A comprehensive antigen production and characterization study for easy-to-implement, highly specific and quantitative SARS-CoV-2 antibody assays

Abstract

Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection. While first-generation antibody tests have primarily provided qualitative results with low specificity, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Here, we describe two quantitative ELISA antibody tests based on the SARS-CoV-2 spike receptor-binding domain and the nucleocapsid protein. Comparative expression in bacterial, insect, mammalian and plant-based platforms enabled the identification of new antigen designs with superior quality and high suitability as diagnostic reagents. Both tests scored excellently in clinical validations with multi-centric specificity and sensitivity cohorts and showed unprecedented correlation with SARS-CoV-2 neutralization titers. Orthogonal testing increased assay specificity to 99.8%, thereby enabling robust serodiagnosis in low-prevalence settings. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19.

SARS-CoV-2 RBD-specific and NP-specific antibody response of healthcare workers in the westernmost Austrian state Vorarlberg: a prospective cohort study

“…Values of ≥5 U/mL were referred to as positive responses. The 5 U/mL cut-off was defined on the basis of criteria suggested by the Youden index and the 99th percentile method 21. In order to meet ongoing concerns about accuracy and cut-offs, values of ≥5 and <8 U/mL for anti-SARS-CoV-2 RBD-specific and anti-SARS-CoV-2 NP-specific antibody responses were referred to as a weak positive response.…”

“…In order to meet ongoing concerns about accuracy and cut-offs, values of ≥5 and <8 U/mL for anti-SARS-CoV-2 RBD-specific and anti-SARS-CoV-2 NP-specific antibody responses were referred to as a weak positive response. Accounting for the prevalence nature of the study, a higher cut-off of ≥8 U/mL was chosen to increase specificity, as proposed by the manufacturer and by a previous study 21. Values of ≥8 U/mL were thus referred to as a strong positive response.…”

Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein Produced in Escherichia Coli

“…Vaccines against SARS-CoV-2 are almost exclusively based on the virus spike protein 8 . Hence, diagnostic markers based on NP will be essential to discriminate between infection-and vaccination-conferred immunity 9 .…”

“…Although NP is post-translationally modified by phosphorylation 22,23 and potentially glycosylation 24 in its native host, it can be expressed in E. coli as a soluble protein with high titers without compromising antigenicity and diagnostic performance in serological assays 9 . The E. coli strains BL21(DE3) and the recently developed enGenes-X-press 25 are all based on T7 expression 26 , whereby with the latter recombinant protein synthesis can be decoupled from cell growth to exclusively utilize metabolic resources for synthesis of the protein of interest.…”

Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein Produced in Escherichia Coli

“…Although NP is post-translationally modified by phosphorylation 22,23 and potentially glycosylation 24 in its native host, it can be expressed in E. coli as a soluble protein with high titers without compromising antigenicity and diagnostic performance in serological assays 9 . The E. coli strains BL21(DE3) and the recently developed enGenes-X-press 25 are all based on T7 expression 26 , whereby with the latter recombinant protein synthesis can be decoupled from cell growth to exclusively utilize metabolic resources for synthesis of the protein of interest.…”