作者：Andrea Padoan, Chiara Cosma, Laura Sciacovelli, Diego Faggian and Mario Plebani*
 
摘要
 
背景：2019新型冠状病毒疾病，简称COVID-19，是一种迅速传播并威胁全球健康的新型传染病；该疾病的临床表现症状从轻微到严重的急性呼吸窘迫综合征（ARDS）不等。此外，还有相当一部分无症状感染者，这增加了诊断工作的不确定性。实验室检测在COVID-19的诊断和治疗中起着关键作用，目前COVID-19诊断的金标准是通过实时逆转录聚合酶链反应（rRT-PCR）的方法对患者的呼吸道标本中的病毒核酸进行检测。但rRT-PCR的诊断准确性受许多分析前、分析中因素的影响。特异性COVID-19抗体（IgG和IgM）的检测可作为疾病检测和治疗中的一个补充的、非侵入性的方法。
Background: Coronavirus disease 2019, abbreviated to COVID-19, represents an emerging health threat world- wide as, it has continued to spread rapidly. The clinical spectrum of the disease varies from mild to severe acute respiratory distress syndrome (ARDS). Moreover, many patients can be asymptomatic, thus increasing the uncertainty of the diagnostic work-up. Laboratory tests play a pivotal role in the diagnosis and management of COVID-19, the current gold standard being real-time reverse transcription polymerase chain reaction (rRT-PCR) on respiratory tract specimens. However, the diagnostic accuracy of rRT-PCR depends on many pre- analytical and analytical variables. The measurement of specific COVID-19 antibodies (both IgG and IgM) should serve as an additional, non-invasive tool for disease detection and management.
 
方法：参考临床和实验室标准化协会（CLSI）EP15-A3方案评估深圳市新产业生物医学工程股份有限公司MAGLUMITM 2000Plus仪器对于检测 2019 新型冠状病毒抗体（IgM和IgG）的不精密度。线性验证与回收率试验通过不同比例地混合高、低浓度血清样本来评估。通过连续收集COVID-19阳性患者不同时段（从
Methods: The imprecision of the MAGLUMITM2000 Plus 2019-nCov IgM and IgG assays (Snibe, Shenzhen, China) was assessed by adopting the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 protocol. Linearity of dilution and recovery was evaluated by means of mixes of high-level pools and low-level pools of serum samples. Immunoglobulin time kinetics were evaluated using a series of serum samples, repeatedly collected from COVID-19-positive patients at different times, from
 
结果：根据临床和实验室标准化协会（CLSI）EP15-A3指南要求进行的分析验证试验结果表明，新冠病毒IgM和IgG抗体检测试剂的不精密度和重复性均在可接受范围内（IgM和IgG的重复性分别为
Results: Findings at the analytical validation of the assay carried out according to the CLSI EP15-A3 guideline demonstrated that imprecision and repeatability were acceptable (repeatability was
 
结论：本研究结果证明了MAGLUMI 2000Plus化学发光免疫分析仪对COVID-19患者血清中特异性IgM和IgG的检测是可靠有效的，并得到COVID-19抗体（IgM和IgG）动力学的有价值的数据。这些数据提示在COVID-19感染患者的诊断和治疗中，合理利用特异性抗体的检测是必须的。
 
Conclusions: The findings of this study demonstrate the validity of the MAGLUMI 2000 Plus CLIA assay for the measurement of specific IgM and IgG in sera of COVID-19 patients, and for obtaining valuable data on the kinetics of both (IgM and IgG) COVID-19 antibodies. These data represent a pre-requisite for the appropriate utilization of specific antibodies for the diagnosis and management of COVID-19 patients.
 
关键词
 
 
性能分析，抗体动力学，COVID-19，COVID IgG 和IgM抗体，实时逆转录聚合酶链式反应，SARS-CoV-2。
 
analytical performances; antibody kinetics; COVID-19; COVID IgG and IgM; rRT-PCR; SARS-CoV-2.
 
简介
 
2019新型冠状病毒病，简称COVID-19，是一种传播迅速的威胁全球健康的新型疾病，世界卫生组织（WHO）的总干事于2020年3月11日将COVID-19的传播定义为广泛流行[1]。引起该疾病的病原体属于冠状病毒科，由于其与引起2003年SARS爆发的同源病毒（即SARS-CoV-1）序列具有高度同源性（达80%），最终被命名为“严重急性呼吸综合征冠状病毒2”（SARS-CoV-2）[2]。
 
Coronavirus disease 2019, abbreviated to COVID-19, is an emerging health threat and, on March 11, 2020, the Director- General of the World Health Organization (WHO) defined the spread of COVID-19 as a pandemic[1]. The responsible pathogen, a virus belonging to the Coronaviridae family, has been finally defined as “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2) for its high sequence identity (i.e. up to 80%) with the homologous virus that caused the 2003 SARS outbreak (i.e. SARS-CoV-1) [2].
 
在中国首次报告该病爆发后，COVID-19已经在全世界范围内传播，几乎在全球所有国家都发现了病例[3]。在意大利，在第一位COVID-19检测呈阳性的患者进入科多格诺医院（Lodi，Lombar-dia）的重症监护室（ICU）的14天内，周边地区陆续诊断出大量的COVID-19病例，其中包括相当比例的危重病人。同时在威尼托地区发现了第二个聚集性疫情，此后，COVID-19患者的数量迅速增加，主要集中在意大利北部，但意大利所有地区都有感染病例的报道[4]。COVID-19感染后的临床表现从轻度到重度急性呼吸窘迫综合征（ARDS）不等。此外，也有很多表现为无症状感染者，这增加了诊断工作的不确定性[5]。
 
及时准确地诊断COVID-19感染是对患者进行恰当治疗的基础，对于限制病毒进一步传播也至关重要，特别是可以有效限制无症状或轻度症状的感染者引起的病毒传播[6]；因此，鉴别疑似病例对应对COVID-19的爆发起着至关重要的作用。目前SARS-CoV-2感染病原学诊断的金标准是呼吸道标本的实时逆转录聚合酶链式反应（rRT-PCR）[7–9]。然而，rRT-PCR检测的质量对于提供准确且可解释的结果至关重要，诊断准确性受多个因素影响，包括分析前因素，如样品类型、采集、运输和储存条件，以及采用的PCR分析仪的质量和、一致性等，这些因素的控制对于实验室提供准确且可解释的结果至关重要[10]；同时，收集鼻咽部的咽拭子样本是一种侵入性的操作，会引起患者咳嗽和打喷嚏等情况，因此产生的气溶胶对医护人员构成潜在的生物安全威胁[11]。
 
特异性抗体的产生，特别是抗SARS-CoV-2的IgM和IgG的产生，应作为一种诊断疾病的非侵入性的补充方法来检测，尤其是针对病毒载量较低但出现症状的患者。然而，血清学检测的时间点和抗体检测结果的解读是其对于COVID-19检测有效的先决条件。因此，本研究的目的在于报道通过一种全自动检测平台，通过化学发光免疫分析方法（CLIA）检测新型冠状病毒抗体的性能验证，并描述COVID-19患者IgM和IgG抗体产生的动力学特征。
After initial reports of disease outbreak in China, COVID-19 has spread worldwide, cases being identified in virtually all countries worldwide [3]. In Italy, after the first patient tested positive on admission to the intensive care unit (ICU) in Codogno Hospital (Lodi, Lombardia), within 14 days, numerous other cases of COVID-19, including a substantial proportion of critically ill patients, were diagnosed in the surrounding area. A second cluster was simultaneously identified in the Veneto area and, since then, the number of COVID-19 patients has rapidly increased, mainly in Northern Italy, but all regions of the country have reported having patients being infected [4]. The clinical spectrum of SARS-CoV-2 infection can vary from mild up to onset of severe acute respiratory distress syndrome (ARDS). Moreover, many patients can be asymptomatic, thus increasing the uncertainty of the diagnostic work-up [5]. The timely and accurate diagnosis of COVID-19 infection is the cornerstone of appropriate treatment for patients, and crucial for limiting further spread of the virus, particularly as asymptomatic or mildly symptomatic subjects may be responsible for virus transmission [6]. Therefore, testing assumes critical relevance for ensuring an effective response to COVID-19 outbreak. The current gold standard for the etiological diagnosis of SARS-CoV-2 infection is (real-time) reverse transcription polymerase chain reaction (rRT-PCR) on respiratory tract specimens[7–9]. However, the quality of rRT-PCR testing remains of paramount importance in providing accurate and interpretable results, its diagnostic accuracy depending on many factors, including pre-analytical variables such as sample types and collection, transportation and storage conditions, as well as the quality and consistency of the PCR assays being used [10]. The collection of nasopharyngeal or throat swab specimens, a relatively invasive and almost uncomfortable procedure, can cause coughing and sneezing, thus generating aerosol, which constitutes a potential health hazard for healthcare workers [11]. The production of specific antibodies, particularly anti-SARS-CoV-2 IgM and IgG, should be used as an additional non-invasive method for detecting the disease, especially in patients who present late, with a low viral load. However, the timing of requests for serological assays and the interpretation of antibody results are pre-requisites of crucial importance in their efficacy. Therefore, the aim of this paper is to report an analytical validation of a novel chemiluminescent immunoassay (CLIA), available on an automatic platform, and to describe the kinetics of IgM and IgG antibodies in COVID-19 patients.
 
材料和方法
 
仪器：高通量（180测试/小时）Maglumi 2000Plus全自动化学发光免疫分析仪（深圳市新产业生物医学工程股份有限公司，中国）
 
试剂：
2019-nCoV IgM（cut off值为1.0 AU/mL)，灵敏度78.65%，特异性97.5%。
 
2019-nCoV IgG（cut off值为1.1 AU/mL)，灵敏度91.21%，特异性97.3%。
 
The MAGLUMI™ 2000 Plus (New Industries Biomedical Engineering Co., Ltd [Snibe], Shenzhen, China) is a chemiluminescent analytical system (CLIA), featuring high throughput (up to 180 tests/h). According to the manufacturer’s inserts (271 2019-nCoV IgM, V2.0, 2020-03 and 272 2019-nCoV IgG, V1.2, 2020-02), the 2019-nCoV IgM cut-off is 1.0 AU/mL, while the 2019-nCoV IgG cut-off is 1.1 AU/mL. Manufacturers claimed that the calculated clinical sensitivities of IgM and IgG were 78.65% and 91.21%, respectively, while specificities of IgM and IgG were 97.50% and 97.3%, respectively.
 
分离胶管干扰评估
 
参照熊彦等的文章[12]，进行病毒抗体检测之前，可以将样本56℃加热30min灭活。我们进行了对于29例样本的系列比较试验，通过对直接对分离胶采血管进行加热和分离出血清加热进行比较，验证是否会存在结果干扰。我们特别对于每一个带分离胶的原始管在病毒灭活之前都进行等量分离血清，随后将分离出的血清与分离胶管同时加热，之后再进行IgM和IgG结果的检测和比较。
According to the procedure recommended by Xiongyan et al. [12], viral activity could be inactivated before antibody determination by heating serum samples to 56 °C for 30 min. To ascertain whether 56 °C dry heat in the primary sample tube containing separator gel caused analytical interferences with respect to heated secondary aliquoted serum, we undertook an experimental series of comparisons in a total of 29 serum samples. In particular, for each primary sample tube with separator gel, an aliquot was prepared before viral inactivation, after which the primary sample tubes and the aliquots were heated together, and IgM and IgG results compared.
 
重复性精密度和中间精密度
 
通过3个不同浓度的人血清样本评估精密度。参照美国临床实验室标准化协会CLSI/EP15-A3要求[13]，每个相同样本等分五份，连续五天进行检测。厂家提供的精密度数据是通过3个血清样本，参照EP5-A3要求[14]进行重复性精密度和中间精密度的验证。精密度的结果与厂家说明书进行比较。精密度的确定符合计量学联合委员会VIM中对5天精密度验证的要求。
 
Precision was evaluated by using three human serum pools of samples with different values. Precision estimations were obtained by means of quintuplicate measurements of aliquots of the same pool, performed for a total of 5 consecutive days, following the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 protocol [13]. The precision data claimed by the manufacturer were verified using three human serum pools, and specifications were estimated with the EP5-A3 protocol [14], by considering repeatability, and between-day variability.The results obtained for precision were compared to those claimed by the manufacturer using the procedure recommended by EP15-A3. Precision estimates were in accordance with the repeatability and intermediate precision conditions specified in the international vocabulary of metrology (VIM, JCGM 100:2012) for precision estimation within a 5-day period.
 
准确度（回收率）
 
使用已知浓度的高值血清样本（其中IgM的为2.18AU/mL，IgG的为2.57AU/mL），混合低值血清（其中IgM的为0.27AU/mL，IgG的为0.093AU/mL）按比例稀释，以得到不同的理论浓度。取稀释后的系列样品，测定浓度。然后计算其平均实测浓度与期望浓度的回收率。
 
回收率（%）＝（测定浓度值-理论浓度值）/理论浓度值×100％

Recovery was assessed using one pool of samples for IgM (2.18 AU/mL) and another pool for IgG (2.57 AU/mL), prepared using human serum samples. These pools were mixed with different amounts of low-level pools of samples (0.27 AU/mL for IgM and 0.093 AU/mL for IgG) in order to obtain different theoretical concentrations of IgM and IgG. Recovery was estimated according to the following formula:
 
线性
 
线性验证参考CLSI/EP06-A第三版第4.3.1节要求，使用4个高值血清样本，用低值血清进行连续稀释。其中IgM的两个高值样本为21.4AU/mL，2.27AU/mL，都用0.33AU/mL的样本进行连续稀释。IgG的一个高值样本为73.72AU/mL，用0.086AU/mL的样本进行连续稀释；IgG的另一个高值样本为2.65AU/mL，用0.107AU/mL的样本进行连续稀释。所有稀释后的样本重复检测三次。
Linearity was assessed by using a series of mixes of four sample pools, prepared with different IgM and IgG values, using serial dilution, as specified in the CLSI EP06 A: 2003 guideline (paragraph 4.3.1). In brief, two serum pools with a measured IgM antibody Linearity was assessed by using a series of mixes of four sample pools, prepared with different IgM and IgG values, using serial dilution, as specified in the CLSI EP06 A: 2003 guideline (paragraph 4.3.1). In brief, two serum pools with a measured IgM antibody value of 21.4 AU/mL and 2.27 AU/mL (high-level pools) were serially diluted with a low IgM antibody value serum pool (0.33 AU/mL). Likewise, two serum pools with a measured IgG antibody value of 73.72 AU/mL and 2.65 AU/mL (high-level pools) were serially diluted with two low IgG antibody value serum pools (0.086 AU/mL and 0.107 AU/mL, respectively). All measurements were performed in triplicate.
 
IgM和IgG抗体时间动力学评价
 
匿名收集37例在3月18日-3月26日期间住院的新冠病毒阳性（通过rRT-PCR检测鼻咽拭子样本确诊）的患者的实验室常规检测之后的剩余血清样本，分装冻存在-80°C。检测时所有样本同批复融并加热灭活。样本通过MAGLUMI 2000Plus化学发光系统检测2019-nCov IgM和2019-nCov IgG。本研究一共通过37个病人收集了87个样本，其中10个患者各抽取了一次样本，6个患者各抽取了两次样本，19个患者各抽取了三次样本，每患者各抽取了4次样本。
Through the time period between March 18 and March 26, from hospital wards with hospitalized COVID-19-positive (confirmed by positive rRT-PCR using nasopharyngeal swab samples) patients, a series of residual serum samples from routine laboratory testing were anonymized, aliquoted and stored at −80 °C. For the analyses, all samples were thawed and heat inactivated (see above) in batch. Samples were then evaluated using the MAGLUMI 2019-nCov IgM and 2019-nCov IgG (CLIA) systems, during the same analytical session. A total of 87 samples were collected from the 37 patients included in the study (one sample from each of 10 patients; two from each of six patients; three from each of 19 patients; four from each of two patients).
 
数据统计
 
使用一致性评价法分析分离胶管采血进行灭活是否干扰结果。使用方差分析法评估重复性、精密度和中间精密度。参照EP5-A3要求使用内部开发的R软件（统计计算基金会，维也纳，奥地利）进行方差分析和计算验证上限。对于时间动力学评估，使用以下策略：对于研究中包含的每个样品，从患者出现新冠临床症状（即发热）开始收集样本并进行抗体测定。定义了以下时间范围（d代表天）：
The possibility to obtain viral activity inactivation in samples collected into serum blood tubes with a gel separator was assessed using Bland-Altman analyses. For evaluation of precision, ANOVA was used to estimate repeatability and intermediate precision. An in-house developed R (R Foundation for Statistical Computing, Vienna, Austria) script for implementing the CLSI EP15-A3 protocol was used for ANOVA and for calculating the upper verification limit. For time kinetic evaluation, the following strategy was used: for each sample included in the study, the collection date was matched with the corresponding date of symptom onset (i.e. fever) and antibodies measured. Using these data, the following time frames were defined (d, days):
 
结果
 
分离胶管干扰评估
 
用Bland-Altman一致性评价方法分析数据，结果显示直接加热灭活分离胶采血管后检测与分离出血清样本加热灭活后检测的IgM 和 IgG的结果相似（IgM 和IgG的p=0.122和0.548）。（补充图1）
Bland-Altman analyses findings demonstrated that the use of a heated primary tube with a gel separator and heated aliquoted serum generated comparable IgM and IgG results (p = 0.122 and p = 0.548, respectively) (Supplementary Figure 1).
 
重复性和中间精密度
 
表1结果显示，参照CLSI/EP15-A3[13]要求检测所得结果与厂家声明的重复性精密度和中间精密度结果（参照CLSI/EP5-A3[14]要求，用三个浓度水平的样本，每天重复测量3次，连续检测5天）检测比较。结果提示低值样本和中值样本的精密度结果比厂家声明的低，符合要求。高值样本的精密度结果比厂家声明的高，参照CLSI/EP15-A3[13]要求进行偏倚计算之后，还是不符合要求。
Table 1 reports repeatability and intermediate precision, calculated using 5-day analysis according to the procedure suggested in CLSI EP-15-A3 [13], compared with the precision value claimed by the manufacturer (obtained using three samples at different concentrations measured in duplicate at three sites on 5 days, with three runs per day, according to the EP5-A3 protocol) [14]. The results obtained for the low and medium values were satisfactory, being lower than those reported by the manufacturer, while at the highest concentration they did not correspond with the manufacturer’s specifications, also after UVL calculation, conducted as suggested by CLSI EP15-A3 [13].
 
表1：精密度结果

a参照CLSI/EP5-A3要求检测所得结果 b试剂盒说明书里的精密度厂家声明值。
c样本的精密度验证值比厂家声明的高，参照CLSI/EP15-A3要求进行偏倚计算之后，还是不符合要求。
 
准确度（回收率）
 
由于缺乏充分验证过的对比试剂，所以用回收率实验来评估该方法的系统误差。检测一系列浓度的样品（包含临界值）的回收率后，结果显示（见表2），IgM的样本回收率明显较高，在103%到123%之间。IgG高值样本的回收率略高，在103%到112%之间；IgG低值样本的回收率偏低，在63%到93%之间。
As a well-validated method was not available for comparison purposes, the recovery study was implemented to estimate possible proportional systematic error of the method. Recovery was calculated in ranges of values covering IgM and IgG cut-offs. Our findings, as shown in Table 2, highlight overestimation for IgM (value range, 103%–123%), overestimation (103%–112%) for higher values and underestimation (63%–93%) for lower values in the case of IgG.
 
表2：回收率结果
线性
 
用Maglumi 2000Plus化学发光免疫分析仪测得的IgM 和 IgG的线性数据结果如图1。除了明显高于临界值的样本检测结果偏离线性，其余所有的样本检测结果都符合线性要求。
Linearity data for IgM and IgG antibody MAGLUMI™ 2000 Plus CLIA are summarized in Figure 1. All tested mixes of sample pools deviated from linearity only at levels significantly higher than the IgM and IgG cut-off.


图1  IgM和IgG线性结果
 
（A）IgM高值样本21.4AU/mL用0.33AU/mL的样本进行连续稀释
（B）IgM高值样本2.27AU/mL用0.33AU/mL的样本进行连续稀释
（C）IgG高值样本73.72AU/mL用0.086AU/mL的样本进行连续稀释
（D）IgG高值样本2.65AU/mL用0.107AU/mL的样本进行连续稀释
 
每个样本测定三次计算平均值。
 
时间动力学
 
以患者感染后开始发热的时间作为起点，采集样本检测IgM和IgG抗体，将感染后间隔相同时间开始发热（即出现临床症状）的样本检测结果计算均值，研究IgM和IgG抗体产生的时间动力学特征，结果如图2所示。研究发现，感染后的第11天，所有患者均能被检测出IgG抗体阳性；感染后的第13天，只有88%的患者被检测出IgM抗体阳性。结果如表3所示。
Figure 2 shows the kinetic results for the study patients at different days from fever onset, divided into time categories. Specifically, the graph shows average values and corresponding standard errors of IgM and IgG for each time category. Overlapping time kinetic trends are shown using
 
spline interpolation. Table 3 shows the number (and percentage) of positive test results of IgM and IgG for each time category. After the 11th day, all patients were found to be positive for IgG (100%), while the higher positivity of IgM (88%) was achieved only after the 13th day.


图2  IgM和IgG抗体的时间动力学（37名患者）
 
表3 不同时间检测患者2019-nCoV IgM和2019-nCoV IgG的阳性率统计
 

 
讨论
 
COVID-19的迅速传播对全球所有国家的医疗系统来说都是一个重大挑战。尽管感染的严重程度可能从轻度到重度不等，但是相当多的患者需要提供呼吸机支持的亚重症和重症监护，这造成了真正的医疗资源紧张。此外，医护人员感染新冠病毒的比例日益上升。在世界卫生组织-中国冠状病毒联合专家考察报告中表明，截至2020年4月3日，在新冠肺炎患者总数112,401名和死亡病例13,241例（死亡率11.7%）的情况下，已有11,251名医护人员感染过新冠病毒。
The rapid spread of COVID-19 represents a major challenge for all national healthcare systems worldwide. Although the degree of infection severity may vary from mild to severe, a considerable percentage of diseased patients need sub-intensive and intensive care with respiratory support, thus causing a real healthcare emergency [15]. Moreover, an increasingly serious issue is the frequency of COVID-19 infection in healthcare workers. In a report from the WHO-China Joint Mission on COVID-19, as many as 11,251 healthcare workers had become infected with COVID-19 by April 3, 2020, with a total of 112,401 cases of COVID-19 and 13,241 associated (11.7%) deaths [16].
 
因此，不仅是医生，还有研究人员、政府决策人员，以及所有国家医疗保健系统的管理者，都将目光持续聚焦在新冠肺炎疾病的诊断检测的讨论上，即使其中只有极少数已经开发的新冠病毒检测试剂被广泛用于临床，因为这个过程相当耗时。
Therefore, not only physicians, but also scientists, policymakers and administrators of all national healthcare systems, are focusing on the ongoing discussion on diagnostic tests for COVID-19 disease, even if only a few of those already developed have been extensively validated for clinical use, because this process is inherently timeconsuming[17].
 
目前新冠肺炎的诊断方法包括通过rRT-PCR在呼吸道样本中鉴定病毒RNA，尽管最近很多报道阐述了该技术分析前和分析中的局限性。首先，rRT-PCR检测方法的敏感性不仅取决于疾病的发展阶段（如样本采集时间），还取决于病情的严重程度。其次，RNA检测的通量有限，因为它不仅工作量大，还需要有熟练的操作人员进行样品制备和检测，同时，RNA检测仪器昂贵，生物安全防护要求高。因此，血清学抗体检测是一种更易操作的，成本更低的新冠病毒检测方法，不仅可用于新冠肺炎的诊断，还可以提示疾病进程以及用于流行病学和疫苗评价。
The current diagnostic method involves the identification of viral RNA in respiratory samples by means of rRT-PCR, though several pre-analytical and analytical limitations have recently been described to plague this technique [18]. First, it was demonstrated that the sensitivity of this test not only depends on the stage of disease (i.e. collection time), but also on the severity [19]. The overall throughput of RNA tests is also limited because it requires high workload, skillful operators for sample preparation and testing, and also requires expensive instrumentation and important biosafety measures. Therefore, less expensive and easy implementable serological tests are needed for detecting 2019-nCov antibodies, not only for diagnosing COVID-19, but also for characterizing the course of disease, as well as for epidemiological and vaccine evaluation studies.
 
因此，我们在MAGLUMI™2000 Plus化学发光免疫分析仪上评估了2019-nCoV IgM和IgG化学发光免疫分析试剂盒的分析性能以及新冠肺炎患者体内抗体出现的时间动力学特征。我们研究的第一个方面是使用带分离胶的血清试管作为原始样本直接进行病毒灭活，而无需制备等量的次级样本。该策略对于操作员安全以及有效的工作量管理极为重要。我们使用分离胶管对29份血清样品进行IgM和IgG检测，结果证实分离胶管适合用于病毒干燥加热灭活后的抗体检测。随后，我们利用EP15-A3文件方案评估了该分析方法的不精密度。
We have hence carried out a study to investigate the analytical performance of the MAGLUMI™ 2000 Plus 2019-nCoV IgM and IgG chemiluminescence immunoassay and the kinetics of appearance of antibodies in COVID-19 patients. The first aspect we investigated was the possibility of using serum tubes with a gel separator as primary samples for direct viral inactivation, without preparing secondary aliquots. This strategy is extremely important for operator safety as well as for effective workload management. The analyses performed on a series of 29 consecutive serum samples with a gel separator confirmed that this type of sampling tube could be suitably used for IgM and IgG measurement after dry heat for viral inactivation. Then, we assessed the imprecision profile of the assay using the CLSI EP15-A3 protocol [12].
 
总体而言，我们的结果表明MAGLUMI™2000 Plus具有很好的检测精密度。IgM和IgG的重复性精密度分别为
Overall, our results show that MAGLUMI™ 2000 Plus has excellent precision characteristics. In fact, repeatability was
 
为了评价该方法线性范围内检测结果是否与IgM和IgG浓度理论值呈线性关系，从而保证线性范围内检测结果的准确性，我们评估了样本的检测线性。我们使用低值样本和高值样本进行了一系列的连续稀释，制备了不同浓度的样本。结果显示，IgM和IgG浓度在0.5 AU / m L–1.5 AU / m L范围内，检测值与理论值呈现良好的正比例关系，但是在高值样本中，线性关系变差，尤其是对于IgG。对于结果的比较，由于缺乏充分验证过的对比试剂，所以用回收率实验来评估准确度。检测范围覆盖了厂家建议的范围，结果表明IgM略被高估，而IgG在高于1.9AU/mL时被高估，在低于1.9AU/mL时被低估，总体而言，IgM回收率更好，因此，建议同时检测IgM和IgG。
Linearity of dilutions was also assessed, in order to evaluate the ability of the method to provide results directly proportional to the concentration of IgM and IgG in tested samples. We performed a series of serial dilutions using high-value pools diluted in low-value pools. The results obtained showed that within the 1.5 AU/mL–0.5 AU/mL range results are linear for both immunoglobulins, whilst linearity seems worse at the highest values, especially for IgG. As a well-validated method for comparing results is currently unavailable, we also performed recovery studies. The range inspected covered the range of values suggested by the manufacturer, and results showed that IgM was slightly overestimated, whereas IgG was overestimated for values above 1.9 AU/mL and slightly underestimated at values below 1.9 AU/mL. Overall, better recovery results were found for IgM, so simultaneous assessment of both IgM and IgG may be advisable.
 
我们在先前建议的时间间隔内还评估了IgM和IgG抗体产生的时间动力学，结果显示在出现发热症状后，IgM和IgG在短时间内均迅速升高。参考到厂家建议的阳性截断值（IgM为1.0 AU / mL，IgG为1.1 AU / mL），可以认为在发热症状出现后6-7天，IgM和IgG会有显著升高。这些发现与近期的一些其他报道一致。例如，有研究人员使用内部研发的ELISA方法，发现在症状发作5天后，几乎所有患者中都可以检测到抗病毒抗体的显著升高，而这一时期通常被认为是感染早期到晚期的过渡。同样，还有研究者使用丽珠诊断的ELISA抗体检测试剂盒，发现在症状发作后7天IgM和IgG显着增加，特别是在重症患者中。表3详细描述了IgM和IgG抗体产生的动力学特征，结果显示IgG需要至少在感染12天后才能达到100％的敏感性，而在整个研究周期间，IgM的最高阳性率为88％。有趣的是，有三名确诊患者的IgM值分别为0.811 AU / mL，0.909 AU / mL和0.863 AU / mL，均低于厂家声称的截断值。这表明，通过进一步优化IgM的cut-off值，对于提高IgM检测的敏感性是很有必要的。
The time kinetics of IgM and IgG was also evaluated during a time interval previously recommended [19]. Our results showed that both IgM and IgG rapidly increased after the onset of fever. Considering the cut-offs suggested by the manufacturer (i.e. 1.0 AU/mL for IgM and 1.1 AU/mL for IgG), the immunoglobulin rise could be considered as significant 6–7 days after fever onset. These findings are in agreement with those recently reported by others. For example, using an ELISA in-house developed method, Zhang et al. found that the increase of antibody against the virus was clearly visible in almost all patients after 5 days of symptom onset, a time period that was usually considered as a transition from an early to a late period of infection [19]. Likewise, using a commercial ELISA kit from Livzon Diagnostics (China), Tan et al. found a marked increase of immunoglobulins 7 days after the onset of symptoms, particularly in patients with severe disease [20]. Table 3 reports in detail the kinetics of IgM and IgG, showing that IgG requires at least 12 days to attain 100% sensitivity, whilst the highest positive rate achieved for IgM was 88% throughout the study period. Interestingly, three patients had IgM values of 0.811 AU/mL, 0.909 AU/mL and 0.863 AU/mL, thus remaining below the cut-off. This suggests that further cut-off refinement would be necessary for increasing IgM sensitivity.
 
本研究有一些明显的局限性。例如，没有可靠的经过验证的方法可用于对照试验，也没有评估检测方法的交叉反应性。此外，评估IgM和IgG抗体时间动力学是以发热症状出现作为标准。我们使用此症状的原因是：
 
（a）适用于我们研究中的所有患者
（b）通常能够由患者和医生准确记录；
（c）已在许多其他研究中使用。例如，某研究者估计的新冠病毒感染患者发烧的潜伏期中位数为5-7天（95％置信区间：4.9–6.8天）。
 
另一个方面，可能需要更长的周期来评估IgM和IgG产生的动力学，以便评估新冠病毒感染的体液免疫反应的整个趋势。
The present study has some notable limitations. For example, no reliably validated method was available for comparison studies, nor were cross-reactivities of the assays tested. Furthermore, the criterion for assessing the time kinetics of IgM and IgG antibodies was the time of fever onset. We used this symptom because: (a) was available for all patients included in our study; (b) is usually accurately recorded by both patients and physicians; (c) has also been used in many other studies. For example, Lauer et al. estimated that the median incubation period to fever onset was 5.7 days (95% CI: 4.9–6.8 days) for COVID-19 patients [5]. Another aspect could be that IgM and IgG kinetics shall be assessed over a longer period in order to estimate the entire trend of humoral immune response to COVID-19 infection.
 
总之，这项研究的结果表明MAGLUMI™2000 Plus 化学发光免疫分析系统是评估新冠病毒肺炎患者免疫反应的可靠的免疫测定方法。我们的结果还证实，同时测量IgM和IgG会对新冠病毒肺炎患者诊断有所帮助，尤其是在早期感染的时候。
In conclusion, the findings of this study show that MAGLUMI™ 2000 Plus CLIA may be a reliable immunoassay for assessing the immunological response in sera of COVID-19 patients. Our results also confirm that simultaneous measurement of IgM and IgG can be helpful, especially from the early phase of infection.