concentration of the analyte in an unknown specimen can be calculated by linking the output signal to a particular value on the calibration curve. (3)
As required by the FDA, quantitative tests are validated using a specific international standard that ensures the results reported correspond to a specific numerical value of antibodies. This harmonizes results reported from different parts of the world allowing comparative analyses. (4) If no comparison to an International Standard is performed, then the assay is semi-quantitative despite implementing a six-point calibration curve.
There is currently only one standard reference for anti-SARS-CoV-2 immunoglobulin (NIBSC code: 20/136) released by the WHO. Based on this International Standard, results can be indicated as Binding Antibody Units (BAU)/mL when using assays to detect binding antibodies. For better comparability of results, results are indicated as International Units (IU)/mL when using assays to detect neutralizing antibodies. As of October 2021, there are currently 17 semi-quantitative assays and 1 quantitative test with an emergency use authorization (EUA).
For COVID-19, semi-quantitative and quantitative tests are crucial for long-term monitoring of patients’ post-infection or following vaccination, to measure antibody-longevity and weaning in symptomatic and asymptomatic individuals, for measuring threshold estimates that can correlate to protection, to quantify seroprevalence in populations to estimate herd immunity, and to collect plasma donations from recovered COVID-19 patients with the highest levels of antibodies. (5) Guidelines to assess the effectiveness of vaccines and to monitor antibody response have not been released in the United States yet. All qualitative, semi-quantitative, and quantitative testing must be monitored by quality control protocols. These quality controls must mimic an actual positive or negative sample as closely as possible.
References
1. Wadhwa V, Rai S, Thukral T, Chopra M. Laboratory quality management system: road to accreditation and beyond. Indian J Med Microbiol. 2012 Apr-Jun;30(2):131-40.
2. Bertin EP. Qualitative and Semiquantitative Analysis. In: Bertin EP, ed. Introduction to X-Ray Spectrometric Analysis. Boston, MA: Springer US; 1978:255-278.
3. Dasgupta A, Wahed A. Chapter 2 – Immunoassay Platform and Designs. In: Dasgupta A, Wahed A, eds. Clinical Chemistry, Immunology and Laboratory Quality Control. San Diego: Elsevier; 2014:19-34.
4. WHO International Standard First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human). https://nibsc.org/documents/ifu/20-136.pdf. Accessed 10/19, 2021.
5. Gundlapalli AV, Salerno RM, Brooks JT, et al. 2021. SARS-CoV-2 Serologic Assay Needs for the Next Phase of the US COVID-19 Pandemic Response. Open Forum Infectious Diseases. 8(1).
Iswariya Venkataraman, PhD, is associate director of scientific affairs at EUROIMMUN US, a PerkinElmer company.
