重磅論文:化學發光法檢測新冠病毒抗體的性能及動力學研究

2021-01-10 騰訊網

作者:Andrea Padoan, Chiara Cosma, Laura Sciacovelli, Diego Faggian and Mario Plebani*

摘要

背景:2019新型冠狀病毒疾病,簡稱COVID-19,是一種迅速傳播並威脅全球健康的新型傳染病;該疾病的臨床表現症狀從輕微到嚴重的急性呼吸窘迫症候群(ARDS)不等。此外,還有相當一部分無症狀感染者,這增加了診斷工作的不確定性。實驗室檢測在COVID-19的診斷和治療中起著關鍵作用,目前COVID-19診斷的金標準是通過實時逆轉錄聚合酶鏈反應(rRT-PCR)的方法對患者的呼吸道標本中的病毒核酸進行檢測。但rRT-PCR的診斷準確性受許多分析前、分析中因素的影響。特異性COVID-19抗體(IgG和IgM)的檢測可作為疾病檢測和治療中的一個補充的、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:參考臨床和實驗室標準化協會(CLSI)EP15-A3方案評估深圳市新產業生物醫學工程股份有限公司MAGLUMITM2000Plus儀器對於檢測 2019 新型冠狀病毒抗體(IgM和IgG)的不精密度。線性驗證與回收率試驗通過不同比例地混合高、低濃度血清樣本來評估。通過連續收集COVID-19陽性患者不同時段(從

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

結果:根據臨床和實驗室標準化協會(CLSI)EP15-A3指南要求進行的分析驗證試驗結果表明,新冠病毒IgM和IgG抗體檢測試劑的不精密度和重複性均在可接受範圍內(IgM和IgG的重複性分別為

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

結論:本研究結果證明了MAGLUMI 2000Plus化學發光免疫分析儀對COVID-19患者血清中特異性IgM和IgG的檢測是可靠有效的,並得到COVID-19抗體(IgM和IgG)動力學的有價值的數據。這些數據提示在COVID-19感染患者的診斷和治療中,合理利用特異性抗體的檢測是必須的。

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.

關鍵詞

性能分析,抗體動力學,COVID-19,COVID IgG 和IgM抗體,實時逆轉錄聚合酶鏈式反應,SARS-CoV-2。

analytical performances; antibody kinetics; COVID-19; COVID IgG and IgM; rRT-PCR; SARS-CoV-2.

簡介

2019新型冠狀病毒病,簡稱COVID-19,是一種傳播迅速的威脅全球健康的新型疾病,世界衛生組織(WHO)的總幹事於2020年3月11日將COVID-19的傳播定義為廣泛流行[1]。引起該疾病的病原體屬於冠狀病毒科,由於其與引起2003年SARS爆發的同源病毒(即SARS-CoV-1)序列具有高度同源性(達80%),最終被命名為「嚴重急性呼吸症候群冠狀病毒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].

在中國首次報告該病爆發後,COVID-19已經在全世界範圍內傳播,幾乎在全球所有國家都發現了病例[3]。在義大利,在第一位COVID-19檢測呈陽性的患者進入科多格諾醫院(Lodi,Lombar-dia)的重症監護室(ICU)的14天內,周邊地區陸續診斷出大量的COVID-19病例,其中包括相當比例的危重病人。同時在威尼託地區發現了第二個聚集性疫情,此後,COVID-19患者的數量迅速增加,主要集中在義大利北部,但義大利所有地區都有感染病例的報導[4]。COVID-19感染後的臨床表現從輕度到重度急性呼吸窘迫症候群(ARDS)不等。此外,也有很多表現為無症狀感染者,這增加了診斷工作的不確定性[5]。

及時準確地診斷COVID-19感染是對患者進行恰當治療的基礎,對於限制病毒進一步傳播也至關重要,特別是可以有效限制無症狀或輕度症狀的感染者引起的病毒傳播[6];因此,鑑別疑似病例對應對COVID-19的爆發起著至關重要的作用。目前SARS-CoV-2感染病原學診斷的金標準是呼吸道標本的實時逆轉錄聚合酶鏈式反應(rRT-PCR)[7–9]。然而,rRT-PCR檢測的質量對於提供準確且可解釋的結果至關重要,診斷準確性受多個因素影響,包括分析前因素,如樣品類型、採集、運輸和儲存條件,以及採用的PCR分析儀的質量和、一致性等,這些因素的控制對於實驗室提供準確且可解釋的結果至關重要[10];同時,收集鼻咽部的咽拭子樣本是一種侵入性的操作,會引起患者咳嗽和打噴嚏等情況,因此產生的氣溶膠對醫護人員構成潛在的生物安全威脅[11]。

特異性抗體的產生,特別是抗SARS-CoV-2的IgM和IgG的產生,應作為一種診斷疾病的非侵入性的補充方法來檢測,尤其是針對病毒載量較低但出現症狀的患者。然而,血清學檢測的時間點和抗體檢測結果的解讀是其對於COVID-19檢測有效的先決條件。因此,本研究的目的在於報導通過一種全自動檢測平臺,通過化學發光免疫分析方法(CLIA)檢測新型冠狀病毒抗體的性能驗證,並描述COVID-19患者IgM和IgG抗體產生的動力學特徵。

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

儀器:高通量(180測試/小時)Maglumi 2000Plus全自動化學發光免疫分析儀(深圳市新產業生物醫學工程股份有限公司,中國)

試劑:

2019-nCoV IgM(cut off值為1.0 AU/mL),靈敏度78.65%,特異性97.5%。

2019-nCoV IgG(cut off值為1.1 AU/mL),靈敏度91.21%,特異性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.

分離膠管幹擾評估

參照熊彥等的文章[12],進行病毒抗體檢測之前,可以將樣本56℃加熱30min滅活。我們進行了對於29例樣本的系列比較試驗,通過對直接對分離膠採血管進行加熱和分離出血清加熱進行比較,驗證是否會存在結果乾擾。我們特別對於每一個帶分離膠的原始管在病毒滅活之前都進行等量分離血清,隨後將分離出的血清與分離膠管同時加熱,之後再進行IgM和IgG結果的檢測和比較。

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.

重複性精密度和中間精密度

通過3個不同濃度的人血清樣本評估精密度。參照美國臨床實驗室標準化協會CLSI/EP15-A3要求[13],每個相同樣本等分五份,連續五天進行檢測。廠家提供的精密度數據是通過3個血清樣本,參照EP5-A3要求[14]進行重複性精密度和中間精密度的驗證。精密度的結果與廠家說明書進行比較。精密度的確定符合計量學聯合委員會VIM中對5天精密度驗證的要求。

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.

準確度(回收率)

使用已知濃度的高值血清樣本(其中IgM的為2.18AU/mL,IgG的為2.57AU/mL),混合低值血清(其中IgM的為0.27AU/mL,IgG的為0.093AU/mL)按比例稀釋,以得到不同的理論濃度。取稀釋後的系列樣品,測定濃度。然後計算其平均實測濃度與期望濃度的回收率。

回收率(%)=(測定濃度值-理論濃度值)/理論濃度值×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:

線性

線性驗證參考CLSI/EP06-A第三版第4.3.1節要求,使用4個高值血清樣本,用低值血清進行連續稀釋。其中IgM的兩個高值樣本為21.4AU/mL,2.27AU/mL,都用0.33AU/mL的樣本進行連續稀釋。IgG的一個高值樣本為73.72AU/mL,用0.086AU/mL的樣本進行連續稀釋;IgG的另一個高值樣本為2.65AU/mL,用0.107AU/mL的樣本進行連續稀釋。所有稀釋後的樣本重複檢測三次。

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抗體時間動力學評價

匿名收集37例在3月18日-3月26日期間住院的新冠病毒陽性(通過rRT-PCR檢測鼻咽拭子樣本確診)的患者的實驗室常規檢測之後的剩餘血清樣本,分裝凍存在-80°C。檢測時所有樣本同批覆融並加熱滅活。樣本通過MAGLUMI 2000Plus化學發光系統檢測2019-nCov IgM和2019-nCov IgG。本研究一共通過37個病人收集了87個樣本,其中10個患者各抽取了一次樣本,6個患者各抽取了兩次樣本,19個患者各抽取了三次樣本,每患者各抽取了4次樣本。

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).

數據統計

使用一致性評價法分析分離膠管採血進行滅活是否幹擾結果。使用方差分析法評估重複性、精密度和中間精密度。參照EP5-A3要求使用內部開發的R軟體(統計計算基金會,維也納,奧地利)進行方差分析和計算驗證上限。對於時間動力學評估,使用以下策略:對於研究中包含的每個樣品,從患者出現新冠臨床症狀(即發熱)開始收集樣本並進行抗體測定。定義了以下時間範圍(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):

結果

分離膠管幹擾評估

用Bland-Altman一致性評價方法分析數據,結果顯示直接加熱滅活分離膠採血管後檢測與分離出血清樣本加熱滅活後檢測的IgM 和 IgG的結果相似(IgM 和IgG的p=0.122和0.548)。(補充圖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).

重複性和中間精密度

表1結果顯示,參照CLSI/EP15-A3[13]要求檢測所得結果與廠家聲明的重複性精密度和中間精密度結果(參照CLSI/EP5-A3[14]要求,用三個濃度水平的樣本,每天重複測量3次,連續檢測5天)檢測比較。結果提示低值樣本和中值樣本的精密度結果比廠家聲明的低,符合要求。高值樣本的精密度結果比廠家聲明的高,參照CLSI/EP15-A3[13]要求進行偏倚計算之後,還是不符合要求。

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:精密度結果

a參照CLSI/EP5-A3要求檢測所得結果 b試劑盒說明書裡的精密度廠家聲明值。

c樣本的精密度驗證值比廠家聲明的高,參照CLSI/EP15-A3要求進行偏倚計算之後,還是不符合要求。

準確度(回收率)

由於缺乏充分驗證過的對比試劑,所以用回收率實驗來評估該方法的系統誤差。檢測一系列濃度的樣品(包含臨界值)的回收率後,結果顯示(見表2),IgM的樣本回收率明顯較高,在103%到123%之間。IgG高值樣本的回收率略高,在103%到112%之間;IgG低值樣本的回收率偏低,在63%到93%之間。

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:回收率結果

線性

用Maglumi 2000Plus化學發光免疫分析儀測得的IgM 和 IgG的線性數據結果如圖1。除了明顯高於臨界值的樣本檢測結果偏離線性,其餘所有的樣本檢測結果都符合線性要求。

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.

圖1 IgM和IgG線性結果

(A)IgM高值樣本21.4AU/mL用0.33AU/mL的樣本進行連續稀釋

(B)IgM高值樣本2.27AU/mL用0.33AU/mL的樣本進行連續稀釋

(C)IgG高值樣本73.72AU/mL用0.086AU/mL的樣本進行連續稀釋

(D)IgG高值樣本2.65AU/mL用0.107AU/mL的樣本進行連續稀釋

每個樣本測定三次計算平均值。

時間動力學

以患者感染後開始發熱的時間作為起點,採集樣本檢測IgM和IgG抗體,將感染後間隔相同時間開始發熱(即出現臨床症狀)的樣本檢測結果計算均值,研究IgM和IgG抗體產生的時間動力學特徵,結果如圖2所示。研究發現,感染後的第11天,所有患者均能被檢測出IgG抗體陽性;感染後的第13天,只有88%的患者被檢測出IgM抗體陽性。結果如表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.

圖2 IgM和IgG抗體的時間動力學(37名患者)

表3 不同時間檢測患者2019-nCoV IgM和2019-nCoV IgG的陽性率統計

討論

COVID-19的迅速傳播對全球所有國家的醫療系統來說都是一個重大挑戰。儘管感染的嚴重程度可能從輕度到重度不等,但是相當多的患者需要提供呼吸機支持的亞重症和重症監護,這造成了真正的醫療資源緊張。此外,醫護人員感染新冠病毒的比例日益上升。在世界衛生組織-中國冠狀病毒聯合專家考察報告中表明,截至2020年4月3日,在新冠肺炎患者總數112,401名和死亡病例13,241例(死亡率11.7%)的情況下,已有11,251名醫護人員感染過新冠病毒。

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,不僅是醫生,還有研究人員、政府決策人員,以及所有國家醫療保健系統的管理者,都將目光持續聚焦在新冠肺炎疾病的診斷檢測的討論上,即使其中只有極少數已經開發的新冠病毒檢測試劑被廣泛用於臨床,因為這個過程相當耗時。

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].

目前新冠肺炎的診斷方法包括通過rRT-PCR在呼吸道樣本中鑑定病毒RNA,儘管最近很多報導闡述了該技術分析前和分析中的局限性。首先,rRT-PCR檢測方法的敏感性不僅取決於疾病的發展階段(如樣本採集時間),還取決於病情的嚴重程度。其次,RNA檢測的通量有限,因為它不僅工作量大,還需要有熟練的操作人員進行樣品製備和檢測,同時,RNA檢測儀器昂貴,生物安全防護要求高。因此,血清學抗體檢測是一種更易操作的,成本更低的新冠病毒檢測方法,不僅可用於新冠肺炎的診斷,還可以提示疾病進程以及用於流行病學和疫苗評價。

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,我們在MAGLUMI2000 Plus化學發光免疫分析儀上評估了2019-nCoV IgM和IgG化學發光免疫分析試劑盒的分析性能以及新冠肺炎患者體內抗體出現的時間動力學特徵。我們研究的第一個方面是使用帶分離膠的血清試管作為原始樣本直接進行病毒滅活,而無需製備等量的次級樣本。該策略對於操作員安全以及有效的工作量管理極為重要。我們使用分離膠管對29份血清樣品進行IgM和IgG檢測,結果證實分離膠管適合用於病毒乾燥加熱滅活後的抗體檢測。隨後,我們利用EP15-A3文件方案評估了該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].

總體而言,我們的結果表明MAGLUMI2000 Plus具有很好的檢測精密度。IgM和IgG的重複性精密度分別為

Overall, our results show that MAGLUMI 2000 Plus has excellent precision characteristics. In fact, repeatability was

為了評價該方法線性範圍內檢測結果是否與IgM和IgG濃度理論值呈線性關係,從而保證線性範圍內檢測結果的準確性,我們評估了樣本的檢測線性。我們使用低值樣本和高值樣本進行了一系列的連續稀釋,製備了不同濃度的樣本。結果顯示,IgM和IgG濃度在0.5 AU / m L–1.5 AU / m L範圍內,檢測值與理論值呈現良好的正比例關係,但是在高值樣本中,線性關係變差,尤其是對於IgG。對於結果的比較,由於缺乏充分驗證過的對比試劑,所以用回收率實驗來評估準確度。檢測範圍覆蓋了廠家建議的範圍,結果表明IgM略被高估,而IgG在高於1.9AU/mL時被高估,在低於1.9AU/mL時被低估,總體而言,IgM回收率更好,因此,建議同時檢測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.

我們在先前建議的時間間隔內還評估了IgM和IgG抗體產生的時間動力學,結果顯示在出現發熱症狀後,IgM和IgG在短時間內均迅速升高。參考到廠家建議的陽性截斷值(IgM為1.0 AU / mL,IgG為1.1 AU / mL),可以認為在發熱症狀出現後6-7天,IgM和IgG會有顯著升高。這些發現與近期的一些其他報導一致。例如,有研究人員使用內部研發的ELISA方法,發現在症狀發作5天後,幾乎所有患者中都可以檢測到抗病毒抗體的顯著升高,而這一時期通常被認為是感染早期到晚期的過渡。同樣,還有研究者使用麗珠診斷的ELISA抗體檢測試劑盒,發現在症狀發作後7天IgM和IgG顯著增加,特別是在重症患者中。表3詳細描述了IgM和IgG抗體產生的動力學特徵,結果顯示IgG需要至少在感染12天後才能達到100%的敏感性,而在整個研究周期間,IgM的最高陽性率為88%。有趣的是,有三名確診患者的IgM值分別為0.811 AU / mL,0.909 AU / mL和0.863 AU / mL,均低於廠家聲稱的截斷值。這表明,通過進一步優化IgM的cut-off值,對於提高IgM檢測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.

本研究有一些明顯的局限性。例如,沒有可靠的經過驗證的方法可用於對照試驗,也沒有評估檢測方法的交叉反應性。此外,評估IgM和IgG抗體時間動力學是以發熱症狀出現作為標準。我們使用此症狀的原因是:

(a)適用於我們研究中的所有患者

(b)通常能夠由患者和醫生準確記錄;

(c)已在許多其他研究中使用。例如,某研究者估計的新冠病毒感染患者發燒的潛伏期中位數為5-7天(95%置信區間:4.9–6.8天)。

另一個方面,可能需要更長的周期來評估IgM和IgG產生的動力學,以便評估新冠病毒感染的體液免疫反應的整個趨勢。

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.

總之,這項研究的結果表明MAGLUMI2000 Plus 化學發光免疫分析系統是評估新冠病毒肺炎患者免疫反應的可靠的免疫測定方法。我們的結果還證實,同時測量IgM和IgG會對新冠病毒肺炎患者診斷有所幫助,尤其是在早期感染的時候。

In conclusion, the findings of this study show that MAGLUMI2000 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.

編輯:Bling 審校:Rose

相關焦點

  • 一滴血15分鐘內測出新冠病毒 但抗體檢測還不能替代核酸檢測
    2月14日,呼吸疾病國家重點實驗室對外透露,近日,在鍾南山院士指導下,實驗室聯合多家研究機構,最新研發出新冠病毒IgM抗體快速檢測試劑盒,並已在實驗室和臨床完成初步評價。與核酸類試劑檢測盒不同,抗體檢測方法目前主要是POCT檢測和化學發光檢測抗原方法。其檢測方式是採集新冠肺炎患者血清或血漿,並測試其中N蛋白或S蛋白等產生的IgM或IgG抗體濃度值。
  • 廈大與萬泰研製的新冠總抗體檢測試劑...
    文中寫道:「萬泰的新冠病毒總抗體檢測試劑(酶聯免疫法)的性能優於目前市場上的其他抗體檢測產品。」荷蘭政府應對新冠疫情特使Feike Sijbesma表示:「這些試劑能同時檢測三種類型的抗體,這使得它格外地有用。」
  • 日本厚勞省:抗體檢測不說明2019年初日本已有新冠病毒
    中新社·華輿訊據中文導報報導 日本厚生勞動省為了尋找出精確的新冠抗體試劑盒等,2020年5月15日發表了日本醫療研究開發機構(AMED)的研究班在日本紅十字會的合作下,進行的《抗體檢查試劑盒等的性能評價》的研究結果。由於各個公司的試劑盒等的檢測結果迥異,厚生勞動省認為目前難以對這些試劑盒的性能做出評價。
  • 萬泰生物:A型肝炎病毒抗體測定試劑盒、新冠病毒檢測試劑盒等檢測...
    11月6日晚間,萬泰生物發布公告,子公司萬泰凱瑞生產的A型肝炎病毒抗體測定試劑盒(化學發光微粒子免疫檢測法)、A型肝炎病毒IgM抗體測定試劑盒(化學發光微粒子免疫檢測法)、戊型肝炎病毒IgG抗體測定試劑盒(磁微粒化學發光法)、戊型肝炎病毒IgM抗體測定試劑盒(磁微粒化學發光法)、梅毒螺旋體抗體測定試劑盒
  • 深企新冠病毒檢測試劑首次出口日本 當地醫療界四大頂尖機構同時採購
    5 月 2 日,一批深圳市亞輝龍生物科技股份有限公司研發生產的新冠病毒檢測試劑從深圳啟運,正式發往日本,供給該國四大高校附屬醫院,助力日本全力開展疫情防控研究。據悉,這是 " 深圳智造 "、" 中國智造 " 化學發光新冠病毒免疫診斷試劑首次出口日本。
  • 興證醫藥健康:新冠病毒檢測方法優缺點全面解析
    -雙抗體夾心法,另一種是檢測患者血液裡特異性針對新型冠狀病毒的IgM抗體-IgM捕捉法。,但化學發光平臺做新冠試劑並沒有優勢,主要因為該方法對設備、操作、使用環境的要求比較高。在這一期間,血清無法檢出IgM與IgG,核酸檢測能檢測處於窗口期的患者是否受到感染。而抗體檢測在取樣過程和檢測時間等方面都有核酸檢測無法相比的優勢。因此在實際檢測過程中需要二者聯合檢測,綜合判讀,彌補不足。根據中國官方文件的建議,核酸檢測用於檢測是否受試者攜帶病毒,而抗體檢測用於核酸檢測結果陰性的疑似患者補充檢測或者聯合核酸檢測。新冠病毒感染檢測由以核酸為主的階段,轉變為核酸、抗體協同檢測階段。
  • 【粵開醫藥】疾病快速檢測方法論:核酸檢測和抗原抗體檢測
    2  新冠病毒檢測兩種方法:核酸法和抗體法 (一)為什麼PCR核酸檢測法最先研製出來?目前,核酸檢測方法主要包括PCR法、恆溫擴增法、測序法、CRISPR檢測法等,目前還研發出基因晶片技術。第二代PCR技術(螢光PCR法)是病毒檢測方法的主流方法,也是目前我國批准上市的新冠病毒檢測試劑盒採用最多的技術。
  • 新生兒高膽紅素血症對血清B肝病毒表面抗原化學發光法檢測及消除
    摘要:目的:將成人高膽紅素血症作為對照,分析新生兒高膽紅素血症對血清B肝病毒表面抗原化學發光法檢測的影響及消除方法。結論:相較於成人來說,新生兒高膽紅素血症有著明顯不同,新生兒間接膽紅素上升,為導致血清B肝病毒表面抗原化學發光法檢測為假陽性的重要因素。在進行藍光照射與高速離心後,利於儘可能降低血清間接膽紅素影響到B肝病毒表面抗原檢測效果。
  • medRxiv:評價九種新冠病毒免疫檢測試劑,國產勝出
    4月9日,丹麥國立血清研究所(Statens Serum Institut)在medRxiv預印本平臺發表研究論文——「Evaluation of nine commercial SARS-CoV-2 immunoassays」(評價九種商用的新冠病毒免疫檢測試劑)。
  • 未來智庫:新冠檢測行業全景分析及重點投資標的
    血清學檢測便是通過檢測血液樣本中的特異性抗體 IgM 和 IgG 的存在及 含量,來間接判斷體內有無病毒及病毒感染情況。目前臨床中常用的抗體血清學 檢測方法有 3 種:酶聯免疫吸附試驗法、化學發光免疫分析法、膠體金免疫層析 法。
  • 傳染病檢測領域,科美診斷光激化學發光技術的「光」究竟有多亮?
    從某種程度上來說,人類的歷史,也是一部與傳染病的鬥爭史……  2020年,全世界人民都在與新冠病毒鬥爭。這也讓我們意識到,傳染病的問題已經不再僅僅是公共衛生問題,而且是關乎國家安全和城市安全體系的重要組成部分。「傳染病」這一概念也重新進入大眾視野。
  • 新冠病毒核酸檢測到底檢測了啥?
    新冠疫情發展至今,已成全球之勢,中國的疫情基本已得到控制,亦有能力幫助其他國家,援助各種醫療資源,例如醫護人員、口罩、檢測試劑、呼吸機等。而3月23日,西班牙媒體報導了一則「西班牙衛生部購買了新冠病毒快速檢測假試紙」的新聞,其中西班牙一家試驗室聲稱其從中國購買的新冠病毒快速檢測試劑有效率僅為30%,跟宣稱的80%準確率相去甚遠。在這風口浪尖,還有企業敢賣假貨?
  • 研究:新冠病毒抗體可能在數月內消失
    就在全球科學家積極研發新冠疫苗的時候,英國的一項研究發現,新冠肺炎患者體內的抗體會在數月內消失,而疫苗提供的抗體可能也會如此,這意味著人們無法通過一次性注射疫苗「一勞永逸」。更糟的是,新冠病毒可能會像感冒一樣讓人們每年重複感染。
  • 義大利3.3%的狗和5.8%的貓檢測到新冠抗體
    該研究發現新冠病毒在雪貂和貓中複製效率較高,而在狗、豬、雞和鴨中複製能力微弱,此外,研究人員還發現新冠病毒可以通過呼吸道飛沫在貓之間傳播。該論文於5月29日在國際頂尖學術期刊 Science 雜誌正式發表。
  • 新冠病毒檢測越快越好嗎?
    目前已經批准的新冠核酸檢測產品: 1、新冠核酸檢測試劑盒(螢光PCR法):檢測新冠病毒的特異性靶基因,一般2-3小時完成 2、新冠核酸檢測試劑盒(恆溫擴增-實時螢光法) :檢測新冠病毒的特異性靶基因,在同一溫度下擴增,相對於螢光PCR法,對設備要求更低,一般1-2小時完成。
  • 新冠病毒快檢技術還能更快嗎?
    3月1日,國家藥品監督管理局應急審批通過3家企業4個新型冠狀病毒檢測產品,包括2個化學發光法抗體檢測試劑、2個螢光PCR法核酸檢測試劑。至此,國家藥監局共批准新冠病毒核酸檢測試劑10個,抗體檢測試劑4個。
  • 梅毒防控重在檢測策略電化學發光法成主流—新聞—科學網
    對性病門診患者沒有必要區分反向流程和傳統流程,應該建議患者同時做這兩類不同的實驗室檢測,這樣可以節約時間。」 電化學發光檢測方法 有效提高梅毒檢測質量 梅毒螺旋體抗原極不穩定,試劑性能的優異取決於更高的設計標準。對於免疫試劑的設計,抗原的篩選和製備工藝需要非常考究。
  • 新冠病毒溯源,有重大發現!
    義大利米蘭國家腫瘤研究所上個月發布的研究顯示,新冠病毒自2019年9月就在義大利傳播。義大利媒體近日又報導稱,米蘭大學的研究人員在一份2019年提取的4歲兒童咽部分泌物中檢測出了新冠病毒,並由此推論,去年11月,新冠病毒已在義大利傳播。目前,相關研究結果已經發表於國際醫學期刊和美國疾控中心網站。
  • Nature子刊:證實貓狗易感新冠病毒,義大利3.3%的狗檢測到新冠
    該研究發現新冠病毒在雪貂和貓中複製效率較高,而在狗、豬、雞和鴨中複製能力微弱,此外,研究人員還發現新冠病毒可以通過呼吸道飛沫在貓之間傳播。該論文於5月29日在國際頂尖學術期刊Science雜誌正式發表。
  • 重磅!北大團隊新發現:某些抗體結合可抑制新冠病毒突變體
    在前期篩選的可抑制新冠病毒的高活性抗體BD-368-2基礎上,團隊進一步發現BD-368-2與新冠病毒Spike三聚體的相互作用機制,並成功解析了二者形成複合體的高分辨冷凍電鏡結構,另外,該抗體與其他抗體形成的抗體「組合」還可應對新冠病毒突變體。