key: cord-0990689-6hep2lin
authors: Lin, Dachuan; Liu, Lei; Zhang, Mingxia; Hu, Yunlong; Yang, Qianting; Guo, Jiubiao; Dai, Youchao; Xu, Yuzhong; Cai, Yi; Chen, Xinchun; Huang, Kaisong; Zhang, Zheng
title: Evaluations of serological test in the diagnosis of 2019 novel coronavirus (SARS-CoV-2) infections during the COVID-19 outbreak
date: 2020-03-30
journal: nan
DOI: 10.1101/2020.03.27.20045153
sha: 266fdda7466e10bc79b8be52a56758ae60608d9a
doc_id: 990689
cord_uid: 6hep2lin

The ongoing SARS-CoV-2 outbreak has killed over twenty-one thousand and sickened over four hundred thousand people worldwide, posing a great challenge to global public health. A sensitive and accurate diagnosis method will substantially help to control disease expansion. Here, we developed a chemiluminescence-immunoassay method based on the recombinant nucleocapsid antigen and the magnetic beads for diagnosis of SARS-CoV-2 infections and surveillance of antibody changing pattern. Serums from 29 healthy individuals, 51 tuberculosis patients, and 79 SARS-CoV-2 confirmed patients were employed to evaluate the performance of this approach. Compared to the IgM testing, the IgG testing was more reliable in which it identified 65 SARS-CoV-2 infections from the 79 confirmed patients and only two false-positive cases from the 80 control group with a sensitivity and specificity reaching 82.28% and 97.5%, respectively. However, only a slight difference (not statistically significant) in the detected cases of SARS-CoV-2 infections was observed between the IgM and IgG testing manner in patients at a different time of onset of disease. A performance comparison between an ELISA kit using the same nucleocapsid antigen and our chemiluminescence method was undertaken. The same false-positive cases were seen in both methods from the paired control group, while ELISA kit can only detect half of the SARS-CoV-2 infections from paired SARS-CoV-2 confirmed patients group than that of the chemiluminescence method, indicating a higher performance for the chemiluminescence-immunoassay approach. Together, our studies provide a useful and valuable serological testing tool for the diagnosis of SARS-CoV-2 infections in the community.

Serums from 29 healthy individuals, 51 tuberculosis patients, and 79 SARS-CoV-2 confirmed patients 25 were employed to evaluate the performance of this approach. Compared to the IgM testing, the IgG 26 testing was more reliable in which it identified 65 SARS-CoV-2 infections from the 79 confirmed 27 patients and only two false-positive cases from the 80 control group with a sensitivity and specificity 28 reaching 82.28% and 97.5%, respectively. However, only a slight difference (not statistically 29 significant) in the detected cases of SARS-CoV-2 infections was observed between the IgM and IgG 30 testing manner in patients at a different time of onset of disease. A performance comparison 31 between an ELISA kit using the same nucleocapsid antigen and our chemiluminescence method was 32 undertaken. The same false-positive cases were seen in both methods from the paired control group, 33 while ELISA kit can only detect half of the SARS-CoV-2 infections from paired SARS-CoV-2 confirmed 34 patients group than that of the chemiluminescence method, indicating a higher performance for the 35 chemiluminescence-immunoassay approach. Together, our studies provide a useful and valuable Introduction 39 Coronavirus, belonging to the family of Coronavirdiae and order of Nidovirales, is a group of 40 enveloped, non-segmented positive-sense RNA virus that has been reported to be able to infect 41 humans and a wide range of animals including cattle, swine, chicken, cat, horse, camels, rodent, bats 42 and snakes and so forth (1) (2) (3) . Based on the genetic properties, coronavirus was further divided into 43 . CC-BY-NC 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 this version posted March 30, 2020 . . https://doi.org/10.1101 /2020 four genera, namely Alphacoronavirus , Betacoronavirus, Gammacoronavirus, and Deltacoronavirus 44 (4). Prior to December 2019, a total of six coronaviruses have been documented to be capable of 45 causing disease in humans. These include two strains from Alphacoronavirus (HCoV-229E and HKU-46 NL63) and four from Betacoronavirus subfamily (HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV) 47 (5-10). Among them, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East 48 Respiratory Syndrome coronavirus (MERS-CoV) are the most well-described as they directly led to two 49 deadly large-scale outbreaks globally, with 8,096 cases infections and roughly 10 percent mortality 50 and 2,494 cases and 34.4 percent mortality, respectively(9, 10). 51 Recently, the outbreak of a severe pneumonia COVID-19 was confirmed to be caused by the 2019 52 novel coronavirus infections (SARS-CoV-2) that was originated from a seafood wholesale market in 53 Wuhan, China(11). So far, this novel coronavirus has spread throughout the whole of China and over 54 198 countries globally, causing over468,905 laboratory-confirmed cases of infections with 21,200 55 people dead posing a great threat to the global public health (http://2019ncov.chinacdc.cn/2019-nCov/). 56 Besides, there are still numerous suspected cases and a myriad of medical monitoring people who 57 were quarantined in specialized hospitals or at homes because of their previous epidemiological link 58 to confirmed SARS-CoV-2 patients. All of these put an extreme burden on the emergency, hospital 59 and public health system particularly the epidemic zone worldwide. Therefore, a timely, sensitive and 60 accurate diagnosis approach is urgently needed and of pivotal importance for surveillances of disease 61 dissemination and the prevention of further expansions. Conventional diagnosis methods such as 62 virus culture and microscopic analysis are generally time-consuming and labor-intensive with limited 63 sensitivity (12, 13). In contrast, the last decade emerged molecular biologic and serologic approaches, 64 . CC-BY-NC 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 certified by peer review)

The copyright holder for this preprint this version posted March 30, 2020 . . https://doi.org/10.1101 /2020 such as TaqMan Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR), Enzyme-linked 65 immunosorbent assay, colloidal gold immunochromatography and direct chemiluminescence 66 immunoassay(CLIA), can be developed into a rapid and effective tool for detections of respiratory 67 pathogens infections, even though in certain circumstances molecular biologic method like RT-PCR 68 had a low sensitivity for specimens from upper respiratory tract (14-17). 69 In this study, we developed a chemiluminescence immunoassay method to specifically detect the 

. CC-BY-NC 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 this version posted March 30, 2020. CC-BY-NC 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 this version posted March 30, 2020. were coupled to these tosyl magnetic beads using the catalytic reagent solution (3M Ammonium 108 sulfate / 0.1M Borate buffer, pH9.5) according to the manufacture's instruction, and the resultant 109 beads were further blocked by 0.05% BSA for six hours at 37 °C. The following testing and detection 110 procedure was automated on a chemical immuno-luminescence analyzer ACCRE6 (Tianshen Tech, 111 Shenzhen, China). It was comprised of those following steps. 50 microliter pure serum was firstly 112 incubated with the magnetic beads that were coupled with antigens for 5 minutes at 37 °C. 113 Subsequently, the unbound substance was gently removed and then washed by Tris buffer for three 114 times. Alkaline phosphatase labeled anti-human immunoglobulin (50µg/ml) was added and further 115 incubated for 5 minutes at 37 °C in the Mes Buffer. After three times washing to remove unbound 116 materials, the lumigen APS-5 substrate (50ug/ml) was eventually added. Ultimately, the light signal 117 was measured by the photomultiplier in ACCRE6 (Tianshen Tech, Shenzhen, China) as relative light 118 units, and the whole testing can be completed in 23 minutes. Confirmed SARS-CoV-2 positive-serum 119 and negative-serum were used as controls in each set test. square and Fisher's exact test was used for comparing the difference between the analyzed groups. 140 The difference was considered significant when a p-value is < 0.05. CC-BY-NC 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 this version posted March 30, 2020. . https://doi.org/10. 1101 /2020 To assess the specificity and sensitivity of the chemiluminescence immunoassay method developed 146 based on the recombinant nucleocapsid antigen, serum from 29 healthy individuals, 51 tuberculosis 147 patients and 79 confirmed SARS-CoV-2 patients were employed and tested. More than six-fold and 148 eight folds higher average RLU (relative light units) values were observed in the SARS-CoV-2 patients 149 group in the IgM testing compared to that of the healthy cohort and tuberculosis patients ( Figure 1A) . (Table 1 ). In contrast, using the IgG testing, we only detected two false-positive cases 160 from the control group, which is in line with the higher specificity for IgG (97.5%) compared to that of 161 the IgM testing (81.25%) as above described. CC-BY-NC 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 this version posted March 30, 2020 . . https://doi.org/10.1101 /2020 To explore whether the onset time was significantly linked with the detection sensitivity by this 165 serological chemiluminescence method, comparison and statistical analysis of the sensitivity rates 166 between different onset time patient categories was undertaken. No statistically significant 167 difference was observed between the IgM and IgG testing results in the patients with the same onset 168 time, although two more cases from 12 cases were detected by IgM testing compared to that of IgG 169 testing in patients less than the one-week onset of disease (Table 2 , p-value ˃0.05). In stark contrast, 170 two more cases SARS-CoV-2 patients were identified by IgG testing than that of IgM testing in patients 171 with more than two weeks onset of disease (p-value ˃0.05). In addition, we also compared the 172 detection rates between the different age groups people, and we found that a significantly lower 173 detection rate in both lgM and lgG testing manner for individual group younger than 18 years old was 174 observed compared to that of people aging from 18 to 65 (p-value < 0.01) or over 65 years old (p-175 value ˃0.05) (Supplementary Table S1 ). No statistically significant differences were observed for male 176 and female groups as well. CC-BY-NC 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 this version posted March 30, 2020 . . https://doi.org/10.1101 /2020 2 in both IgM and IgG testing manners was seen in our chemiluminescence method, suggesting a 186 higher sensitivity of our approach compared to the tested ELISA kit (Table 3, that a single IgG testing is feasible in the clinical diagnosis for SARS-CoV-2, as a higher specificity and 199 sensitivity were observed in our chemiluminescence method. In the humoral immune response, the 200 antibody IgM was generally produced earlier than the IgG isotype as the IgM can be expressed 201 without the isotype switching. Unexpectedly, we only observed a slight detection rate difference (not 202 statistically significant) between these two antibody isotype testing manner in patients in the first 203 week or more than two weeks after onset of disease. However, compared to the IgG approach, our 204 IgM method showed a lower specificity (higher false-positive cases) in our testing. As the IgM and IgG 205 . CC-BY-NC 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 this version posted March 30, 2020 . . https://doi.org/10.1101 /2020 using the same pure recombinant antigen and coupling condition (supplementary figure S1), the 206 detection specificity difference is more likely linked to intrinsic antibody traits and concentration 207 differences in the patients' blood. 208 We noted that four patients with clinical symptoms less than four days were simultaneously detected 209 by both IgM and IgG testing. A close examination of their medical record reveals that all of them had 210 previous contact with confirmed SARS-CoV-2 individuals in at least 16 days ago, pointing to the 211 possibility that they were probably asymptomatically infected by SARS-CoV-2 for certain time already. 212 Fourteen cases (17.7%) from 79 SARS-CoV-2 confirmed patients were not identified by our serological 213 testing method (both the IgM and IgG manner). Interestingly, of them, seven people were younger 214 than 8-year-old or over 70-year-old. These people generally have low immunity in which a clinical 215 symptom may occur rapidly upon exposure to the SARS-CoV-2, and we speculate that the antibodies 216 in these people may not develop well yet when testing. More investigations are warranted to 217 uncover the real situations. When comparing the detection rates in different age groups by our 218 method, we noted that a significantly lower detection rate in both lgM and lgG testing manner for the 219 individual group younger than 18 years old was observed compared to that of people aging from 18 to 220 65. An in-depth look at the days after onset for these 12 individuals younger than 18-years-old, the 221 symptom onset time for all the 12 people are less than 14 days with six people even less seven days 222 (Supplementary Table S2 ). The lower detection rate for these 12 people younger than 18-years-old 223 was likely associated with no or less production of antibodies in them yet when we collected the 224 serums.

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The copyright holder for this preprint this version posted March 30, 2020. .

In our parallel performance testing, the same antigen of nucleocapsid protein was used in both the 226 commercially available ELISA kit and our chemiluminescence immunoassay. Unexpectedly, a 227 significantly higher sensitivity was observed in our method compared to the ELISA kit. This sensitivity 228 difference may be partially attributed to the difference in the serum amount for the first incubation CC-BY-NC 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 certified by peer review)

The copyright holder for this preprint this version posted March 30, 2020. . Table 1 Evaluations of a chemiluminescence immunoassay method for diagnosis of SARS-CoV-2 by detections of specific IgM and IgG in the patient's serum. 

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