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The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells by endocytosis from the cell surface and fusion of the viral envelope with the membrane of late endosomes. The viral envelope glycoproteins undergo antigenic changes both upon virion endocytosis and upon fusion of the viral envelope with the endosomal membrane. These changes in virion antigenicity during virus entry were first described by immunofluorescence of infected cells. Although immunofluorescence provides valuable information on the subcellular distribution of the viral glycoproteins, the quantification of immunofluorescence signals in a large number of cells is not only dependent on relatively expensive microscopy equipment, but is also relatively time-consuming. In order to quantify the antigenicity of MuHV-4 virions entering NMuMG epithelial cells in a reliable, as well as time- and cost-effective way, we have developed an ELISA with infected cells as the solid phase. In this assay, cells are grown on 96-well tissue culture plates, exposed to virions at 4 °C, followed by incubation at 37 °C allowing virion endocytosis. Cells are fixed either directly after virion binding at 4 °C or after incubation at 37 °C. After subsequent permeabilization, the cells are incubated with monoclonal antibodies specific for the viral envelope glycoproteins, followed by detection with an alkaline phosphatase-coupled secondary antibody. Upon incubation of cells with p-nitrophenyl phosphate substrate, the absorbance is measured on a conventional ELISA microplate reader. The different ways of data interpretation are discussed.

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ELISA on Virus-Infected Cells
病毒感染细胞的酶联免疫吸附试验

微生物学 > 微生物-宿主相互作用 > 病毒
作者: Daniel L. Glauser
Daniel L. GlauserAffiliation: Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK
For correspondence: daglaus@outlook.com
Bio-protocol author page: a1363
 and Philip G. Stevenson
Philip G. StevensonAffiliation: Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK
Bio-protocol author page: a1364
Vol 4, Iss 10, 5/20/2014, 3111 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1127

[Abstract] The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells by endocytosis from the cell surface and fusion of the viral envelope with the membrane of late endosomes. The viral envelope glycoproteins undergo antigenic changes both upon virion endocytosis and upon fusion of the viral envelope with the endosomal membrane. These changes in virion antigenicity during virus entry were first described by immunofluorescence of infected cells. Although immunofluorescence provides valuable information on the subcellular distribution of the viral glycoproteins, the quantification of immunofluorescence signals in a large number of cells is not only dependent on relatively expensive microscopy equipment, but is also relatively time-consuming. In order to quantify the antigenicity of MuHV-4 virions entering NMuMG epithelial cells in a reliable, as well as time- and cost-effective way, we have developed an ELISA with infected cells as the solid phase. In this assay, cells are grown on 96-well tissue culture plates, exposed to virions at 4 °C, followed by incubation at 37 °C allowing virion endocytosis. Cells are fixed either directly after virion binding at 4 °C or after incubation at 37 °C. After subsequent permeabilization, the cells are incubated with monoclonal antibodies specific for the viral envelope glycoproteins, followed by detection with an alkaline phosphatase-coupled secondary antibody. Upon incubation of cells with p-nitrophenyl phosphate substrate, the absorbance is measured on a conventional ELISA microplate reader. The different ways of data interpretation are discussed.
Keywords: Herpesvirus(疱疹病毒), Cell entry(细胞出入), Viral glycoproteins(病毒糖蛋白), Murid herpesvirus 4(鼠疱疹病毒4), ELISA(ELISA)

[Abstract]

Materials and Reagents

  1. NMuMG cells (ATCC, catalog number: CRL-1636 )
  2. BAC-derived MuHV-4 strain 68 (Adler et al., 2000)
  3. Glutamine (PAA Laboratories GmbH, catalog number: E15-883 )
  4. Fetal bovine serum (FBS) (Amimed, catalog number: 2-01F10-I )
  5. 100 U/ml penicillin and 100 µg/ml streptomycin (PAA Laboratories GmbH, catalog number: P11-010 )
  6. Trypsin-EDTA (PAA Laboratories GmbH, catalog number: L11-003 )
  7. Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
  8. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  9. Glycine (Sigma-Aldrich, catalog number: 50046 )
  10. Triton-X100 (Sigma-Aldrich, catalog number: X100 )
  11. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  12. Bovine serum albumin (BSA) (Fraction V) (Amresco, catalog number: 0332 )
  13. MuHV-4 envelope glycoprotein-specific monoclonal antibodies (hybridoma supernatants) (Glauser et al., 2012b; Glauser et al., 2013)
  14. Alkaline phosphatase-conjugated goat anti-mouse IgG (γ chain specific) polyclonal antibody (SouthernBiotech, catalog number: 1030-04 )
  15. p-nitrophenyl phosphate tablets (Sigma-Aldrich, catalog number: N2770 )
  16. Complete medium (see Recipes)
  17. 10x PBS (pH 7.4) (see Recipes)
  18. 1x PBS (pH 7.4) (see Recipes)
  19. Trypsin-EDTA (see Recipes)
  20. PBS (pH 7.4)
    1. PBS containing 4% formaldehyde (see Recipes)
    2. PBS containing 0.1 M glycine (see Recipes)
    3. PBS containing 0.1% Triton-X100 (see Recipes)
    4. PBS containing 0.1% Tween 20 and 2% BSA (see Recipes)
    5. PBS containing 0.1% Tween 20 (see Recipes)
  21. ELISA substrate (p-nitrophenyl phosphate) (see Recipes)

Equipment

  1. 150 cm2 tissue culture flasks (TPP Techno Plastic Products, catalog number: 90150 )
  2. 96-Well tissue culture plates (F-base) (TPP Techno Plastic Products, catalog number: 92096 )
  3. Standard tissue culture equipment
  4. Cooled tabletop microfuge
  5. Fume hood
  6. 37 °C, 5% CO2 incubator
  7. 4 °C cold room
  8. Sunrise microplate reader (Tecan Trading AG)
  9. Neubauer improved hemocytometer
  10. 10, 20, 200, and 1,000 µl volume pipettes and tips
  11. 200 µl volume 12-channel pipette
  12. 5 ml Bijoux tubes (Sigma-Aldrich, catalog number: Z645346 )
  13. Disposable sterile buffer reservoirs for multichannel pipettes
  14. Polystyrene boxes

Software

  1. Microsoft Excel 2010

Procedure

Part I. Considerations for the experimental design

  1. Cells are fixed at 3 different stages in the viral entry process: (i) after virion binding at 4 °C, (ii) after cell entry at 37 °C for 1 h, and (iii) after cell entry at 37 °C for 2 h. For each of these conditions, a separate tissue culture plate is used.
  2. At least 6 wells should be infected for each experimental condition (virus, treatment, antibody etc.).
  3. On each plate, at least 6 wells should be left uninfected in order to determine the background signal for each virus-specific monoclonal antibody.
  4. If the assay is used to study the entry process of viruses pretreated with neutralizing antibodies, it might be necessary to use IgG subclass-specific secondary antibodies in order to avoid binding of the secondary antibody to the neutralizing antibody (Glauser et al., 2012a).

Part II. Experimental procedure
Day 1

  1. Passage and seed cells
    1. Grow NMuMG cells in a 150 cm2 tissue culture flask until they form a 90-100% confluent monolayer. One 150 cm2 tissue culture flask yields approximately 2-3 x 107 cells, the experiment described below requires 6 x 106 cells. Wash cells 1x with 10 ml sterile PBS, then overlay with 5 ml Trypsin-EDTA and incubate for 10 min at 37 °C. Detach the cells by hitting the flask, then add 15 ml complete medium. Re-suspend cells by pipetting up and down, centrifuge 3 min at 350 x g at 4 °C and discard the supernatant. Re-suspend cells in 10 ml fresh complete medium, mix a small aliquot 1:1 with trypan blue and count with hemocytometer.
    2. Seed cells into 96-well tissue culture plates (20,000 cells in 100 µl complete medium/well) and incubate overnight at 37 °C.


Day 2

  1. Binding of virus to cells (work in 4 °C cold room)
    1. Dilute virus (MOI of 5 PFU or 15 eGFP units/cell) in complete medium (83 µl/well) in 5 ml Bijoux tubes.
    2. Pre-cool the 96-well tissue culture plates containing the cells and the tubes containing the diluted viruses for 1 h on ice at 4 °C.
    3. Gently tap off the cell medium and overlay cells with 83 µl/well diluted viruses or complete medium (uninfected controls), incubate for 2 h on ice at 4 °C.
    4. Gently wash the cells 3x with ice-cold PBS (approximately 300 µl/well).
  2. Fixation of cells after cell binding at 4 °C
    1. Take one plate and overlay the cells with ice-cold PBS containing 4% formaldehyde (100 µl/well), incubate for 1 h at room temperature (RT).
    2. In order to stop the fixation, gently tap off the fixative and incubate the cells with PBS containing 0.1 M glycine (100 µl/well) for 15 min at RT.
    3. Wash the cells 3x with PBS (approximately 300 µl/well).
    4. Permeabilize the cells by incubation with PBS containing 0.1% Triton-X100 (100 µl/well) for 30 min at RT.
    5. Block the cells by overnight incubation with PBS containing 0.1% Tween 20 and 2% BSA (100 µl/well) at 4 °C.
  3. Fixation of cells after cell entry at 37 °C
    1. Take the remaining two plates and overlay the cells with ice-cold complete medium (83 µl/well), then incubate one plate for 1 h and the other for 2 h at 37 °C.
    2.  Wash the cells 1x with ice-cold PBS (approximately 300 µl/well), then fix and treat as described in steps C1-5.


Day 3

  1. Detection of virions by ELISA
    Remark: For the primary antibodies use a concentration which gives a strong signal in immunofluorescence (IF). In our experience, hybridoma supernatants diluted 1:2 give good signals in IF and ELISA.
    1. Dilute primary antibodies (hybridoma supernatants) in PBS containing 0.1% Tween 20 and 2% BSA, add to cells (50 µl/well) and incubate 3 h at RT.
    2. Wash cells 3x with PBS containing 0.1% Tween 20 (approximately 300 µl/well).
    3. Centrifuge secondary antibody (alkaline phosphatase conjugate) for 10 min at 16,000 x g in a cooled tabletop microfuge (4 °C) to remove any potential antibody aggregates.
    4. Dilute secondary antibody 1:1,000 in PBS containing 0.1% Tween 20 and 2% BSA, add to cells (100 µl/well) and incubate 3 h at RT.
    5. Wash cells 6x with PBS containing 0.1% Tween 20 (approximately 300 µl/well).
    6. Dilute p-nitrophenyl phosphate in H2O according to manufacturer's instructions, add to cells (100 µl/well) and incubate overnight at RT and protected from light.
    7. Measure absorbance at 405 nm on an ELISA microplate reader. If the microplate reader can measure at a reference wavelenght, use 650 nm.

Part III. Data interpretation

  1. The absorbance values should not be considered an absolute measure of virion antigenicity, instead they should only be used to compare the antigenicity of virions between the different stages of virus entry, i.e., virus binding at 4 °C and virus entry at 37 °C for 1 or 2 h.
  2. The absorbance signal which is measured on uninfected cells can be considered the background signal of the respective monoclonal antibody and can be subtracted from the absorbance values measured on infected cells (for an example, see Figure 1A-B).
  3. If the aim of the experiment is to compare the virion antigenicity of a single virus at different entry stages, the absolute absorbance values can be shown (Glauser et al., 2012b).
  4. If the aim of the experiment is to compare the entry kinetics of different virus mutants (Glauser et al., 2013), the absorbance values for each virus mutant should be normalized to the values measured after virion binding at 4 °C (for an example, see Figure 1C).
  5. If the aim of the experiment is to compare the effect of different neutralizing antibodies on virus entry (Glauser et al., 2012a), the absorbance values for each virus-antibody combination should be normalized to the values measured after virion binding at 4 °C, because virus neutralization can either increase or decrease cell binding.


    Figure 1. Wild-type, mutant 1, mutant 2, and mutant 1 revertant MuHV-4 (MOI 15 eGFP units/cell) were bound to NMuMG cells for 2 h at 4 °C. After 3 washes with ice-cold PBS to remove unbound virions, the cells were either fixed directly or after further incubations at 37 °C to allow virion endocytosis. The cells were then incubated with mAb BN-1A7 recognizing the pre-fusion conformation of MuHV-4 glycoprotein B and bound antibody detected with an alkaline phosphatase-conjugated secondary antibody and incubation with p-nitrophenyl phosphate substrate. The bars show mean values from 6 wells ± standard error of the mean. (A) Mean absorbance readings at 405 nm (raw data). (B) The signal measured on uninfected cells was taken as background and subtracted from the signals measured on the infected cells. (C) After subtraction of the background signal, all values were normalized to the values measured after virus binding at 4 °C. This allows better comparison of the entry kinetics different virus mutants.

Recipes

  1. Complete medium
    DMEM, 4.5 g/L glucose, with stable glutamine
    500 ml
    Heat-inactivated fetal bovine serum
    50 ml
    100x Penicillin-Streptomycin
    5.5 ml
    Stored at 4 °C

  2. 10x PBS (pH 7.4)
    Deionized H2O
    800 ml
    NaCl
    80 g
    KaCl
    2 g
    Na2HPO4.7H2O
    26.6 g
    KH2PO4
    2.4 g
    Stir with magnetic stirrer until completely dissolved
    Adjust to pH 7.4 with HCl
    Adjust volume to 1 liter with H2O
    Sterilize by autoclaving
    Stored at RT
  3. 1x PBS (pH 7.4)
    Deionized H2O
    900 ml
    10x PBS (pH 7.4)
    100 ml
    Sterilize by autoclaving
    Stored at RT
  4. Trypsin-EDTA
    Sterile PBS (pH 7.4)
    90 ml
    10x Trypsin-EDTA
    10 ml
    Stored at 4 °C
  5. PBS containing 4% formaldehyde
    PBS (pH 7.4)
    100 ml
    Paraformaldehyde
    4 g
    Boil PBS in microwave oven
    Caution: Do not close the lid of the bottle.
    Let PBS cool down for approximately 5 min, then add paraformaldehyde to warm PBS.
    Caution: Work in fume hood and wear gloves and eye protection.
    Stir with magnetic stirrer until completely dissolved
    Stored at -20 °C
  6. PBS containing 0.1 M glycine
    PBS (pH 7.4)
    100 ml
    Glycine
    0.75 g
    Mix until completely dissolved
    Sterilize by autoclaving
    Stored at RT
  7. PBS containing 0.1% Triton-X100
    PBS (pH 7.4)
    100 ml
    Triton-X100
    0.1 ml
    Mix until completely dissolved
    Stored at RT
  8. PBS containing 0.1% Tween 20 and 2% BSA
    PBS (pH 7.4)
    100 ml
    Tween 20
    0.1 ml
    BSA
    2 g
    Mix until completely dissolved
    Sterilize by filtration (0.2 µm filter)
    Stored at 4 °C
  9. PBS containing 0.1% Tween 20
    PBS (pH 7.4)
    1,000 ml
    Tween 20
    1 ml
    Mix until completely dissolved
    Stored at RT
  10. ELISA substrate (p-nitrophenyl phosphate)
    Deionized H2O 20 ml
    Buffer tablet
    1 pc.
    Substrate tablet
    1 pc.
    Mix until completely dissolved
    Prepare immediately before use

Acknowledgments

This protocol is adapted from Glauser et al. (2012a); Glauser et al. (2012b); and Glauser et al. (2013).

References

  1. Adler, H., Messerle, M., Wagner, M. and Koszinowski, U. H. (2000). Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome. J Virol 74(15): 6964-6974.
  2. Glauser, D. L., Gillet, L. and Stevenson, P. G. (2012a). Virion endocytosis is a major target for murid herpesvirus-4 neutralization. J Gen Virol 93(Pt 6): 1316-1327.
  3. Glauser, D. L., Kratz, A. S. and Stevenson, P. G. (2012b). Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion. PLoS One 7(1): e30152.
  4. Glauser, D. L., Milho, R., Frederico, B., May, J. S., Kratz, A.-S., Gillet, L. and Stevenson, P. G. (2013). Glycoprotein B cleavage is important for murid herpesvirus 4 to infect myeloid cells. J Virol 87(19): 10828-10842.

材料和试剂

  1. NMuMG细胞(ATCC,目录号:CRL-1636)
  2. BAC衍生的MuHV-4株68(Adler等人,2000)
  3. 谷氨酰胺(PAA Laboratories GmbH,目录号:E15-883)
  4. 胎牛血清(FBS)(Amimed,目录号:2-01F10-I)
  5. 100U/ml青霉素和100μg/ml链霉素(PAA Laboratories GmbH,目录号:P11-010)。
  6. 胰蛋白酶-EDTA(PAA Laboratories GmbH,目录号:L11-003)
  7. 台盼蓝溶液(Sigma-Aldrich,目录号:T8154)
  8. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  9. 甘氨酸(Sigma-Aldrich,目录号:50046)
  10. Triton-X100(Sigma-Aldrich,目录号:X100)
  11. 吐温20(Sigma-Aldrich,目录号:P1379)
  12. 牛血清白蛋白(BSA)(级分V)(Amresco,目录号:0332)
  13. MuHV-4包膜糖蛋白特异性单克隆抗体(杂交瘤上清液)(Glauser等人,2012b; Glauser等人,2013)
  14. 碱性磷酸酶结合的山羊抗小鼠IgG(γ链特异性)多克隆抗体(SouthernBiotech,目录号:1030-04)
  15. 磷酸硝基苯酯片剂(Sigma-Aldrich,目录号:N2770)
  16. 完整介质(见配方)
  17. 10x PBS(pH 7.4)(参见配方)
  18. 1x PBS(pH 7.4)(参见配方)
  19. 胰蛋白酶-EDTA(见配方)
  20. PBS(pH 7.4)
    1. 含有4%甲醛的PBS(参见配方)
    2. 含有0.1M甘氨酸的PBS(参见配方)
    3. 含有0.1%Triton-X100的PBS(参见配方)
    4. 含有0.1%吐温20和2%BSA的PBS(参见Recipes)
    5. 含有0.1%吐温20的PBS(参见配方)
  21. ELISA底物(磷酸硝基苯酯)(参见配方)

设备

  1. 150cm 2组织培养瓶(TPP Techno Plastic Products,目录号:90150)
  2. 96孔组织培养板(F-base)(TPP Techno Plastic Products,目录号:92096)
  3. 标准组织培养设备
  4. 冷却的台式微型离心机
  5. 通风橱
  6. 37℃,5%CO 2培养箱
  7. 4°C冷室
  8. 日射酶标仪(Tecan Trading AG)
  9. Neubauer改善血细胞计数器
  10. 10,20,200和1000μl体积移液器和提示
  11. 200μl体积12通道移液器
  12. 5ml Bijoux管(Sigma-Aldrich,目录号:Z645346)
  13. 用于多通道移液器的一次性无菌缓冲液容器
  14. 聚苯乙烯盒

软件

  1. Microsoft Excel 2010

程序

第一部分。实验设计的注意事项

  1. 细胞在病毒进入过程中的3个不同阶段固定:(i)在4℃下病毒体结合后,(ii)在37℃下细胞进入1小时后,和(iii)在37℃下进入细胞后 2小时。 对于这些条件中的每一种,使用单独的组织培养板
  2. 对于每个实验条件(病毒,治疗,抗体等),至少应该感染6个孔。
  3. 在每个板上,至少6个孔应该保持未感染,以确定每种病毒特异性单克隆抗体的背景信号。
  4. 如果该测定用于研究用中和抗体预处理的病毒的进入过程,则可能有必要使用IgG亚类特异性第二抗体,以避免第二抗体与中和抗体的结合(Glauser等人, ,2012a)。

第二部分。 实验程序
第1天

  1. 通道和种子细胞
    1. 在150cm 2组织培养瓶中生长NMuMG细胞,直到它们形成90-100%铺满的单层。一个150cm 2组织培养瓶产生约2-3×10 7个细胞,下述实验需要6×10 6个细胞。用10ml无菌PBS洗涤细胞1x,然后用5ml胰蛋白酶-EDTA覆盖并在37℃孵育10分钟。通过击打烧瓶分离细胞,然后加入15毫升完全培养基。通过上下吹吸重新悬浮细胞,在4℃下以350×g离心3分钟,弃去上清液。重悬细胞在10ml新鲜完全培养基,混合小等分1:1与台盼蓝和血细胞计数器计数。
    2. 将细胞接种到96孔组织培养板中(20,000个细胞在100μl完全培养基/孔中),并在37℃下孵育过夜。


第2天

  1. 病毒与细胞的结合(在4℃冷室中工作)
    1. 在5ml Bijoux管中的完全培养基(83μl/孔)中稀释病毒(5PFU的MOI或15 eGFP单位/细胞)。
    2. 将含有细胞的96孔组织培养板和含有稀释的病毒的管在冰上于4℃预冷1小时。
    3. 用83μl/孔稀释的病毒或完全培养基(未感染的对照)轻轻敲打细胞培养基并覆盖细胞,在冰上在4℃孵育2小时。
    4. 用冰冷PBS(约300μl/孔)轻轻冲洗细胞3次
  2. 细胞在4℃下结合后固定细胞
    1. 取一块板,用含有4%甲醛(100μl/孔)的冰冷PBS覆盖细胞,在室温(RT)下孵育1小时。
    2. 为了停止固定,轻轻取出固定剂,并用含有0.1M甘氨酸(100μl/孔)的PBS在室温孵育细胞15分钟。
    3. 用PBS(约300μl/孔)洗涤细胞3次
    4. 通过与含有0.1%Triton-X100的PBS(100μl/孔)在室温下孵育30分钟来使细胞透化。
    5. 通过与含有0.1%Tween 20和2%BSA(100μl/孔)的PBS在4℃下孵育过夜来封闭细胞。
  3. 细胞在37℃下固定细胞
    1. 取剩余的两个板,用冰冷的完全培养基(83μl/孔)覆盖细胞,然后在37℃孵育一个板1小时,另一个孵育2小时。
    2.  用冰冷的PBS(约300μl/孔)洗涤细胞1次,然后按照步骤C1-5中所述进行固定和处理。


第3天

  1. 通过ELISA检测病毒粒子
    注释:对于初级抗体使用在免疫荧光(IF)中产生强信号的浓度。 根据我们的经验,以1:2稀释的杂交瘤上清液在IF和ELISA中给出良好的信号
    1. 稀释在含有0.1%吐温20和2%BSA的PBS中的一抗(杂交瘤上清液),加入细胞(50μl/孔)并在室温下孵育3小时。
    2. 用含有0.1%吐温20的PBS(约300μl/孔)洗涤细胞3次
    3. 在冷却的台式微量离心机(4℃)中以16,000×g离心二级抗体(碱性磷酸酶缀合物)10分钟,以除去任何潜在的抗体聚集体。
    4. 在含有0.1%吐温20和2%BSA的PBS中稀释稀释二抗1:1,000,加入细胞(100μl/孔)并在室温下孵育3小时。
    5. 用含有0.1%吐温20的PBS(约300μl/孔)洗涤细胞6次
    6. 根据制造商的说明,将H 2 O中的磷酸硝基苯酯磷酸盐加入到细胞(100μl/孔)中并在室温下孵育过夜并避光保存。< br/>
    7. 在ELISA酶标仪上测量405nm处的吸光度。如果酶标仪可以在参考波长处测量,请使用650 nm

第三部分。 数据解释

  1. 吸光度值不应被认为是病毒粒子抗原性的绝对量度,而是应该仅用于比较病毒进入的不同阶段之间的病毒粒子的抗原性,即在4℃下的病毒结合, 病毒在37℃进行1或2小时
  2. 在未感染细胞上测量的吸光度信号可以被认为是相应单克隆抗体的背景信号,并且可以从在感染细胞上测量的吸光度值中减去(例如,参见图1A-B)。
  3. 如果实验的目的是比较不同进入阶段的单个病毒的病毒粒子抗原性,可以显示绝对吸光度值(Glauser等人,2012b)。
  4. 如果实验的目的是比较不同病毒突变体的进入动力学(Glauser等人,2013),每个病毒突变体的吸光度值应当归一化为在病毒体结合后测量的值4℃(例如,参见图1C)。
  5. 如果实验的目的是比较不同中和抗体对病毒进入的影响(Glauser等人,2012a),则应将每种病毒 - 抗体组合的吸光度值标准化为测量值在病毒体在4℃结合后,因为病毒中和可以增加或减少细胞结合

    图1.野生型,突变体1,突变体2和突变体1回复突变MuHV-4(MOI 15 eGFP单位/细胞)在4℃与NMuMG细胞结合2小时。用冰冷的PBS洗涤以除去未结合的病毒体,将细胞直接固定或在37℃下进一步温育以允许病毒颗粒内吞作用。然后将细胞与识别MuHV-4糖蛋白B的融合前构象的mAb BN-1A7和用碱性磷酸酶缀合的二抗检测的结合抗体孵育,并与磷酸硝基苯酯底物孵育。条形图显示来自6个孔的平均值±平均值的标准误差。 (A)405nm处的平均吸光度读数(原始数据)。 (B)将在未感染细胞上测量的信号作为背景,并从在感染细胞上测量的信号中扣除。 (C)减去背景信号后,将所有值标准化为在4℃下病毒结合后测量的值。这允许更好地比较不同病毒突变体的进入动力学

食谱

  1. 完全培养基
    DMEM,4.5g/L葡萄糖,具有稳定的谷氨酰胺 500 ml
    热灭活的胎牛血清
    50 ml
    100x青霉素 - 链霉素 5.5 ml
    储存在4°C

  2. 10x PBS(pH 7.4)
    去离子H 2 O 2 / 800 ml
    NaCl
    80克
    KaCl
    2克
    lt; sub> 4< sub> 7H O
    26.6克
    KH 2 PO 4
    2.4 g
    用磁力搅拌器搅拌直至完全溶解
    用HCl
    调节至pH 7.4 使用H sub 2 O调整体积到1升
    高压灭菌
    灭菌 存储在RT
  3. 1×PBS(pH 7.4)
    去离子H 2 O 2 / 900 ml
    10x PBS(pH 7.4)
    100 ml
    高压灭菌
    灭菌 存储在RT
  4. 胰蛋白酶-EDTA
    无菌PBS(pH 7.4)
    90 ml
    10x胰蛋白酶-EDTA
    10 ml
    储存在4°C
  5. 含有4%甲醛的PBS
    PBS(pH 7.4)
    100 ml
    多聚甲醛
    4克
    在微波炉中煮PBS PBS
    注意:请勿关闭瓶盖。
    让PBS冷却约5分钟,然后加入多聚甲醛加温PBS。
    注意:在通风橱中工作,戴上手套和保护眼睛。
    用磁力搅拌器搅拌直至完全溶解
    储存于-20°C
  6. 含有0.1M甘氨酸的PBS
    PBS(pH 7.4)
    100 ml
    甘氨酸
    0.75克
    混合直至完全溶解
    高压灭菌
    灭菌 存储在RT
  7. 含有0.1%Triton-X100的PBS
    PBS(pH 7.4)
    100 ml
    Triton-X100
    0.1 ml
    混合直至完全溶解
    存储在RT
  8. 含有0.1%吐温20和2%BSA的PBS
    PBS(pH 7.4)
    100 ml
    吐温20
    0.1 ml
    BSA
    2克
    混合直至完全溶解
    过滤灭菌(0.2μm过滤器)
    储存在4°C
  9. 含有0.1%吐温20的PBS
    PBS(pH 7.4)
    1000 ml
    吐温20
    1 ml
    混合直至完全溶解
    存储在RT
  10. ELISA底物(磷酸硝基苯酯)
    去离子H 2 O 20ml
    缓冲区
    1 pc。
    基材平板电脑
    1 pc。
    混合直至完全溶解
    使用前立即准备

致谢

该协议改编自Glauser等人(2012a); Glauser et al。(2012b); 和Glauser 等人(2013)。

参考文献

  1. Adler,H.,Messerle,M.,Wagner,M。和Koszinowski,U.H。(2000)。 将鼠γ疱疹病毒68基因组克隆和诱变为感染性细菌人工染色体。 em> J Virol 74(15):6964-6974。
  2. Glauser,D.L.,Gillet,L。和Stevenson,P.G。(2012a)。 病毒颗粒内吞是鼠疱疹病毒-4中和的主要靶点。 J Gen Virol 93(Pt 6):1316-1327。
  3. Glauser,D.L.,Kratz,A.S。和Stevenson,P.G。(2012b)。 疱疹病毒糖蛋白在膜融合之前经历多种抗原变化。 em> 7(1):e30152。
  4. Glauser,D.L.,Milho,R.,Frederico,B.,May,J.S.,Kratz,A.-S.,Gillet,L.and Stevenson,P.G.(2013)。 糖蛋白B裂解对于疟原虫疱疹病毒4感染骨髓细胞很重要。 em> J Virol 87(19):10828-10842。
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How to cite this protocol: Glauser, D. L. and Stevenson, P. G. (2014). ELISA on Virus-Infected Cells. Bio-protocol 4(10): e1127. DOI: 10.21769/BioProtoc.1127; Full Text



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