The analysis of characteristics of anti-SARS-CoV-2 antibodies in clinically COVID-19 patients

Background: The novel coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had spread all of China and many other countries. The COVID-19 patients exhibit typical clinical symptoms, and most cases could be diagnosed by nucleic acid testing and imaging. Recent studies have reported that serum antibody testing can be used for diagnostic screening of SARS-CoV-2 infection. However, the rules and standards of antibody production in serum of discharged COVID-19 patients remain ambiguous. 
Methods: We examined both nucleic acid and antibody detection of 139 non-severe (mild and common) patients who were diagnosed with COVID-19. 
Results: Although the nucleic acid of the discharged patient has turned to negative, some patients have not detected antibodies, including IgG (n=11) and IgM (n=27) .19.4% (27/139) discharged patients’ IgM detection was negative. In the IgM positive discharged patients, 72.8% (83/114) have an onset time over 30 days. And, there was no statistically significant difference in IgM concentration of discharged patients from 4 to 9 weeks after the onset of disease. In addition, we also detected IgM and IgG in nucleic acid test positive patients (n=12) within two weeks after the onset of disease. The concentration of IgM was peaked about on the sixth day and then decreased, while the IgG concentration was continuously increasing. 
Conclusions: Our study demonstrated the rules and standards of antibody production in serum of discharged COVID-19 patients which will provide more ideas for COVID-19 research.

Although COVID-19 has been effectively controlled and cured in China, the global situation is still grim [9,10].
Detecting the specifi c anti-SARS-CoV2 antibodies could effectively and quickly identify the COVID-19 patients [11][12][13]. Antibody testing has been incorporated into COVID-19 shtml). As known, during virus infection, the body produces IgM antibody fi rstly, and then produces IgG antibody with high adaptive affi nity, which are important for long-term immunity and immune memory [14]. Based on the experience in combating MERS and SARS-CoV infection, we have known that IgM could be detected in patient blood after 3-6 days and the IgG could be detected after 8 days [15][16][17]. So we speculate that rapid detection of IgG and IgM antibodies will play an important role in assessing the disease status of admitted patients and the prognosis of discharged COVID-19 patients.
Herein, we detected the nucleic acid and antibody of 139

Patients and sample collection
According to the discharge criteria:1) Normal temperature for over 3 days; 2) Respiratory symptoms improved markedly;

Detection of joint IgM/IgG anti-SARS-CoV-2 antibodies
Wondfo SARS-CoV-2 Antibody Test Kit (Fluorescence immunochromatography) to detect joint IgM/IgG antibodies in serum. After reading the ID chip, pipette 10 L of serum into the sample dilution solution, then add 75 μL of the mixed solution into the sample well. After 10 minutes of incubation at room temperature, read the value on the Wondfo immunofl uorescence detector (FS113). In our lab, we valued the Wondfo SARS-CoV-2 antibody Test Kit's precision/sensitivity and accuracy to make sure effective of our results in this work.

Data analysis
The result of antibody detection can be obtained directly on the machine, where the negative result of antibody detection shows <1.0. All data graph was processed in GraphPad Prism5.0. Negative nucleic acid tests for COVID-19 from 2 consecutive respiratory specimens one day apart. We confi rmed that their nucleic acid tests were negative. At the same time, we also performed antibody detection. As shown in Figure 1A were not detected ( Figure 1B). Citation: Guo

Discussion
Here identifi cation and subsequent processing [18,19]. compared with detection of IgG [24]. As reported, the IgM was still at low level at 100 days after the onset of infection, although a previous study showed IgM disappeared after 12 weeks [24][25][26]. A recent research reported that both IgM and IgG antibodies were detected 5 days after infection with SARS-CoV-2, so using serology antibody detection may facilitate the diagnosis of COVID-19 [27,28]. In our study, we found that 19.42% patients did not detected IgM when they discharged.  Figure   3, which demonstrated that the concentration of IgM reached its peak on the sixth day.
As shown, we only detected the antibody levels of discharged patients ranging from 30-60 days. This level throughout the disease cycle may be at its peak, rising or falling, and it does not indicate the time latitude of the antibody change in COVID-19 patients. In order to understand more clearly the law of antibody production in infected patients, we collected 12 nucleic acid positive patients diagnosed on admission. Within a week of infection, both IgM and IgG increased, and the concentration of IgM reached its fi rst peak on the sixth day. Based on the above results, we speculated that the decreased IgM after one week may have a second increase, reaching a second peak in a certain period. Therefore, this also suggests that we need to carry out large-scale epidemiological investigations, especially in patients with non-severe COVID-19 infection, which will have a better understanding of disease assessment and the immune law of SARS-CoV-2 in the population. Based on the diverse, some coronavirus proteins are conserved through the different clades while others, like Spike proteins, are quite species-specific [29].Of the 61 samples of presumably fully seroconverted COVID-19+ patients (i.e. two weeks after symptom onset) tested on the five antigens, 100 % crossreacted with SARS-CoV-1 Nucleocapsid protein and 45.9 % also cross-reacted with SARS-CoV-1 Spike protein. Notably, only 2 (3.3%) of the 61 samples cross-reacted with MERS-CoV Nucleocapsid [30]. So, antibody detection may give falsepositive reactions in some coronaviruses, which is also one of the urgent points to be resolved.
In conclusion, we detected the antibody (IgG and IgM) in discharged COVID-19 patients. There are some patients whose