key: cord-0808750-9fnlt1wp
authors: Cai, Xue-fei; Chen, Juan; Hu, Jie-li; Long, Quan-xin; Deng, Hai-jun; Fan, Kai; Liao, Pu; Liu, Bei-zhong; Wu, Gui-cheng; Chen, Yao-kai; Li, Zhi-jie; Wang, Kun; Zhang, Xiao-li; Tian, Wen-guang; Xiang, Jiang-lin; Du, Hong-xin; Wang, Jing; Hu, Yuan; Tang, Ni; Lin, Yong; Ren, Ji-hua; Huang, Lu-yi; Wei, Jie; Gan, Chun-yang; Chen, Yan-meng; Gao, Qing-zhu; Chen, A-mei; He, Chang-long; Wang, Dao-Xin; Hu, Peng; Zhou, Fa-Chun; Huang, Ai-long; Liu, Ping; Wang, De-qiang
title: A Peptide-based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Coronavirus Disease 2019 (COVID-19)
date: 2020-05-08
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa243
sha: 676bad1105e527b64741e1b63c48f240385a37e1
doc_id: 808750
cord_uid: 9fnlt1wp

SARS-CoV-2, a novel ß-coronavirus, cause severe pneumonia and has spread throughout the globe rapidly. The disease associated with SARS-CoV-2 infection is named COVID-19. To date, real-time RT-PCR is the only test able to confirm this infection. However, the accuracy of RT-PCR depends on several factors; variations in these factors might significantly lower the sensitivity of detection. Here, we developed a peptide-based luminescent immunoassay that detected immunoglobulin G (IgG) and IgM. The assay cut-off value was determined by evaluating the sera from healthy and infected patients for pathogens other than SARS-CoV-2. To evaluate assay performance, we detected IgG and IgM in the sera from confirmed patients. The positive rate of IgG and IgM was 71.4% and 57.2%, respectively. Therefore, combining our immunoassay with real-time RT-PCR might enhance the diagnostic accuracy of COVID-19.

The coronavirus disease 2019 (COVID-19), once an unknown acute respiratory disease, is caused by a novel coronavirus (SARS-CoV-2). Given the substantial increase in daily confirmed global cases, on January 30 th , 2020, the World Health Organization (WHO) officially declared the COVID-19 outbreak a public health emergency of international concern. Furthermore, on March 12 th , 2020, the WHO [1, 2] . In this study, we developed a magnetic chemiluminescence enzyme immunoassay (MCLIA), which showed high specificity and sensitivity in detecting serum immunoglobulin G (IgG) and IgM against SARS-CoV-2. 

The cut-off value of the test was determined as the mean luminescence value of the 200 normal sera standard deviation (SD) plus 5-fold cross validation. Sera from 276 patients with COVID-19 and 167 patients with foreign pathogens were used to evaluate the performance of the assay.

Twenty synthetic peptides derived from the amino acid sequence of ORF1a/b, S, and N proteins were used to develop the MCLIA for detecting IgG and IgM antibodies against SARS-CoV-2. To screen these peptides, 5 sera from confirmed patients with COVID-19 and 10 normal sera were used to react with these peptides, respectively.

Among the tested peptide, one from the S protein showed the best performance. We used the assay based on this peptide for the following study. To determine the cut-off value of this assay, serum samples from 200 healthy blood donors who donated blood 1-2 years before the COVID-19 outbreak were tested first. The mean S/co values for IgG and IgM were 0.152 ± 0.109 and 0.151 ± 0.107, respectively (Fig. 1) A c c e p t e d M a n u s c r i p t pneumonia, K. pneumonia, A. baumannii, C. albicans, and S. aureus were tested. The mean CL values for IgG and IgM in non-SARS-CoV-2 infected patients were 0.121 ± 0.062 and 0.120 ± 0.065, respectively (Fig. 1A-B) . These results showed that no cross-reactivity was observed for these 20 pathogens, indicating a high specificity.

To test the stability of this MCLIA based serological diagnosis method, serum samples with different concentrations were measured 10 times ( Fig. 2A-D) . The

samples were all below 6% (Fig. 2) , implying successful assay stability for IgG/IgM detection. Furthermore, series dilutions for 6 serum samples (3 for IgG, 3 for IgM) were performed, and S/co values were collected. Regression analysis revealed the S/co value ranged from 1 to 200 linear reflected serum antibody concentration (IgG, R 2 = -0.902, p < 0.001, Fig. 3A ; IgM, R 2 = -0.946, p < 0.001, Fig. 3B ), which assured the rationality for further quantitative comparison based on S/co values. (Table 1) . There was a small cohort of patients that showed negative results for IgG or IgM. We classified these patients with a clear record of fever onset into IgG-or IgM-positive group and IgG-or IgM-negative group and compared the intervals between fever onset and antibody testing between the groups. As shown in Fig. 4 , both IgG positive and IgM positive groups had longer intervals than that of IgG negative and IgM negative groups, with median intervals 13 days (IQR,10-17) vs. 10 days (IQR, 5-12) for IgG and 13 days (IQR, 10-17) vs. 11 days (IQR, 7-14) for IgM, respectively.

We developed a luminescent immunoassay for IgG and IgM against SARS-CoV-2, which, to our knowledge, was the first such assay allowing us to study the antibody response to the newly identified coronavirus. This assay was based on a peptide from the S protein, which was screened out from 20 candidate peptides deduced from the genomic sequence. Using a synthetic peptide as an antigen enhanced the stability and repeatability of the assay, and theoretically would be more specific than using a virus as an antigen. Moreover, this peptide showed high specificity in our assay; for example, none of the 167 sera from patients infected with pathogens other than SARS-CoV-2 reacted with this peptide.

Till now, real-time RT-PCR was the only test able to confirm SARS-CoV-2 infection.

In this study, we detected both IgM and IgG in the same sera from the 276 (Fig. 4) .

IgG can be detected as early as 2 days after the onset of fever. IgM was not detected earlier than IgG, similar to the situation in MERS [4] , which limits its diagnostic utility. It was reported that 20%-50% of patients with SARS could not be confirmed by RT-PCR [5] , and this elicited speculation that there might be a comparable aspect of SARS-CoV-2 infection that also cannot be detected by real-time RT-PCR. Failure of detection by real-time RT-PCR can be caused by issues from sampling, RNA extraction, and PCR amplification, while detecting antibodies in a serum sample may avoid these issues.

M a n u s c r i p t 

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

Viral Shedding and Antibody Response in 37

Patients With Middle East Respiratory Syndrome Coronavirus Infection

Evaluation of reverse transcription-PCR assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus