Evaluation of Orthogonal Testing Algorithm for Detection of SARS-CoV-2 IgG Antibodies

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody testing is an important tool in assessment of pandemic progress, contact tracing, and identification of recovered coronavirus disease 2019 (COVID-19) patients. We evaluated an orthogonal testing algorithm (OTA) to improve test specificity in these use cases.

Methods: A two-step OTA was applied where individuals who initially tested positive were tested with a second test. The first-line test, detecting IgG antibodies to the viral nucleocapsid protein, was validated in 130 samples and the second-line test, detecting IgG antibodies to the viral spike protein in 148 samples. The OTA was evaluated in 4333 clinical patient specimens. The seropositivity rates relative to the SARS-CoV-2 PCR positivity rates were evaluated from our entire patient population data (n = 5102).

Results: The first-line test resulted in a clinical sensitivity of 96.4% (95% CI; 82.3% to 99.4%), and specificity of 99.0% (95% CI; 94.7% to 99.8%), whereas the second-line test had a sensitivity of 100% (95% CI; 87.1% to 100%) and specificity of 98.4% (95% CI; 94.2% to 99.5%). Using the OTA, 78/98 (80%) of initially positive SARS-CoV-2 IgG results were confirmed with a second-line test, while 11/42 (26%) of previously diagnosed COVID-19 patients had no detectable antibodies as long as 94 days post PCR diagnosis.

Conclusion: Our results show that an OTA can be used to identify patients who require further follow-up due to potential SARS CoV-2 IgG false positive results. In addition, serological testing may not be sufficiently sensitive to reliably detect prior COVID-19 infection.

Fig. 1.

Distribution of all initially positive (n = 98) Abbott SARS-CoV-2 IgG results (S/C > 1.4). Discordant samples (n = 20) were negative on MUSC ELISA, and Positive (n = 78) were positive on both tests.

Fig. 2.

Distribution of all MUSC results sent to RL for analysis: (A) all MUSC discordant results (n = 20) and (B) a subset of results positive by both MUSC tests (n = 20). All results are shown relative to Abbott SARS-CoV-2 IgG Index results (S/C > 1.4).