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

Clin Chem. 2020 Dec 1;66(12):1531-1537. doi: 10.1093/clinchem/hvaa210.


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.

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Algorithms
  • Antibodies, Viral / blood*
  • Antibodies, Viral / immunology
  • COVID-19 / blood
  • COVID-19 / diagnosis*
  • COVID-19 Serological Testing / methods*
  • Cohort Studies
  • Coronavirus Nucleocapsid Proteins / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Immunoglobulin G / blood*
  • Immunoglobulin G / immunology
  • Middle Aged
  • Phosphoproteins / immunology
  • SARS-CoV-2 / chemistry*
  • Sensitivity and Specificity
  • South Carolina
  • Spike Glycoprotein, Coronavirus / immunology
  • Young Adult


  • Antibodies, Viral
  • Coronavirus Nucleocapsid Proteins
  • Immunoglobulin G
  • Phosphoproteins
  • Spike Glycoprotein, Coronavirus
  • nucleocapsid phosphoprotein, SARS-CoV-2
  • spike protein, SARS-CoV-2