key: cord-0924579-i9do06ty
authors: Zhang, Shun; Ning, Yingjun; Rong, Yejing; Nie, Yaxing; Xiong, Zi; Li, Rui; Jin, Tong; Cai, Ting
title: Rapid immunoassay and clinical evaluation of the SARS‐CoV‐2 antibody assay on the real express‐6 analyzer
date: 2021-07-29
journal: J Med Virol
DOI: 10.1002/jmv.27201
sha: c2f128266be7cd2ff8219ee1ca73380abfee3373
doc_id: 924579
cord_uid: i9do06ty

We developed a rapid and simple magnetic chemiluminescence enzyme immunoassay on the Real Express‐6 analyzer, which could simultaneously detect immunoglobulin G and immunoglobulin M antibodies against SARS‐CoV‐2 virus in human blood within 18 min, and which could be used to detect clinical studies to verify its clinical efficacy. We selected blood samples from 185 COVID‐19 patients confirmed by polymerase chain reaction and 271 negative patients to determine the clinical detection sensitivity, specificity, stability, and precision of this method. Meanwhile, we also surveyed the dynamic variance of viral antibodies during SARS‐CoV‐2 infection. This rapid immunoassay test has huge potential benefits for rapid screening of SARS‐CoV‐2 infection and may help clinical drug and vaccine development.

Near the end of 2019, many cases of unexplained pneumonia occurred in Wuhan City, Hubei Province. The illness spread quickly throughout the city and eventually over the entire country. 1 By early January 2020, it was confirmed that it was an acute respiratory infection that was caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with the disease being named coronavirus disease 2019 . 2 However, the virus soon found its way around the world, and by the beginning of March 2020, the World Health Organization (WHO) officially labeled the disease as a pandemic. 3 As of April 2021, SARS-CoV-2 had spread to 223 countries, and there have been 147 539 302 confirmed cases of SARS-CoV-2, including 3 116 444 deaths. 4 SARS-CoV-2 occurred by human-to-human transmission and mostly affected elderly and immunocompromised persons. 5 The rapid spread of SARS-CoV-2 has caused considerable damage to public health and the economy. 67 In the absence of treatment for this virus, accurate and rapid diagnosis of SARS-CoV-2 is the cornerstone of the efforts to control the epidemic, and save people's lives. Currently, the detection of viral nucleic acid real-time polymerase chain reaction (RT-PCR) has become the current standard diagnostic method for the diagnosis of COVID-19. 8, 9 However, the performance of RT-PCR depends on many factors, such as the sample collection skill, sample type, different disease progression, and the quality and consistency of the PCR assay used. 10, 11 Therefore, there is an urgent need for a rapid, simple to use, sensitive, and accurate test to identify infected patients of SARS-CoV-2 to prevent virus transmission.

Early diagnosis, isolation, and treatment are essential to cure the disease and control the epidemic. Antibody detection is of great significance in the diagnosis of infected patients, and helps to identify the stage of the infectious. 12 Based on these, we developed a magnetic chemiluminescence enzyme immunoassay test product, which could detect IgG and IgM simultaneously in human blood within 18 min. Here, we retrospectively described 456 serum samples through IgG/IgM antibody detection. All samples are from HwaMei Hospital, University of Chinese Academy of Sciences. This study may provide a reference for the clinical profile of SARS-CoV-2 patients confirmed by antibody detection, and further to investigate the potential relationship between immune antibodies and disease progression. 

The SARS-CoV-2 antibodies (IgG and IgM) of the subjects were de- 

To establish the stability of the kit, two samples were stored for 0, 1, 4, 7, 15, 21, 28, and 60 days at room temperature, 2-8 and −20℃.

We redetected the IgG and IgM concentrations of known positive and negative plasma samples with the SARS-CoV-2 antibody test kit.

A negative samples pool, approximately 30 ml, was prepared by combing leftover antibody-negative samples (IgG < 278.8 U/ml, IgM < 6.6 U/ml). Similarly, critical positive (278.8 U/ml< IgG <320 U/ml, 6.60 < IgM < 7.59 U/ml), medium/strong (IgG > 320 U/ml, IgM < 7.59 U/ml) positive pool, approximately 30 ml, were prepared by diluting a positive clinical sample with a partial negative sample. Aliquots of 400 μl were prepared from each pool and frozen at −20℃. Two controls and three samples containing different concentrations of analysis were assayed in duplicate, with two runs per day, one lot of reagent for each run, and two replicates per run. Repeatability and Between-Lot precision study was performed by assaying each sample and one lot of reagent 10 times. A between-day precision study was performed by thawing out each respective aliquot to room temperature and running over 20 days. The mean and SD were calculated for each sample, and the coefficient of variation (CV) was determined as CV (%) = (SD × 100)/mean.

In total, some samples from patients with Influenza A virus anti- 

The patients underwent chest CT examinations on admission. All CT images were reviewed independently by two experienced radiologists. The image features included lesion distribution, local or bilateral patchy shadowing, lesion density, and interstitial abnormalities. Additionally, the CT scan was obtained every 5 days or in case of deterioration during hospitalization. 

To evaluate the stability of the SARS-CoV-2 antibody test kit, we tested the IgG and IgM levels of two samples (n = 2) at three different temperatures (Figure 2) . When stored at room temperature, these samples were stable for 7 days, and their IgG and IgM levels were the same as at room temperature. In addition, the samples were stable for at least 60 days when stored at −20℃, and were consistent with the IgG and IgM levels at the first two temperatures.

The cross-reactivity study for SARS-CoV-2 IgG and IgM test kits were designed to evaluate potential cross-reactants and were shown in Table 1 . The cross-reaction of the IgG and IgM test kit with Influenza A virus antibodies was 8.33%, which was lower than of colloidal gold 25.00%, whereas the IgG presented a crossreaction of 0.00% with respiratory syncytial virus antibodies, and lower than the colloidal gold and IgM 6.67%. The cross-reaction of both IgG and IgM were 11.76% with EBV VCA IgM, and lower than the colloidal gold 17.65%. Similarly, the cross-reaction of the IgG and IgM were 10.00% and 0.00% for the CMV IgG, when compared with that of colloidal gold 20.00%. In addition, the cross-reaction of both IgG and IgM were 7.14% and 14.29% for the C. pneumoniae IgM, which were significantly lower than that of colloidal gold 21.43%. Taking together, these results indicate the low cross-reactivity between the IgG and IgM, when compared with the colloidal gold.

To investigate the precision of the SARS-CoV-2 IgG and IgM test kit, we detected three aspects: repeatability, between-lot, and between-day. The results are summarized in the following Table 2 . tively. The stability of the SARS-CoV-2 IgG/IgM test kit is better and less affected by temperature. In the cross-reaction, we used the SARS-CoV-2 IgG/IgM test kit and the colloidal gold to detect the IgG and IgM levels of 10 viruses. We found that the positive rate of the IgG/IgM kit was lower, which was lower than the colloidal gold result. For precision, the negative detection rate of negative samples was 100%, the positive detection rate of borderline positive samples was more than 95%, and the positive detection rate of medium/ strong positive samples was 100% and CV ≤ 15%. Meanwhile, the IgG positive rate was always higher than IgM, and this phenomenon was also observed in a study by Zhang et al. 13 Based on the above, we analytically and clinically evaluated the qualitative and report that it performs reliably, precisely, consistent with manufacturer specifications Currently, virus nucleic acid RT-PCR, CT imaging, and hematology parameter are the primary tools for clinical diagnosis of the infection. 14 Chest CT has been proposed as an ancillary approach for screening individuals with suspected COVID-19 pneumonia during the epidemic period and monitoring treatment response according to the dynamic radiological changes. 15 Although detection of the RNA by either RT-PCR or sequencing is the gold standard for COVID-19 diagnosis, it still suffers from some limitations such as being labor-intensive and timeconsuming. 16, 17 Testing the SARS-CoV-2 specific antibodies in the blood of patients is a good choice for rapid, simple, and highly sensitive diagnosis of SARS-CoV-2. 18 Serologic tests could provide much-needed insight into the adaptive immune response against SARS-CoV-2, the exposure history of an individual, transmission patterns, and potential donors of convalescent plasma. 19 Therefore, we also study the dynamic variance of viral antibodies during SARS-CoV-2 infection. We found that the IgG seroconversion was earlier than that of IgM and this is similar to Long et al. 20 On the contrary, the antibody levels increased rapidly during the first two weeks. Studies have found that IgM antibodies appear about 2 weeks after infection, while IgG antibodies last for months or even years. 21 Another study showed that the IgM antibody appeared within 1 week after SARS-CoV-2 infection, and this antibody was present in the body for 1 month or even longer, the IgG antibody is usually produced in about 10 days. 12 antibody response which is crucial for the clearance of the initial virus infection has been widely used to help diagnose virus infection. 22 The IgM and IgG could be used to understand the epidemiology of SARS-CoV-2 infection and to help to determine the level of humoral immunity in patients. 23 

We developed a rapid SARS-CoV-2 IgG/IgM antibody test using magnetic chemiluminescence enzyme immunoassay technology. 

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Rapid immunoassay and clinical evaluation of the SARS-CoV-2 antibody assay on the real express-6 analyzer

The authors declare that there are no conflict of interests.

Ting Cai designed the study, Zhang Shun analyzed the data and