A SARS-CoV-2 Label-Free Surrogate Virus Neutralization Test and a Longitudinal Study of Antibody Characteristics in COVID-19 Patients

Abstract

Methods designed to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humoral response include virus neutralization tests to determine antibody neutralization activity. For ease of use and universal applicability, surrogate virus neutralization tests (sVNTs) based on antibody-mediated blockage of molecular interactions have been proposed. A surrogate virus neutralization test was established on a label-free immunoassay platform (LF-sVNT). The LF-sVNT analyzes the binding ability of SARS-CoV-2 spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) after neutralizing RBD with antibodies in serum. The LF-sVNT neutralizing antibody titers (50% inhibitory concentration [IC₅₀]) were determined from serum samples (n = 246) from coronavirus disease 2019 (COVID-19) patients (n = 113), as well as the IgG concentrations and the IgG avidity indices. Although there was variability in the kinetics of the IgG concentrations and neutralizing antibody titers between individuals, there was an initial rise, plateau, and then in some cases a gradual decline at later time points after 40 days after symptom onset. The IgG avidity indices, in the same cases, plateaued after an initial rise and did not show a decline. The LF-sVNT can be a valuable tool in research and clinical laboratories for the assessment of the presence of neutralizing antibodies to COVID-19. This study is the first to provide longitudinal neutralizing antibody titers beyond 200 days post-symptom onset. Despite the decline of IgG concentration and neutralizing antibody titer, IgG avidity index increases, reaches a plateau, and then remains constant up to 8 months postinfection. The decline of antibody neutralization activity can be attributed to the reduction in antibody quantity rather than the deterioration of antibody quality, as measured by antibody avidity.

Keywords: SARS-CoV-2; label-free technology; longitudinal study; neutralizing antibody titer; surrogate neutralization virus test.

FIG 1

Illustration of the LF-sVNT protocol and example sensorgrams. (A) The first cycle measuring the binding ability of RBD to ACE2 after neutralization. (B) The second cycle measuring the full binding ability of RBD without neutralization. (C and D) The first-cycle sensorgrams (C) and second-cycle sensorgrams (D) of a dilution series of one serum sample (1:50, 1:100, 1:250, 1:500, 1:1,000, and 1:2,000).

FIG 2

Comparison of LF-sVNT neutralizing antibody titers with IgG concentrations (A) and IgG avidity indices (n = 190) (B). (C) Comparison of LF-sVNT and the cVNT neutralizing antibody titers obtained from 30 serum samples. (D) LF-sVNT neutralizing antibody titers plotted against days since onset of symptoms. All titers of <50 (n = 39) were plotted at 50 and titers of >2,000 (n = 4) were plotted at 2,000. For panels A, B, and C, only one sample was included per subject per week.

FIG 3

IgG concentration (A), IgG avidity index (B), and LF-sVNT neutralizing antibody titer (C) of paired serum samples collected at week 4 and 3 to 8 months post-symptom onset (n = 20). The horizontal dashed line indicates the limit of detection for the LF-sVNT.

FIG 4

Kinetics of LF-sVNT neutralizing antibody titer, IgG concentration, and IgG avidity for 9 patients by days after symptom onset. All cases with ≥4 serial time points and enough remaining sample volume to perform all tests were included.