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Suggested new breakpoints of anti-MERS-CoV antibody ELISA titers: performance analysis of serologic tests.

ABSTRACT: To provide optimal cut-off values of anti-Middle East respiratory syndrome coronavirus (MERS-CoV) serologic tests, we evaluated performance of ELISA IgG, ELISA IgA, IFA IgM, and IFA IgG using 138 serum samples of 49 MERS-CoV-infected patients and 219 serum samples of 219 rRT-PCR-negative MERS-CoV-exposed healthcare personnel and patients. The performance analysis was conducted for two different purposes: (1) prediction of neutralization activity in MERS-CoV-infected patients, and (2) epidemiologic surveillance of MERS-CoV infections among MERS-CoV-exposed individuals. To evaluate performance according to serum collection time, we used 'days post onset of illness (dpoi)' and 'days post exposure (dpex)' assessing neutralization activity and infection diagnosis, respectively. Performance of serologic tests improved with delayed sampling time, being maximized after a seroconversion period. In predicting neutralization activity, ELISA IgG tests showed optimal performance using sera collected after 21 dpoi at cut-off values of OD ratio 0.4 (sensitivity 100% and specificity 100%), and ELISA IgA showed optimal performance using sera collected after 14 dpoi at cut-off value of OD ratio 0.2 (sensitivity 85.2% and specificity 100%). In diagnosis of MERS-CoV infection, ELISA IgG exhibited optimal performance using sera collected after 28 dpex, at a cut-off value of OD ratio 0.2 (sensitivity 97.3% and specificity 92.9%). These new breakpoints are markedly lower than previously suggested values (ELISA IgG OD ratio 1.1, sensitivity 34.8% and specificity 100% in the present data set), and the performance data help serologic tests to be practically used in the field of MERS management.

SUBMITTER: Ko JH

PROVIDER: S-EPMC7087918 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.

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Suggested new breakpoints of anti-MERS-CoV antibody ELISA titers: performance analysis of serologic tests.

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