A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative...

Area of Science:

• Immunology
• Virology
• Biotechnology

Background:

• Antibody tests are crucial for assessing humoral immunity after SARS-CoV-2 infection or vaccination.
• Existing first-generation tests often provide only qualitative results, limiting their use in detailed seroprevalence studies.
• Quantitative, sensitive, and specific antibody tests are needed for accurate monitoring of immune responses over time.

Purpose of the Study:

• To develop and validate quantitative, easy-to-implement antibody tests for SARS-CoV-2.
• To identify optimal antigen designs for reliable serodiagnosis.
• To establish optimal thresholds for broad clinical applications, including seroprevalence and convalescent plasma donor qualification.

Main Methods:

• Development of two quantitative SARS-CoV-2 antibody tests utilizing the spike receptor binding domain and nucleocapsid protein.
• Comprehensive evaluation of antigens from various biotechnological platforms.
• Cut-off modeling using large, heterogeneous, multicentric validation cohorts.

Main Results:

• Both developed serotests demonstrated performance comparable to fully-automated CE-marked systems.
• A sensitivity-improved orthogonal test approach achieved high specificity (99.8%), enabling robust serodiagnosis in low-prevalence settings.
• Inclusion of a calibrator allowed accurate quantitative monitoring of antibody concentrations and identified thresholds correlating with SARS-CoV-2 neutralization.

Conclusions:

• Antigen source and purity significantly influence serotest performance.
• Biotechnology-assisted antigen selection and in-depth assay characterization overcame limitations of simple ELISA formats.
• The developed assays achieve performance comparable to fully-automated platforms, offering improved quantitative insights into humoral immunity.