Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus that causes Coronavirus Disease 2019 (COVID-19), has recently emerged to cause a human pandemic.
Currently, detection of SARS-CoV-2 with RT-PCR testing from upper or lower respiratory specimens is gold standard method for the confirmation of suspected COVID-19 patients [1] [2] [3] .
Besides SARS-CoV-2 RT-PCR testing, serological testing is emerging as additional option in COVID-19 diagnostics [4] . Preliminary data show a potential use of specific SARS-CoV-2 antibody tests to aid in the diagnosis of suspected COVID-19 patients [5, 6] . Furthermore, there is great interest in antibody testing of healthcare workers to evaluate hospital transmission and in addition antibody tests could ultimately help to see how far the transmission is in the general population [4] .
Recently, Epitope Diagnostics Inc. has developed the EDI TM Coronavirus COVID-19 IgM and IgG Enzyme Linked Immunosorbent Assay (ELISA) Kits. To our knowledge there is only very limited peer reviewed published data in the literature on the performance of these novel immunoassays [7] . Before these novel immunoassays can potentially be implemented in clinical routine, they need thorough evaluation in appropriate sized "positive" and "negative" cohorts.
Therefore, the aim of this study was to perform an analytical and clinical evaluation of the EDI TM ELISAs for the detection of SARS-CoV-2 IgM and IgG antibodies in human plasma.
This work was performed at the Konventhospital Barmherzige Brueder Linz and Ordensklinikum Linz Barmherzige Schwestern in Linz, Austria. The study protocol was approved by the local ethics committee in accordance with the Declaration of Helsinki,. Using VACUETTE® polyethylene terephthalate glycol blood collection tubes (Greiner Bio-One, Kremsmuenster, Austria) EDTA and lithium-heparin anticoagulated blood were collected and plasma aliquots were frozen at -80°C until further analysis. Data were statistically analyzed with the MedCalc 13.1.2.0 (MedCalc Software) and SPSS 23.0 (SPSS Inc.).
We measured SARS-CoV-2 IgM and IgG antibodies fully-automated on an Immunomat instrument (Serion Diagnostics) with the EDI TM Novel Coronavirus COVID-19 IgM and IgG enzyme linked immunosorbent assay (ELISA) kits (Epitope Diagnostics Inc.). The EDI TM Novel Coronavirus COVID-19 IgM and IgG ELISA kits are based on the nucleocapsid protein of SARS-CoV-2 (N), are IVD CE-marked, and are approved for the qualitative detection of SARS-CoV-2 IgM and IgG antibodies in human plasma. The measurement of the SARS-CoV-2 IgM and IgG antibodies was performed following the manufacturer´s instruction. The following interpretation rules of the patient results (single run) for the SARS-CoV-2 IgM and IgG assays were used: If the patient sample OD was below the negative cutoff the result was reported negative (-); If the patient sample OD was above the negative cut off but below the positive cutoff the result was reported borderline (+-); If the patients sample OD was above the positive cutoff the patient was reported as positive (+).
To evaluate the precision of the SARS-CoV-2 IgM and IgG assays in our laboratory, we performed a replication study according to the Clinical and Laboratory Standards Institute (CLSI) guideline EP5-A [8] . One negative patient lithium heparin plasma pool and one positive lithium patient heparin plasma pool were aliquoted into 10 1.5 mL plastic tubes for each concentration level and frozen at -80°C. We analyzed these samples in duplicates in one run per day for 10 days on an Immunomat instrument. Within-run and total analytical imprecision (CV) was calculated with the CLSI single-run precision evaluation test [8] .
The detection limits for the SARS-CoV-2 IgM and IgG assays were determined by assaying a diluted lithium heparin plasma sample of a healthy individual in replicates of 20 and was calculated as 3 SD added to the mean response of the diluted sample. The diluted sample was prepared by mixing a fresh plasma sample in a 1:10 ratio with sample dilution buffer. This prediluted plasma sample was then treated equally with all other patient samples with respect to the assay procedure. The 20 replicates were assayed on the same microwell plate.
For the clinical evaluation we measured SARS-CoV-2 IgM and IgG antibodies in three different cohorts: First in a "positive cohort" of patients with SARS-CoV-2 RT-PCR confirmed 5 COVID-19 with serial blood samples at different time points from symptom onset. In these confirmed COVID-19 patients SARS-CoV-2 IgM and IgG antibodies were clinically suspected.
Second a cohort of healthy blood donors and third a cohort of patients admitted to an intensive care unit (ICU). The healthy blood donors and the ICU patients were recruited prior to the COVID-19 outbreak (December 3 rd 2019) and serve as "negative cohorts" were no SARS-CoV-2 IgM and IgG antibodies were clinically suspected. 
The SARS-CoV-2 IgM assay had a within-run CV of 7% and a total CV of 10% at a mean optical density (OD) of 0.103 (negative patient pool), and within-run CV of 4% and a total CV of 10% at a mean OD of 0.297 (positive patient pool). The SARS-CoV-2 IgG assay had a within-run CV of 4% and a total CV of 9% at a mean OD of 0.173 (negative patient pool), and within-run CV of 7% and a total CV of 9% at a mean OD of 0.537 (positive patient pool). The detection limit was an OD of 0.095 for the SARS-CoV-2 IgM and an OD of 0.083 for the SARS-CoV-2 IgG assay.
3.2. Rates of "true" vs. "false" positivity
The final study population consisted of 64 patients (53 males, 11 females) with SARS-CoV-2 RT-PCR confirmed COVID-19 with serial blood samples (n=104) collected at different time points from symptom onset. The median age was 65 years (range 14-95, IQR 56-87, years). Table 1 shows low "true" positivity rates (i.e. seroconversion rates) for IgM (5.9%) and IgG (2.9%) SARS-CoV-2 antibodies within the first 5 days after symptom onset in patients with COVID-19. Between day 5 and day 10 the "true" positivity rates were 37.1% for IgM and 37.1% for IgG. The "true" positivity rates rose to 76.4% for IgM and 82.4% for IgG after 10-15 days. After 15-22 days the "true" positivity rate was 94.4% for IgM and 100% for IgG SARS-CoV-2 antibodies (Table 1) . Of the 64 patients with confirmed COVID-19 two patients (3%) were immune compromised. One patient had an active hemato-onoclogical disease (a diffuse large B-cell lymphoma). This patient did not develop antibodies against SARS-CoV-2 until 13 days after symptom onset and eventually died of COVID 19. The second confirmed COVID-19 patient suffered from immune thrombocytopenia and was treated with immune modulating/suppressing therapies. This patient developed SARS-CoV-2 antibodies on day 13 and fully recovered from COVID-19.
In the cohort of 200 healthy blood donors the "false" positivity rate was 0.5% for IgM and 1%
for IgG SARS-CoV-2 antibodies ( Table 2 ). The "false" positivity rate in the cohort of 256 ICU patients (was 1.6% for IgM and 1.2% for IgG SARS-CoV-2 antibodies (Table 3 ).
In the supplementary data we report the quantitative values of OD and the ratio OD/negative control in the cohort of patients with SARS-CoV-2 RT-PCR confirmed COVID-19, in the healthy blood donors as well as in the ICU patients ( Supplementary Table 1-3 ).
Our evaluation of the EDI TM Novel Coronavirus COVID-19 ELISAs for the qualitative detection of SARS-CoV-2 IgM and IgG antibodies in human plasma confirms that these novel immunoassays meet the quality specification for laboratory medicine. The CVs were ≤10%, which is adequate for ELISAs. The detection limits of the SARS-CoV-2 IgM and IgG assays were below the negative control and therefore also appropriate. Furthermore, the clinical evaluation study demonstrated high "true" positivity rates in the SARS-CoV-2 RT-PCR confirmed COVID-19 patients with symptom onset after 15 days with 94.4% for IgM and 100% for IgG SARS-CoV-2
antibodies. Finally, we reported low "false" positivity rates in the healthy blood donors with 0.5% for IgM and 1% for IgG as well as in the ICU patients with 1.6% for IgM and 1.2% for IgG SARS-CoV-2 antibodies.
Prior approaches to serologic detection of infection with emerging coronaviruses including SARS and Middle East Respiratory Syndrome (MERS) have focused on the spike glycoprotein and the nucleocapsid protein, which are considered the immunodominant antigens for these viruses [11] . The EDI TM SARS-CoV-2 IgM and IgG assays are based on the nucleocapsid protein of SARS-CoV-2 (N). In agreement with the proposed SARS-CoV-2 antibody response after symptom onset we found very low seroconversion rates before day 5. Whereas after 15 days we found very high seroconversion rates in our SARS-CoV-2 confirmed COVID-19 patients [5, 6, [12] [13] [14] . In line with our findings, it has recently been reported that serological diagnosis is especially important for patients who may present late, beyond the first 2 weeks of illness onset. At this late stage of disease SARS-CoV-2 RT-PCR might already be negative [15] . It would be tempting to only use SARS-CoV-2 IgG antibody tests to aid in the diagnosis of suspected COVID-19 patients. However, when using IgM in addition to IgG assays, this might allow differentiating an early from a late stage or even past infection. In addition, the combined "false" positivity rates for the EDI TM SARS-CoV-2
IgM and IgG assays we reported in the healthy blood donors (<2%) and intensive care patients (<3%) was rather low.
Of note, with the EDI TM SARS-CoV-2 IgM and IgG immunoassays we only report antibody binding to the recombinant nuceleocapsid protein of the SARS-CoV-2 and we did not perform neutralization assays in our SARS-CoV-2 RT-PCR confirmed patients.
Since the EDI TM SARS-CoV-2 IgM and IgG is currently approved as qualitative assay, we did not test the linearity of the assays. However, when interpreting the quantitative data (OD values 8 and the ratios OD/negative control) in supplementary Table 1 , we found a clear antibody response in SARS-CoV-2 antibody positive COVID-19 patients from <5 days until >15 days with increasing median OD and the values ratios OD/negative control, demonstrating that the EDI TM SARS-CoV-2
IgM and IgG assays are suitable for serial measurements.
This study is the first thorough evaluation of the EDI TM SARS-CoV-2 IgM and IgG ELISAs and we reported high "true" vs. low "false" positivity rates, indicating the suitability of these novel immunoassays for clinical routine.
Competing interests: None declared.