Serological analysis was performed using a commercially available, CE-marked, IgGAM enzyme-linked immunosorbent assay (ELISA) that is optimized for seroprevalence studies and measures the total antibody response against the spike glycoprotein (product code: MK654; The Binding Site [TBS]). Briefly, this assay simultaneously measures any IgG, IgA, or IgM directed against the spike glycoprotein, facilitating detection of any antibody response against the antigen. Development of this assay was undertaken by the authors (AMS, SEF, AMC, MTD, AGR) at the University of Birmingham in collaboration with The Binding Site. Detailed descriptions of this assay, including its construction, validation, and verification, have been published previously (Cook et al. 2021 (link); Faustini et al. 2021 (link)). The assay demonstrates 98.3% (95% confidence interval [CI], 96.4%–99.4%) specificity and 98.6% sensitivity (95% CI, 92.6%–100%) in detecting serological responses to the SARS-CoV-2 spike glycoprotein following polymerase chain reaction (PCR)–positive, nonhospitalized, mild-to-moderate coronavirus disease 2019 (COVID-19). Internal quality control material demonstrates an interassay coefficient of variance of 7.2% at the cutoff. Samples are run at a standard dilution of 1/40.
To provide greater detail on the composition of the total serological response and insight into the correlates of protective humoral immunity (rather than seroprevalence), the IgG and IgA responses against the spike glycoprotein were measured individually. To do this, the IgGAM ELISA protocol was modified. The antigen coating layer, serum dilution, and washing steps were unchanged from the original IgGAM protocol (above), but the detection layer employed polyclonal sheep-anti-human horseradish peroxidase (HRP)–conjugated antibodies directed against IgG (1:16,000) or IgA (1:2,000) individually, rather than in combination. For these assays, a cutoff ratio of 1.0 relative to the existing TBS cutoff calibrators was determined by plotting the pre-2019 negatives (n = 90) in a frequency histogram chart. Once the ratio cutoff was determined from the pre-2019 negatives, a cutoff multiplier of 1.0 and 0.71 was established for IgG and IgA, respectively. Further comparison of the properties and comparative performance of these assays relative to the IgGAM assay and others has also been published (Shields, Faustini, Perez-Toledo, Jossi, Allen, et al. 2020 (link); Mohanraj et al. 2021 (link)).
To provide greater detail on the composition of the total serological response and insight into the correlates of protective humoral immunity (rather than seroprevalence), the IgG and IgA responses against the spike glycoprotein were measured individually. To do this, the IgGAM ELISA protocol was modified. The antigen coating layer, serum dilution, and washing steps were unchanged from the original IgGAM protocol (above), but the detection layer employed polyclonal sheep-anti-human horseradish peroxidase (HRP)–conjugated antibodies directed against IgG (1:16,000) or IgA (1:2,000) individually, rather than in combination. For these assays, a cutoff ratio of 1.0 relative to the existing TBS cutoff calibrators was determined by plotting the pre-2019 negatives (n = 90) in a frequency histogram chart. Once the ratio cutoff was determined from the pre-2019 negatives, a cutoff multiplier of 1.0 and 0.71 was established for IgG and IgA, respectively. Further comparison of the properties and comparative performance of these assays relative to the IgGAM assay and others has also been published (Shields, Faustini, Perez-Toledo, Jossi, Allen, et al. 2020 (link); Mohanraj et al. 2021 (link)).
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