key: cord-0923853-lm9pay2b
authors: Liu, W.; Liu, L.; Kou, G.; Zheng, Y.; Ding, Y.; Ni, W.; Wang, Q.; Tan, L.; Wu, W.; Tang, S.; Xiong, Z.; Zheng, S.
title: Evaluation of Nucleocapsid and Spike Protein-based ELISAs for detecting antibodies against SARS-CoV-2
date: 2020-03-20
journal: nan
DOI: 10.1101/2020.03.16.20035014
sha: 43102c2a4af46ae690f823d77401913f113f092d
doc_id: 923853
cord_uid: lm9pay2b

Background: At present, PCR-based nucleic acid detection cannot meet the demands for coronavirus infectious disease (COVID-19) diagnosis. Methods: 214 confirmed COVID-19 patients who were hospitalized in the General Hospital of Central Theater Command of the People's Liberation Army between January 18 and February 26, 2020, were recruited. Two Enzyme-Linked Immunosorbent Assay (ELISA) kits based on recombinant SARS-CoV-2 nucleocapsid protein (rN) and spike protein (rS) were used for detecting IgM and IgG antibodies, and their diagnostic feasibility was evaluated. Results: Among the 214 patients, 146 (68.2%) and 150 (70.1%) were successfully diagnosed with the rN-based IgM and IgG ELISAs, respectively; 165 (77.1%) and 159 (74.3%) were successfully diagnosed with the rS-based IgM and IgG ELISAs, respectively. The positive rates of the rN-based and rS-based ELISAs for antibody (IgM and/or IgG) detection were 80.4% and 82.2%, respectively. The sensitivity of the rS-based ELISA for IgM detection was significantly higher than that of the rN-based ELISA. We observed an increase in the positive rate for IgM and IgG with an increasing number of days post-disease onset (d.p.o.), but the positive rate of IgM dropped after 35 d.p.o. The positive rate of rN-based and rS-based IgM and IgG ELISAs was less than 60% during the early stage of the illness 0-10 d.p.o., and that of IgM and IgG was obviously increased after 10 d.p.o. Conclusions: ELISA has a high sensitivity, especially for the detection of serum samples from patients after 10 d.p.o, it can be an important supplementary method for COVID-19 diagnosis.

The ongoing outbreak of coronavirus infectious disease 2019 (COVID-19) (1), 47 which emerged in Wuhan, China, is caused by a novel coronavirus named severe 48 acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2-4). As of March 1, 2020, 49 more than 80,000 laboratory-confirmed cases have been reported in China (5), and the 50 disease has spread over 58 countries in Asia, Australia, Europe, and North America identified it as a Beta coronavirus and showed it is closely related (with 96% identity) 56 to Bat CoV RaTG13, but distinct from SARS-CoV (6). SARS-CoV-2 has a 57 receptor-binding domain (RBD) structure similar to that of SARS-CoV. Functionally 58 important ORFs (ORF1a and ORF1b) and major structural proteins, including the 59 spick (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, are also well 60 annotated (7). According to previous reports, the M and E proteins are necessary for 61 virus assembly (8, 9) . The S protein is important for attachment to host cells, where 62 the RBD of S protein mediates the interaction with angiotensin-converting enzyme 2 63 (ACE2) (6). The S protein is located on the surface of the viral particles and has been 64 reported to be highly immunogenic (10). The N protein is one of the major structural 65 proteins of the virus and is involved in the transcription and replication of viral RNA, 66 packaging of the encapsidated genome into virions (11, 12) , and interference with cell 67 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03. 16 Therefore, with the current tests it is difficult to achieve a meaningful assessment of 85 the proportion of symptomatic patients that are infected, and a rapid and accurate 86 detection method of COVID-19 is urgently needed. Serological assays are accurate 87 and efficient methods for the screening for many pathogens, as specific IgM and IgG 88 antibodies can be detected with ELISA, which has relatively high throughput capacity 89 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint and less stringent specimen requirements (uniformly serum collection) than 90 RNA-based assays.

The present study was conducted to evaluate the performance of rN-based and CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. When A450 was below the cutoff value, the test was considered negative, and when 123 A450 was greater than or equal to the cutoff value, the test was considered positive.

For IgG detection, ELISA plates were coated with rN protein. Serum sample (5 μL) 125 diluted in 100 μL dilution buffer was added to the plates. After incubation and 126 washing, HRP-conjugated monoclonal mouse anti-human IgG antibody was added to 127 the plates for detection. The other operation steps were performed as described above 128 for IgM detection. The cutoff value was calculated by summing 0.130 and the average 129 A450 of negative control replicates. When A450 was below the cutoff value, the test was 130 considered negative, and when A450 was greater than or equal to the cutoff value, the 131 test was considered positive.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. When A450 was below the cutoff value, the test was considered negative, and when 147 A450 was greater than or equal to the cutoff value, the test was considered positive. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint 156 The serum samples were collected from 214 COVID-19 patients who were is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint IgM and/or IgG positive rate was 88.9% at 11-15 d.p.o., and more than 90% at later 177 stages of the disease (Table 1) .

For rS-based ELISA, a similar trend of IgM and IgG positive rates was observed 179 (Fig. 2b) . The positive rate of IgM is a little higher than that of IgG at different 180 disease stages, except >35 d.p.o., possibly due to the higher sensitivity of rS-based 181 IgM detection (77.1%) than IgG detection (74.3%) ( Table 1) .

To verify the specificity of the ELISA assays, 100 samples from healthy blood 

The sensitivity of the rS-based IgM ELISA was significantly higher than that of 188 the rN-based ELISA (P < 0.05). No significant difference between rS-and rN-based 189 ELISAs was observed for detecting IgG and total antibodies (IgM and IgG) (Fig. 1) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint significant difference between the two antigen-based ELISAs was observed for IgM 199 detection in other groups. For IgG detection, there was no significant difference 200 between the two kits in all groups.

In this study, the rN-and rS-based ELISAs for detecting IgM and IgG in 214 202 COVID-19 patients were evaluated (Table 2 ). In total, the detected positive rate 203 (174/214, 81.3%) for IgM by the two kits was significantly higher than that of the is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint amount of data on the serologic diagnosis using antibodies against SARS-CoV-2 are 221 available.

In this study, we evaluated immunoassays for the detection of antibodies against 223 SARS-CoV-2. Specifically, we evaluated IgM and IgG production and their diagnostic 224 value. rN-and rS-based ELISAs were used to detect IgM and IgG in serum samples 225 of confirmed COVID-19 patients. The results revealed that the rS-based ELISA is 226 more sensitive than the rN-based one in the detection of IgM antibodies (Fig. 1) . We 227 speculate that this difference is due to the relatively high sensitivity and early 228 response to the S antigen compared to the N antigen in patients with COVID-19. We is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint could not collect the samples from these patients after discharge. Combined, rN-and CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint 268 The authors declare that no conflict of interest exists. 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint 10 11 Figure 2 12 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.16.20035014 doi: medRxiv preprint

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