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Oct 2021
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Identification of SARS-CoV-2 Neutralizing Antibody with Pseudotyped Virus-based Test on HEK-293T hACE2 Cells
基于 HEK-293T hACE2 细胞的假型病毒试验鉴定 SARS-CoV-2 中和抗体   

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Abstract

Neutralizing antibodies (NAbs) are of particular importance because they can prevent binding of the receptor binding domain (RBD) of the spike protein (S protein) to the angiotensin-converting enzyme 2 (ACE2) receptor present at the surface of human cells, preventing virus entry into the host cells. The gold standard method for detection of NAbs is the plaque reduction neutralization test (PRNT). Based on the measurement of cell lysis due to viral infection, this test is able to detect antibodies that prevent cell infection (Muruato et al., 2020; Lau et al., 2021). This technique requires the use of live pathogens, i.e., SARS-CoV-2 in this case, and must be done in a biosafety level 3 (BL3) laboratory. In addition, it requires expensive installations, skillful and meticulous staff, and a high workload, which prevents its wide implementation even in research laboratories. A SARS-CoV-2 pseudovirus will express the S protein responsible for cell entrance, but will not express the pathogenic genetic material of the virus, making them less dangerous for laboratory staff and the environment.


Graphic abstract:



Keywords: COVID-19 (COVID-19), SARS-CoV-2 ( SARS-CoV-2), Neutralizing antibody (中和抗体), Pseudo-type virus (假型病毒), Immune response (免疫反应)

Background

The gold standard method for detection of NAbs is the plaque reduction neutralization test (PRNT) (Perera et al., 2020). Based on the measurement of cell lysis due to viral infection, this test is able to detect antibodies that prevent the cell infection (Muruato et al., 2020; Lau et al., 2021). Such technique requires the use of live pathogens, i.e., SARS-CoV-2 in this case, and must be done in a biosafety level 3 (BL3) laboratory. In addition, it requires expensive installations, skillful and meticulous staff, and a high workload, which prevent its wide implementation even in research laboratories (Muruato et al., 2020; Lee et al., 2021). Such facilities are not widely available, and only very specialized institutions can offer access to BL3 laboratories and trained staff. Quite similar neutralization techniques based on pseudoviral particles (called pseudo-virus neutralization tests, pVNT) have been developed, and can be performed in BL2 laboratories, allowing higher throughput (Nie et al., 2020a). A SARS-CoV-2 pseudovirus will express the S protein responsible for cell entrance, but will not express the pathogenic genetic material of the virus, making them less dangerous (Nie et al., 2020a, 2020b).

Materials and Reagents

  1. Sterile white 384-well µClear flat bottom cell culture plate with lid (Greiner Bio-One, Kremsmünster, Austria, catalog number: 781098)

  2. Sterile 384-well flat bottom assay plate with lid (Corning, NY, USA, catalog number: 3701)

  3. Pipette tip 200 µL (Thermo Fisher Scientific, Waltham, MA, USA, catalog number: AM12650)

  4. Eppendorf tube (Sigma-Aldrich, Saint-Louis, MO, USA, catalog number: T2795)

  5. 50 mL reagent reservoir sterile polystyrene (Merck, Overijse, Belgium, catalog number: CLS4870)

  6. HEK-293T hACE2 (Invivogen, San Diego, CA, USA, catalog number: HKB-hACE2)

  7. SARS-CoV-2 Pseudoviral Particles (E-enzyme, Gaithersburg, MD, USA, catalog number: SCV2-PsV-001)

  8. Dulbecco’s Modified Eagle Medium (DMEM), with L-glutamine and glucose (Lonza, Bâle, Switzerland, catalog number: LO BE12-604F)

  9. FireFly Luciferase kit (E-enzyme, Gaithersburg, MD, USA, catalog number: CA-L165-10)

  10. Tryptan blue (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA, catalog number: T10282)

Equipment

  1. Spectramax 3 iD (Molecular Devices, LLC, CA, USA)

  2. Laminar flow hood (Thermo Fisher Scientific, Waltham, MA, USA, MSC Advantage 1.8 catalog number: 51025413)

  3. Electronic multichannel 5–125 µL pipette (Brand, Transferpette -12 electronic, catalog number: 705453)

  4. Monochannel 5–50 µL pipette (Socorex, Ecubens, Switzerland, catalog number: 825.0050)

  5. Centrifuge 5702 (Eppendorf, Hamburg, Deutschland, catalog number: 5702000320)

  6. Neubauer counting slide (Hecht Assistant, Altnau, Switzeland, catalog number: 40441)

  7. Julobo ED Water bath (Sigma Aldrich, Saint-Louis, MO, USA, catalog number: Z615498)

Software

  1. GraphPad Prism software (version 9.1.0, San Diego, CA, USA)

Procedure

  1. Cell inoculation on a 384-well plate

    To determine the quantity of cell suspension necessary, a calculation of this type must be made:

    1. A volume of 15 μL of cell suspension within ± 8.5 × 103 cells are seeded in each well of the 384-wells plate. The quantity of cells suspension to prepare is 15 µL × 384 × X, where X is the number of plates to prepare.

    2. To prepare this cell suspension, after counting and centrifugation, add the volume required to have 566 cells per µL.

    3. To prepare the counting slide, the slat is stuck with water to the slide.

      1. In an Eppendorf, add 50 µL of cell solution to put in the wells and 50 µL of trypan blue. Mix.

      2. Place 10 µL of this mix in each part of the counting slide. Under a microscope, the living cells inside the squares are counted. Living cells appear transparent and dead cells appear blue.

      3. To calculate the number of cells per milliliter, the following formula must be used, where n is the number of cells counted using a Neubauer counting slide.



    4. The new cell suspension is maintained in a scotch bottle with constant agitation at moderate power. The 384-well plates are then filled with the cell suspension at a volume of 15 µL per well, with an electronic multichannel micropipette.

    5. The plates are then annotated with "cell type—# of passages—operator's initials" and incubated in a calibrated oven for cell culture at 37°C during 24 h.


  2. Serum dilutions

    1. Heat inactivate the serums in a water bath at 56°C for 30 min.

    2. Twenty-six sera can be diluted on a 384-well plate. Dilutions are made in line, and start at a 1:2 dilution, up to a 1:5120 dilution. If further dilutions are required, a second 384-well plate should be used.

    3. Before making the serum dilutions, each well must be filled with 30 µL of dilution medium (DMEM + 10% HyClone FetalClone Serum) using an electronic multichannel micropipette, except for columns 2 and 12 which are filled with 50 µL.

    4. Add 10 µL of serum in the first well, using a monochannel pipette 5–50 µL. Serial dilution of the sera can then be started, and proceeds as follows:

      1. Flush 15 times in the aliquot.

      2. Take a volume of 30 µL and place it in the first well.

      3. Flush 15 times in the first well.

      4. Change tips to collect liquid from the first to the second well.

      5. Repeat the previous steps until the end of the serial dilution.

        In the last well, 30 µL must be removed, so that all wells contain the same volume.

    5. A cell control (CC) and a viral check (VC) must be performed. The cell control (CC) is an assay in which cells are incubated with culture medium. The viral check consists of the incubation of viruses without any sera, in step C.1, 17.9 µL of SARS-CoV-2-PP must be incubated with 7.1 µL of culture medium.

    6. Centrifugate at 161 × g during 5 min.


  3. Interaction between antibodies and Pseudoviral Particles

    1. Dilute the pseudovirus three times in culture medium, to obtain the necessary volume for the analysis.

    2. In each well of a 384-well plate, add 17.9 µL of diluted SARS-CoV-2-Pseudoviral Particles with an electronic multichannel micropipette, and 7.1 µL of dilution serums previously perfomed with a manual multichannel micropipette (5 µL–50 µL). Each sample is carried out in duplicate. For one dilution serum, two tests are carried out.

    3. The plates are then annotated with "operator's initial" and incubated in a calibrated oven for cell culture at 37°C during 2 h.


  4. Innoculation of the virus on cells

    1. First, start by emptying the culture medium from the 384-well plate containing the cells. Once this is done, transfer 17.5 µL of each column from the plate containing serum dilutions and virals particles to the cells palte with a manual multichannel micropipette (5 µL–50 µL). Repeat the procedure, changing tips between each serum.

    2. Add 7.5 µL of DMEM + 10% FC into each well.

    3. Let incubate for 42 h at 37°C.


  5. Signal Measurement

    1. Remove the supernatant and add 20 µL of eEnzyme’s luciferase assay reagent into each well with an electronic multichannel micropipette.

    2. Read in a luminescence plate reader. There must be a proportional relationship between luminescence and dilutions, the higher the dilutions, the higher the signal. Indeed, the luciferase enables the detection of infected cells, the more there is of antibody, the less the cells will be infected.

Data analysis

Based on the relative light units (RLU) values from each sample, a percentage of inhibition can be calculated. The following formula must be applied to each dilution for each sample:



The different percentages of inhibition are used to plot the evolution of the relative inhibition as a function of the serum dilution. By intrapolation of the sigmoid curves obtained, it is possible to determine the dilution at which 50% inhibition is achieved, called the RI50. The results obtained via statistical software give us the logarithm of the dilution in comparison to the 1:10 dilution, considered our initial condition. This logarithm is then transformed into a numerical dilution within the range achieved. A sample is considered negative if the RI50 value of this sample is below the 1:20 dilution. An example of the expected results is shown in Figure 1.



Figure 1. Percentage of relative inhibition as a function of the log10 of the dilution compared to the 1:10 dilution.

Acknowledgments

We acknowledge Nie et al. (2020b) for the paper “Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay” in Nature Protocol, our protocol was direved from.

Competing interests

All authors have none to declare.

Ethics

The protocol was in accordance with the Declaration of Helsinkiand was approved by the Medical Ethical Committee of Saint-Luc Bouge (Bouge, Belgium,approval number B0392020000005). The informed consent was obtained from all subject.

References

  1. Lau, E. H. Y., Tsang, O. T. Y., Hui, D. S. C., Kwan, M. Y. W., Chan, W. H., Chiu, S. S., Ko, R. L. W., Chan, K. H., Cheng, S. M. S., Perera, R., et al. (2021). Neutralizing antibody titres in SARS-CoV-2 infections. Nat Commun 12(1): 63.
  2. Lee, W. T., Girardin, R. C., Dupuis, A. P., Kulas, K. E., Payne, A. F., Wong, S. J., Arinsburg, S., Nguyen, F. T., Mendu, D. R., Firpo-Betancourt, A., et al. (2021). Neutralizing Antibody Responses in COVID-19 Convalescent Sera. J Infect Dis 223(1): 47-55.
  3. Muruato, A. E., Fontes-Garfias, C. R., Ren, P., Garcia-Blanco, M. A., Menachery, V. D., Xie, X. and Shi, P. Y. (2020). A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation. Nat Commun 11(1): 4059.
  4. Nie, J., Li, Q., Wu, J., Zhao, C., Hao, H., Liu, H., Zhang, L., Nie, L., Qin, H., Wang, M., Lu, Q., Li, X., Sun, Q., Liu, J., Fan, C., Huang, W., Xu, M. and Wang, Y. (2020a). Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect 9(1): 680-686.
  5. Nie, J., Li, Q., Wu, J., Zhao, C., Hao, H., Liu, H., Zhang, L., Nie, L., Qin, H., Wang, M., Lu, Q., Li, X., Sun, Q., Liu, J., Fan, C., Huang, W., Xu, M. and Wang, Y. (2020b). Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay. Nat Protoc 15(11): 3699-3715.
  6. Perera, R. A., Mok, C. K., Tsang, O. T., Lv, H., Ko, R. L., Wu, N. C., Yuan, M., Leung, W. S., Chan, J. M., Chik, T. S., et al. (2020). Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), March 2020. Euro Surveill 25(16): 2000421.

简介

中和抗体 (NAb) 特别重要,因为它们可以阻止刺突蛋白 (S 蛋白) 的受体结合域 (RBD) 与人体细胞表面的血管紧张素转换酶 2 (ACE2) 受体结合,从而防止病毒进入宿主细胞。检测 NAbs 的金标准方法是斑块减少中和试验 (PRNT)。基于对病毒感染引起的细胞裂解的测量,该测试能够检测出预防细胞感染的抗体(Muruato 等人,2020;Lau 等人,2021)。该技术需要使用活病原体,即在这种情况下为 SARS-CoV-2,并且必须在生物安全 3 级 (BL3) 实验室中进行。此外,它需要昂贵的安装、熟练和细致的工作人员以及高工作量,即使在研究实验室中也无法广泛实施。 SARS-CoV-2 假病毒会表达负责细胞进入的 S 蛋白,但不会表达病毒的致病遗传物质,从而降低它们对实验室工作人员和环境的危害。

图文摘要:




背景

NAbs的金标准方法是斑块减少中和试验 (PRNT) ( Perera 等人,2020 年)。基于病毒感染引起的细胞溶解测量,该测试能够检测防止细胞感染的抗体( Muruato 等人,2020;刘等人,2021 )。这种技术需要使用活病原体,即在这种情况下为 SARS-CoV-2,并且必须在生物安全 3 级 (BL3) 实验室中进行。此外,它需要昂贵的设备、熟练和细致的工作人员以及高工作量,这使得即使在研究实验室也无法广泛实施( Muruato 等人,2020;李等人,2021 年)。此类设施并不普遍,只有非常专业的机构才能提供进入 BL3 实验室和训练有素的工作人员的机会。已经开发出非常相似的基于假病毒颗粒的中和技术(称为假病毒中和试验, pVNT ),并且可以在 BL2 实验室中进行,从而实现更高的通量( Nie 等人,2020a )。一种 SARS-CoV-2假病毒会表达负责细胞进入的 S 蛋白,但不会表达病毒的致病遗传物质,从而降低了它们的危险性( Nie 等人,2020a,2020b )。

关键字:COVID-19, SARS-CoV-2, 中和抗体, 假型病毒, 免疫反应

材料和试剂
带盖的无菌白色384孔µClear平底细胞培养板(Greiner Bio-One, Kremsmünster ,Austria,目录号:781098)
带盖的无菌384孔平底测定板(Corning,NY,USA,目录号:3701)
移液器吸头 200 µL(Thermo Fisher Scientific,Waltham,MA,USA,目录号:AM12650)
Eppendorf管(Sigma-Aldrich,Saint-Louis,MO,USA,目录号:T2795)
50 mL试剂容器无菌聚苯乙烯(Merck, Overijse ,Belgium,目录号:CLS4870)
HEK-293T hACE2( Invivogen ,San Diego,CA,USA,目录号:HKB-hACE2)
SARS-CoV-2 Pseudoviral Particles(E-enzyme,Gaithersburg,MD,USA,目录号:SCV2-PsV-001)
Dulbecco's Modified Eagle培养基(DMEM),含有L-谷氨酰胺和葡萄糖(Lonza, Bâle ,Switzerland,目录号:LO BE12-604F)
FireFly萤光素酶试剂盒(E-enzyme,Gaithersburg,MD,USA,目录号:CA-L165-10)
色氨酸蓝(Invitrogen by Thermo Fisher Scientific, Waltham,MA,USA,目录号:T10282)




设备


Spectramax 3 iD( Molecular Devices, LLC, CA, USA )
层流罩(Thermo Fisher Scientific,Waltham,MA,USA,MSC Advantage 1.8 目录号:51025413 )
电子多通道 5–125 µL 移液器(品牌, Transferpette -12 electronic,目录号:705453)
单通道 5–50 µL 移液器(Socorex,Ecubens,瑞士,目录号:825.0050)
Centrifuge 5702(Eppendorf,Hamburg,Deutschland,目录号:5702000320)
Neubauer 计数玻片(Hecht Assistant, Altnau , Switzeland ,目录号:40441)
Julobo ED水浴(Sigma Aldrich,Saint-Louis,MO,USA,目录号:Z615498)




软件


GraphPad Prism 软件(版本 9.1.0,San Diego,CA,USA)




程序


在 384 孔板上接种细胞


为了确定所需的细胞悬液量,必须进行这种类型的计算:
在 384 孔板的每个孔中接种15 μ L体积为± 8.5 × 10 3 个细胞的细胞悬液。要制备的细胞悬液量为 15 µL × 384 × X,其中 X 是要制备的板数。
要制备这种细胞悬浮液,在计数和离心后,添加每微升 566 个细胞所需的体积。
为了准备计数幻灯片,板条用水粘在幻灯片上。
在Eppendorf中,加入 50 μL 的细胞溶液放入孔中,并加入 50 μL 的台盼蓝。混合。
将 10 μL 的这种混合物放在计数幻灯片的每个部分。在显微镜下,正方形内的活细胞被计数。活细胞呈透明状,死细胞呈蓝色。
要计算每毫升的细胞数,必须使用以下公式,其中 n 是使用 Neubauer 计数玻片计数的细胞数。


Cells/ml=(n × 10 000)/((4×1)/2)


新的细胞悬液保存在苏格兰威士忌瓶中,并以中等功率持续搅拌。然后使用电子多通道微量移液器以每孔 15 µL 的体积将细胞悬液填充到 384 孔板中。
-传代次数-操作员姓名首字母”对板进行注释,并在校准的烘箱中在 37°C下孵育 24 小时以进行细胞培养。




血清稀释


在 56°C 的水浴中加热灭活血清 30 分钟。
26 份血清可在 384 孔板上稀释。稀释是在线进行的,从 1:2 稀释开始,直至 1:5120 稀释。如果需要进一步稀释,则应使用第二个 384 孔板。
在进行血清稀释之前,每孔必须填充 30 µL 稀释培养基(DMEM + 10% HyClone FetalClone Serum)使用电子多通道微量移液器,第 2 列和第 12 列除外,它们填充了 50 µL。
单通道移液器 5–50 µL在第一口井中加入 10 µL 血清。然后可以开始血清的连续稀释,并进行如下:
在等分试样中冲洗 15 次。
取 30 μL 的体积并将其放入第一口井中。
在第一口井中冲洗 15 次。
更改提示以从第一口井收集液体到第二口井。
重复前面的步骤,直到连续稀释结束。
在最后一口井中,必须去除 30 μL,以便所有井包含相同的体积。
必须执行细胞控制(CC) 和病毒检查(VC)。细胞对照 (CC) 是一种将细胞与培养基一起孵育的试验。病毒检查包括在没有任何血清的情况下孵育病毒,在步骤 C.1 中,17.9 µL SARS-CoV-2-PP 必须与 7.1 µL 培养基一起孵育。
以 161 × g离心5 分钟。


抗体与假病毒颗粒之间的相互作用


稀释假病毒 在培养基中3次,以获得分析所需的体积。
在 384 孔板的每个孔中,加入 17.9 μL 稀释的 SARS-CoV-2-Pseudoviral Particles 和电子多通道微量移液器,以及 7.1 μL 先前使用手动多通道微量移液器(5 μL–50 μL)执行的稀释血清。每个样品一式两份进行。对于一种稀释血清,进行两次测试。
然后将板用“操作员姓名首字母”注释并在校准的烘箱中孵育,以便在 37°C 下进行细胞培养。 2 小时。


接种到细胞上


首先,首先从含有细胞的 384 孔板中清空培养基。完成此操作后,使用手动多通道微量移液器 (5 µL–50 µL)将含有血清稀释液和病毒颗粒的板中的每列 17.5 µL 转移到细胞盘中。重复该过程,在每种血清之间更换提示。
在每口井中加入 7.5 μL 的 DMEM + 10% FC。
在 37°C下孵育42 小时。


信号测量


取出上清液,用电子多通道微量移液器将 20 μL 的eEnzyme荧光素酶检测试剂添加到每个孔中。
在发光板阅读器中阅读。 发光和稀释度之间必须存在比例关系,稀释度越高,信号越高。事实上,萤光素酶可以检测受感染的细胞,抗体越多,受感染的细胞就越少。








数据分析


根据每个样品的相对光单位 (RLU) 值,可以计算抑制百分比。以下公式必须适用于每个样品的每次稀释:


Relative inhibition= □((RLU sample X-RLU negative control)/(RLU viral control-RLU cell control))


不同的抑制百分比用于绘制作为血清稀释函数的相对抑制的演变。通过对获得的 S 曲线进行内插,可以确定实现 50% 抑制的稀释度,称为 RI50 。通过统计软件获得的结果为我们提供了稀释度与 1:10 稀释度相比的对数,考虑了我们的初始条件。然后将该对数转换为所达到范围内的数值稀释。如果该样品的 RI50 值低于 1:20 稀释度,则认为该样品为阴性。预期结果的示例如图 1 所示。


 


与 1:10 稀释相比,作为 log 10函数的相对抑制百分比。
致谢


我们承认聂 等人。 (2020b) 论文“通过基于假型病毒的检测方法对 SARS-CoV-2 中和抗体进行量化”,我们的协议源自。




利益争夺


所有作者都无需声明。




伦理


该方案符合赫尔辛基宣言,并获得了 Saint-Luc Bouge医学伦理委员会(比利时Bouge ,批准号 B0392020000005)的批准。所有受试者均获得知情同意。






参考


Lau,EHY,Tsang,OTY,Hui,DSC,Kwan,MYW,Chan,WH,Chiu,SS,Ko,RLW,Chan,KH,Cheng,SMS,Perera,R.,等。 (2021 年)。中和 SARS-CoV-2 感染中的抗体滴度。 国家通讯12(1): 63。
Lee,WT,Girardin,RC,Dupuis,AP,Kulas,KE,Payne,AF,Wong,SJ,Arinsburg,S.,Nguyen,FT,Mendu,DR,Firpo-Betancourt,A.,等。 (2021 年)。中和 COVID-19 恢复期血清中的抗体反应。 感染病杂志 223(1):47-55 。
Muruato , AE, Fontes- Garfias , CR, Ren, P., Garcia-Blanco, MA, Menachery , VD, Xie , X. 和 Shi, PY (2020)。用于 COVID-19 诊断和疫苗评估的高通量中和抗体测定。 国家通讯11(1):4059 。
Nie , J., Li, Q., Wu, J., Zhao, C., Hao, H., Liu, H., Zhang, L., Nie , L., Qin, H., Wang, M., Lu, Q.、Li, X.、Sun, Q.、Liu, J.、Fan, C.、Huang, W.、Xu, M. 和 Wang, Y. (2020a)。 SARS-CoV-2 假病毒中和试验的建立和验证。 新兴微生物感染9(1):680-686 。
Nie , J., Li, Q., Wu, J., Zhao, C., Hao, H., Liu, H., Zhang, L., Nie , L., Qin, H., Wang, M., Lu, Q.、Li, X.、Sun, Q.、Liu, J.、Fan, C.、Huang, W.、Xu, M. 和 Wang, Y. (2020b)。通过基于假型病毒的测定法定量 SARS-CoV-2 中和抗体。 国家协议15(11):3699-3715 。
Perera ,RA, Mok ,CK,Tsang,OT, Lv ,H.,Ko,RL,Wu,NC,Yuan,M.,Leung,WS,Chan,JM, Chik ,TS,等。 (2020 年)。严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的血清学检测,2020 年 3 月。 欧洲监测25(16): 2000421。


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引用:Gillot, C., Favresse, J., Maloteau, V., Dogné, J. M. and Douxfils, J. (2022). Identification of SARS-CoV-2 Neutralizing Antibody with Pseudotyped Virus-based Test on HEK-293T hACE2 Cells. Bio-protocol 12(7): e4377. DOI: 10.21769/BioProtoc.4377.
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