Increasing test specificity without impairing sensitivity - lessons learned from SARS-CoV-2 serology

Abstract

Background SARS-CoV-2 antibody tests have undergone a remarkable improvement in performance. However, due to the low seroprevalence in several areas, very high demands are made on their specificity. Furthermore, the low antibody-response in some individuals requires high test sensitivity to avoid underestimating true seroprevalence. Optimization of testing has been reported through lowering manufacturer cut-offs to improve SARS-CoV-2 assay sensitivity or by combining two tests to improve specificity at the cost of sensitivity. However, these strategies have thus far been used in isolation of each other. Methods To increase sensitivity, cut-offs of three commercially available SARS-CoV-2 automated assays (Roche, Abbott, and DiaSorin) were reduced according to published values in a pre-pandemic specificity cohort (n=1117) and a SARS-CoV-2 positive cohort (n=64). All three testing systems were combined in an orthogonal approach with a confirmatory test, which was one of the remaining automated assays or one of two commercial ELISAs directed against the spike protein receptor binding-domain (RBD) or the nucleocapsid antigen (NP). Results The modified orthogonal test strategy resulted in an improved specificity of at least 99.8%, often even 100%, in all 12 tested combinations with no significant decline in sensitivity. In our cohort, regardless of whether the assays were used for screening or confirmation, combining Roche and Abbott delivered the best overall performance (+~10% sensitivity compared to the single tests and 100% specificity). Conclusion Here we propose a novel orthogonal assay strategy that approaches 100% specificity while maintaining or even significantly improving the screening test's sensitivity.

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

“…2C ). As a classical OTA might negatively affect sensitivities an adaptive, sensitivity-improved (SI-OTA) was applied ( 10 ). To this end, the above-described validation cohorts were screened with the tRBD ELISA.…”

“…Abbot, Specificity: 97.5%) (27). For the Meduni Wien Biobank cohort we had performance data with CE-marked automated test systems available (10) (10). While we cannot rule out minor cross-reactivity between hCoV-specific antibodies and SARS-CoV-2 antigens, they appeared to have a limited effect on assay performances (Fig.…”

“…Abbot, Specificity: 97.5%)( 27 ). For the Meduni Wien Biobank cohort we had performance data with CE-marked automated test systems available ( 10 ) to directly compare with our ELISAs at the high specificity cut-off criterion (8.000 U/mL). With an AUC of 0.987 [0.979-0.992] and a specificity of 99.1%, the NP ELISA presented with comparable performance to the Abbott SARS-CoV-2 chemiluminescence microparticle assay (AUC: 0.993 [0.987-0.997], Fig.…”

“…With an AUC of 0.987 [0.979-0.992] and a specificity of 99.1%, the NP ELISA presented with comparable performance to the Abbott SARS-CoV-2 chemiluminescence microparticle assay (AUC: 0.993 [0.987-0.997], Fig. S5 , Sp 99.2%) ( 10 ), that also relies on the NP antigen. The tRBD ELISA even outperformed the DiaSorin LIAISON ® SARS-CoV-2 S1/S2 IgG chemiluminescence assay (tRBD ELISA: AUC/Sp/Sen=0.993/99.2%/84.9% vs DiaSorin:0.976/98.2%/82.8%, see Fig.…”

Evaluation of four commercial, fully automated SARS-CoV-2 antibody tests suggests a revision of the Siemens SARS-CoV-2 IgG assay

“…While singular tests have been optimized for maximal specificity, a combinatorial testing approach could allow to lower the thresholds to recognize borderline reactive samples without impairing specificity (38). With the lower cut-offs according to the ROC analysis, the combined sensitivity improved to >92% for any combination.…”