SARS-CoV-2 Surrogate Virus Neutralization Test


Methods designed to measure the humoral response of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include virus-neutralization tests to determine antibody-neutralizing activity. For ease of use and universal applicability, surrogate virus neutralization tests (sVNTs) based on antibody-mediated blockade of molecular interactions have been proposed. A surrogate virus neutralization assay was established on a label-free immunoassay (LF-sVNT) platform.

The LF-sVNT tests for the binding ability of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein to angiotensin-converting enzyme 2 (ACE2) after neutralizing RBDs with serum antibodies. LF-sVNT neutralizing antibody titers (50% inhibitory concentration [IC50]) were determined from serum samples (n = 246) from patients with coronavirus disease 2019 (COVID-19) (n = 113), as well as IgG concentrations and IgG avidity indices. Although there was variability in the kinetics of IgG concentrations and neutralizing antibody titers between individuals, there was an initial rise, a plateau, and then, in some cases, a gradual decline at later time points after 40 days after the start of dosing the symptoms.

IgG avidity indices, in the same cases, stagnated after an initial increase and did not show a decrease. LF-sVNT can be a valuable tool in clinical and research laboratories for the evaluation of the presence of neutralizing antibodies against COVID-19. This study is the first to provide longitudinal neutralizing antibody titers beyond 200 days after symptom onset. Despite the decrease in IgG concentration and neutralizing antibody titer, the IgG avidity index increases reach a plateau and then remains constant up to 8 months after infection. The decrease in antibody-neutralizing activity can be attributed to the reduction in the number of antibodies rather than deterioration in the quality of the antibodies, as measured by the avidity of the antibodies.

Materials And Methods

  • Ethical review.

All serum samples used in the analysis were carryover samples obtained after routine clinical laboratory testing. The study protocol was approved by the institutional review board of the University of California, San Francisco. The committee considered that no written consent was required for the use of the remaining specimens.

  • Samples and reagents.

Individual and serial (n = 246) serum samples were obtained from patients diagnosed with COVID-19 (confirmed by PCR) (n = 113) for testing. Patients were 63% male and 75% Hispanic, with a median age of 51 years. Fifty-eight patients (51%) were hospitalized and 55 patients (49%) were outpatients. Of the hospitalized patients, 33 (57%) were admitted to the intensive care unit (ICU), 25 (43%) received mechanical ventilation, and 2 died while in the ICU. The sampling time span of the patients ranged from 5 to 225 days after the onset of symptoms.

Recombinant RBD and ACE2 were purchased from Sino Biological (Wayne, PA). A human monoclonal anti-RBD IgG1 antibody was obtained from Absolute Antibody (Oxford, UK) and a goat anti-human IgG (anti-IgG) antibody was obtained from Jackson Immunoresearch (West Grove, PA). The TFI Labelless Immunoassay Analyzer and RBD-coated detection probes were manufactured by Gator Bio (Palo Alto, CA). The Pylon three-dimensional (3D) fluorescence immunoassay analyzer was manufactured by ET Healthcare (Palo Alto, CA).

  • Label-free IgG avidity assay and fluorescence IgG concentration assay.

A label-free IgG avidity assay was also established using TFI technology. The detection probes and the execution buffer were the same as those of the LF-sVNT. Each serum sample was diluted 10-fold in a running buffer for analysis. The IgG avidity assay protocol included the following steps: (i) immersing the detection probe in a running buffer to obtain a baseline, (ii) forming an RBD-IgG complex on the detection probe, (iii) ) dissociating weakly bound IgG using a running buffer or 3 M urea in running buffer and (iv) formation of the RBD-IgG-anti-IgG complex using 10 µg/ml anti-IgG in running buffer.

The signal increase in the final step, which is proportional to the amount of RBD-IgG-anti-IgG complex in the detection probe, was measured. The IgG avidity index was calculated as the ratio between the reading with the dissociating agent (urea) and the reference (running buffer) presented as a percentage. A fluorescence IgG concentration assay was carried out on the Pylon 3D fluorescence immunoassay analyzer.

Briefly, a detection probe coated with affinity-purified goat anti-human IgM (IgM capture) or protein G (IgG capture) was immersed in a diluted patient sample (15µl). After a wash sequence, the probe was immersed in the assay reagent containing biotinylated RBD and nucleocapsid protein (NP). Following a wash sequence, the probe is incubated with a Cy5-streptavidin polysaccharide conjugate reagent (Cy5-SA). The polysaccharide carries multiple copies of Cy5-SA, allowing for cyclic amplification of the fluorescence signal. The background corrected signal is reported as relative fluorescence units (RFU), which is proportional to the number of specific antibodies in the sample.

  • Conventional virus neutralization test.

A cVNT was used for method comparison with the LF-sVNT on a subset of serum samples spanning the assay range. The experiment was implemented at Colorado State University. A doubling dilution series of each serum sample was prepared in Hanks balanced salt medium (BA-1). Each dilution was mixed with an equal volume of SARS-CoV-2 virus suspension and incubated for 60 min at 37 °C with 5% CO2. The mixture was then added to a Vero cell suspension and incubated for 45 min.

After incubation, a first overlay layer (2 times minimal essential medium [MEM] with 1% agarose) was added to each well and the plates were placed back in the incubator. After 24 h, a second layer with 0.05% neutral red dye was added and the plates were incubated at 37 °C. Viral plaques were counted the next day and the 50% cut-off was calculated based on the negative control plaque counts. The 50% reduction in plaque count titer (PRNT50) reported is the reciprocal of the highest dilution of serum that inhibits ≥50% of plaques relative to control.

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