key: cord-0872715-llxhmfsw
authors: Dou, Xiaowen; Wang, Enyun; Hu, Jiwen; Zong, Zengyan; Jiang, Ruiwei; Wang, Mengmeng; Kan, Lijuan; Zhang, Xiuming
title: Comparison of three automatic chemiluminescent immunoassays for monitoring dynamic profile of SARS‐CoV‐2 IgG and IgM
date: 2020-12-19
journal: J Clin Lab Anal
DOI: 10.1002/jcla.23681
sha: 7f75dd9cab1a2607fe8aa75fb8961bfd29a5b508
doc_id: 872715
cord_uid: llxhmfsw

BACKGROUND: Seldom performance evaluation and diagnosis comparison studies were reported for different chemiluminescent immunoassay (CLIA) kits approved under an emergency approval program for SARS‐CoV‐2 infection. METHODS: A total of 100 and 105 serum separately from non‐infected populations and COVID‐19 patients were detected with SARS‐CoV‐2 IgM and IgG kits. The characteristics including precision, functional sensitivity, linearity, and accuracy were evaluated for Axceed, iFlash, and Maglumi CLIA kits. RESULTS: Maglumi and iFlash had the best analytical sensitivity for IgM and IgG, respectively. Axceed kits had a linearity response in the detected concentration. The clinical sensitivity of Axceed, iFlash, and Maglumi was 68.0%, 64.9%, and 63.9% with a specificity of 99.0%, 96.0%, and 100% for IgM, 85.6%, 97.9%, and 94.8% with a specificity of 97.0% for IgG. ROC analysis indicated all kits had a diagnostic power greater than 0.9. Notably, either IgM or IgG kits obtained a poor agreement (Kappa value from 0.397 to 0.713) with others. Among 38 recovered patients, 94.7% had an effective immune response, and both seropositive IgM and IgG accounted for the biggest proportion (medium, 42 days onset), then followed by the single seropositive IgG (medium, 50 days onset) in Ab profile. CONCLUSION: The performance of CLIA kits satisfied the diagnosis of SARS‐CoV‐2 infection. Both positive of IgG and IgM contributes to improve the specificity, and a positive one will enhance the sensitivity.

A spread of SARS-CoV-2 virus infection occurred in the whole world.

The novel coronavirus (COVID- 19) pandemic brings a severe threat to human health. As of August 27, there are now more than 24 million reported cases of COVID-19 and 820,000 deaths. 1 Symptoms such as fever, cough, sore throat, body aches, headaches, and other acute respiratory syndromes commonly appeared in most of COVID-19 patients. However, it is worth noting that a grow number of patients were found to be asymptomatic or false-negative results in nucleic acid tests, thus increasing the difficulty of patient diagnosis and the risk of virus transmission. 2 Laboratory tests play a pivotal role in the diagnosis and management of COVID-19. To response the serious COVID-19 pandemic in United States and improve diagnostic efficiency for SARS-CoV-2 infection, a study on protocol to validate the sufficiency of home-collected samples to detection of SARS-CoV-2 RNA and antibodies was reported. Although the nucleic acid testing is the gold standard for COVID-19 diagnosis, the accuracy is generally restricted by specimen collection, transportation, and RNA extracted operation.

Besides, the method was proved to be time-consuming, labor-intensive, and at high risk of infection. 3, 4 The SARS-CoV-2 infected patients were confirmed by a positive nucleic acid result, but a negative result cannot absolutely exclude the infection possibility because of 50% positive rate found in nucleic acid testing. 5 Less expensive and easy implementable serological tests are urgently needed for an accurate diagnosis of SARS-CoV-2 infection, contact tracing, and epidemiological studies. Excitedly, the immune system produces virus-specific antibody, including immunoglobulin M (IgM) and immunoglobulin G (IgG) in response to COVID-19.

Serological antibody detection emerging as an auxiliary diagnostic method for COVID-19 has attracted extensive concerns. 6 The assay of SARS-CoV-2 IgM and IgG should be applied as an additional non-invasive method for the infection. It was reported that the positive virus-specific IgG was 100% within three weeks and IgM reached 94.1% within 20-22 days after symptom onset. 7 The profile of IgM and IgG can not only make an assisted diagnosis, but also characterize the disease course, investigate epidemiologic features, as well as accelerate vaccine development. [8] [9] [10] Except the diagnostic efficacy of IgM and IgG has been proved in SARS-CoV-2 infection, the performance of commercial immunoassays was compared aiming at a better choice for a detection tool. 11, 12 The diagnostic sensitivity, specificity, and concordance of SARS-CoV-2 assay between Abbott and Euroimmun was compared, the former had fewer false-positive and false-negative results than the later. 13 Most of above previous researches focused on the clinical significance of different antibody kits for SARS-CoV-2 infection, the diagnostic characteristics just included sensitivity, specificity, and receiver operator curve (ROC) among various immunoassays.

Analytical performance involving testing process of SARS-CoV-2 antibody kits was closely related to the diagnostic accuracy. There were only several studies reported the analytical performance evaluation in serological antibody kits. Besides of the sensitivity and specificity, the performance containing imprecision, linearity of dilution, and accuracy were evaluated in the MAGLUMI ™ 2000

Plus 2019-nCov IgM and IgG assays (Snibe, Shenzhen, China). 14 To date, more than 10 antibody kits for SARS-CoV-2 infection have been approved by the National Medical Products Administration (NMPA). 15 The antibody was mainly detected by chemiluminescence, colloidal gold immunochromatographic, and enzyme-linked immunosorbent assay. A report showed variable performance values were acquired among six SARS-CoV-2 immunoassays containing Abbott Architec, Diasorin Liasison, Euroimmun, Acro, Xiamen Biotime, etc, in comparison with microneutralization. 16 

The patients were enrolled from hospital clinics, wards and quaran- 

Upper respiratory nasopharyngeal swabs were collected. Specimens acquisition, preservation, transportation, and detection were strictly in accordance with standard operating procedure. The system NP968-C (TianLong medtl., China) was used to extract RNA from PCR cell media and then detected with registered kits from BioGerm 

The analytical performance consisting of precision, functional sensitivity, and linearity was evaluated following CLSI protocol EP15-A2 and EP17-A 17,18 and NCCLS protocol EP12-A. 19 Considering repeatability and between-day variability for IgG and IgM detection, the precision was evaluated by using three serum pools at low (negative), medium (around the cutoff value) and high levels. Each level sample was measured four repetitions per day within a five-day period. The repeatability and within-laboratory precision were calculated and expressed as coefficient of variation (CV%). 

The imprecision of repeatability and within-laboratory precision tests for three IgG and IgM kits was summarized in Table S1 . The CV% was between 0.62% to 5.54% for IgG kits at three levels. The impression ranged from 0.70% to 5.58% for IgM kits, except Axceed kits obtained a high CV% of 12.97% and 16.82% at the low level (0.02 ± 0.00 S/CO). Most of the measured CV% was less than 6.0% or a manufacturer declaration.

The positive probabilistic curves for SARS-CoV-2 IgM and IgG kits were shown in Figure 1 . The highest dilution times produced 95%

positive results (C 95 ) reflecting the excellent detection limit. The 

In Figure S1 , the good linearity was obtained with Axceed kits in the concentration range of 0.06-25.9 S/CO (R 2 = 0.9991) and 0.07-11.37 S/CO (R 2 = 0.9854) for IgM and IgG antibody, respectively. The iFlash IgM kit was obtained a linear concentration between 4.87 AU/mL and 258.72 AU/mL, while a non-linearity was observed for iFlash IgG kit ranging from 6.75 AU/mL to 134.2 AU/mL. Both of Maglumi IgG and IgM kits exhibited a non-linear trend in the investigated concentration. 

The serological antibodies profile in confirmed COVID-19 patients detected with three CLIA kits and RNA testing was listed in Table 1 . The χ 2 test attested that there were no remarkable dif- The concentration of serological antibodies in all included serum was dramatically scattered in two groups ( Figure 3A,B) . The U test indicated the mean concentration in patients was significantly different from that in non-infected group. Those tested serum consisted of two time periods after symptom onset, eight samples within two days and 97 serum from 20 days to 70 days. In Figure 3C ,D, the an- 

This study presents an analytical performance evaluation and diagnosis comparison for three COVID-19 chemiluminescent immunoassays for monitoring dynamic profile of SARS-CoV-2 IgG and

IgM on a panel of 105 samples from individuals with confirmed SARS-CoV-2. Two asymptomatic SARS-CoV-2-infected cases were F I G U R E 3 IgM and IgG antibodies response against SARS-CoV-2 from patients in comparison with that from control population (A and B) , a Mann-Whitney U test was used and P value below 0.05 was considered statistically significant. Kinetics of antibody responses against SARS-CoV-2 following infection from patients (C and D), the x-axis represents the days of ill onset and synchronous date of IgM and IgG detection, the y-axis shows the response value detected by Axceed detected by immunoassay and confirmed with RNA. In the early infection period (≤two days), viral RNA testing maintained a higher positive rate than SARS-CoV-2 IgG and IgM detection (25%~38%).

In agreement with previous publications, 9, 20 Safety operation with the infected serum also was important, and previous report showed the heat inactivation at 56°C significantly decreased the level of SARS-CoV-2 antibodies. 23 In the study, au- positive IgG by iFlash and Maglumi kits with high titers. However, the patient was found only a slight elevation of the amplification curve in repetitive RNA tests prior to confirmation. Six recovered patients maintained SARS-CoV-2 IgM and IgG negative status after discharge. The phenomenon was also reported in previous study. 24 It is worth noting that the IgM clearance was not always accompanied by the rising of IgG. 7 The study also had limitations. Due to lack of standard serological serum plates, the performance evaluation replied on a serial of home-made diluted serum, the real concentration remained unknown. The serum collected focused on early-infected and recovered patients, a whole seroconversion phase was restricted to be followed up.

In summary, thanks to automatic operation, the CLIA kits were valuable to rapidly and effectively screen the infected patients in con- 

The authors state that there are no financial, personal, or professional conflicts of interests that may hinder this work.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Zengyan Zong https://orcid.org/0000-0003-2332-4211

Xiuming Zhang https://orcid.org/0000-0002-7971-6444

World Health Organization. WHO Coronavirus Disease (COVID-19)

The Incubation Period of Coronavirus Disease 2019 (COVID-19) From Publicly Reported Confirmed Cases: Estimation and Application

SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Chest CT for Typical 2019-nCoV Pneumonia: relationship to Negative RT-PCR Testing

Challenges in Laboratory Diagnosis of the Novel Coronavirus SARS-CoV-2

Antibody responses to SARS-CoV-2 in patients with COVID-19

Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19)

Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019

Characteristics of patients with coronavirus disease (COVID-19) confirmed using an IgM-IgG antibody test

Performance characteristics of five immunoassays for SARS-CoV-2: a head-to-head benchmark comparison

Antibody tests in detecting SARS-CoV-2 infection: a meta-analysis

Clinical Performance of Two SARS-CoV-2 Serologic Assays

Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics

?table Id= 2 6 & t a b l e N a m e = T A B L E 2 6 & t i t l e = % E 5 % 9 B % B D % E 4 % B A % A 7 % E 5 % 9 9 % A 8 % E 6 % A 2 % B 0 & b c I d = 11810 30586 17027 08383 87067 01567 &CbSlD lH0 =qAksq a qSWs lSWsl SWw2U.yyWmp yLmvM ytJyf eXC2G aEqqkl

Performance of six SARS-CoV-2 immunoassays in comparison with microneutralisation

User verification of precision and estimation of bias: Approved guideline-Third edition

Protocols for determination of limits of detection and limits of quantita-tion. approved guideline-second edition

Protocol for Evaluation of Quantitate Test of Performance; Approved guideline

Virological assessment of hospitalized patients with COVID-2019

Serological chemiluminescence immunoassay for the diagnosis of SARS-CoV-2 infection

Analysis of COVID-19 convalescent plasma for SARS-CoV-2 IgG using two commercial immunoassays

Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2

Symptoms and immunoglobulin development in hospital staff exposed to a SARS-CoV-2 outbreak

Additional supporting information may be found online in the Supporting Information section. How to cite this article