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INF-gamma Release ELISpot Assay
酶联免疫斑点法(ELISpot)测定INF-γ释放水平

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Abstract

IFN-γ (Interferon-gamma) is produced mainly by activated T cells and NK cells. Production of IFN-γ (Interferon-gamma) by helper T cells as well as cytotoxic T cells is a hallmark of the TH1-type phenotype, thus, high-level production of IFN-γ (Interferon-gamma) is typically associated with effective host defense against intracellular pathogens. The Enzyme-Linked ImmunoSpot (ELISpot) assay is commonly used to assess the function of antigen specific T cells by detecting IFN-γ release. The ELISpot assay is a very sensitive immunoassay, allowing the detection of a secreted cytokine at the single cell level. With detection levels that can be as low as one cell in 100,000, the ELISpot is one of the most sensitive cellular assays available. Depending on the substance analyzed, the ELISpot assay is between 20 and 200 times more sensitive than a conventional ELISA.

Materials and Reagents

  1. Mouse IFN-γ ELISpot kit (ALP) (Mabtech, catalog number: 3321-2A )
  2. Human IFN-γ ELISpot kit (ALP) (Mabtech, catalog number: 3420-2A )
    Note: The above ELISpot kits have been tested by the author and may be substituted with the kits desired by users.
  3. SIGMAFAST™ BCIP®/NBT (Sigma-Aldrich, catalog number: B5655 )
  4. Phosphate buffered saline (PBS)
  5. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
  6. Ionomycin (Sigma-Aldrich, catalog number: I9657 )
  7. Wash buffer (Santa Cruz Biotechnology, catalog number: sc-29113 ) (see Recipes)

Equipment

  1. AID EliSpot Reader classic (Autoimmun Diagnostika GmbH)
  2. ELISpot plate
  3. 37 °C humidified incubator

Procedure

Day 1: Preparation of ELISpot plate (sterile conditions)

  1. Dilute the coating antibody (AN18) to 2-15 μg/ml in sterile PBS, pH 7.4 (16 μl antibody in 8 ml PBS, 1:500).
  2. Remove the ELISpot plate from the package and add 75 μl/well of the antibody solution and incubate overnight at 4-8 °C.

Day 2: Incubation of cells in plate (sterile conditions)

  1. Remove excess antibody and wash plate 5 times with sterile wash buffer (PBS-T), 250 μl/well.
  2. Add 250 μl/well of medium containing 10% of the same serum as used for the cell suspensions (usually FBS). Incubate for at least 2 h at room temperature (RT).
  3. Remove the medium and add the cells suspension including possible stimulatory agents. Typical groups include cells with no stimulant, cells with antigens (protein or peptides) and cells with PMA (10 ng/ml) and Ionomycin (500 ng/ml) (positive control).
  4. The number of cells in each well depends on the frequency of the IFN-γ releasing cells in your sample, usually from 2 x 104 to 2.5 x 105. If target cells are used such as T2 pulsed with peptide or tumor cells, the T cells/target cells ratio needs to be determined to achieve the optimal detection.
  5. Put the plate in a 37 °C humidified incubator with 5% CO2 and incubate for 12-48 h. Do not move the plate during this time and take measures to avoid evaporation.

Day 3: Incubation of cells in plate (sterile conditions)

  1. Remove the cells by emptying the plate and add 250 μl/well H2O and keep it on ice for 10 min.
  2. Wash the plate 5 times with wash buffer, 250 μl/ well.
  3. Dilute the detection antibody (R4-6A2-biotin) to 0.5-1 μg/ml (4 μl antibody in 8 ml PBS, 1:2000) in PBS containing 0.5% FBS. Add 75 μl/ well and incubate for 2 h at 37 °C.
  4. Wash plate 5 times with wash buffer, 250 μl/ well.
  5. Dilute the Streptavidin-ALP (1:1,000) in PBS-0.5% FBS and add 100 μl/well. Incubate for 1 h at RT.
  6. Wash plate 5 times with wash buffer, 250 μl/ well.
  7. Prepare the substrate solution by dissolving one BCIP/NBT tablet in 10 ml ddH2O and add 100 μl/ well.
  8. Develop the plate in the dark until distinct spots emerge.
  9. Stop color development by washing extensively in tap water. If desirable, remove the plate from the tray or the inderdrain and rinse the underside of the membrane.
  10. Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection microscope (not recommended).
  11. Store plate in the dark at RT.

Recipes

  1. Wash buffer
    1x PBS
    0.1% Tween-20

Acknowledgments

This protocol was previously used in Jager et al. (2000) and Shang et al. (2004).

References

  1. Jager, E., Gnjatic, S., Nagata, Y., Stockert, E., Jager, D., Karbach, J., Neumann, A., Rieckenberg, J., Chen, Y. T., Ritter, G., Hoffman, E., Arand, M., Old, L. J. and Knuth, A. (2000). Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers. Proc Natl Acad Sci U S A 97(22): 12198-12203.
  2. Shang, X. Y., Chen, H. S., Zhang, H. G., Pang, X. W., Qiao, H., Peng, J. R., Qin, L. L., Fei, R., Mei, M. H., Leng, X. S., Gnjatic, S., Ritter, G., Simpson, A. J., Old, L. J. and Chen, W. F. (2004). The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients. Clin Cancer Res 10(20): 6946-6955.

简介

119.txt

材料和试剂

  1. 小鼠IFN-γELISpot试剂盒(ALP)(Mabtech,目录号:3321-2A)
  2. 人IFN-γELISpot试剂盒(ALP)(Mabtech,目录号:3420-2A)
    注意:以上ELISpot套件已经由作者测试,可以替换为用户所需的套件。
  3. SIGMAFAST TM BCIP /NBT(Sigma-Aldrich,目录号:B5655)
  4. 磷酸盐缓冲盐水(PBS)
  5. 佛波醇12-豆蔻酸酯13-乙酸酯(PMA)(Sigma-Aldrich,目录号:79346)
  6. 离子霉素(Sigma-Aldrich,目录号:I9657)
  7. 洗涤缓冲液(Santa Cruz Biotechnology,目录号:sc-29113)(参见Recipes)

设备

  1. AID EliSpot Reader classic(Autoimmun Diagnostika GmbH)
  2. ELISpot牌
  3. 37℃培养箱

程序

第1天:酶联免疫斑点板的制作(无菌条件下)

  1. 稀释涂料抗体(AN18),以2-15微克在无菌PBS/毫升,pH 7.4的(在8ml PBS中16微升抗体,1:500)。
  2. 从包装中取出酶联免疫斑点板,并添加75微升/孔的抗体溶液,在4-8℃下孵育过夜。

第2天:在平板中孵育细胞(无菌条件)

  1. 除去过量的抗体,用无菌洗涤缓冲液(PBS-T),250μl/孔洗涤板5次。
  2. 加入250μl/孔的含有10%与用于细胞悬浮液(通常为FBS)相同的血清的培养基。在室温(RT)下孵育至少2小时。
  3. 取出培养基,加入细胞悬浮液,包括可能的刺激剂。典型的组包括没有刺激物的细胞,具有抗原的细胞(蛋白质或肽)和具有PMA(10ng/ml)和离子霉素(500ng/ml)(阳性对照)的细胞。
  4. 每个孔中的细胞数目取决于样品中IFN-γ释放细胞的频率,通常为2×10 4至2.5×10 5个。如果使用靶细胞,例如用肽或肿瘤细胞脉冲的T2,则需要确定T细胞/靶细胞比率以实现最佳检测。
  5. 将板置于37℃,5%CO 2的湿润培养箱中孵育12-48小时。在此期间不要移动平板,并采取措施避免蒸发

第3天:在平板中孵育细胞(无菌条件)

  1. 通过排空板并加入250μl/孔H 2 O 2并在冰上保持10分钟来除去细胞。
  2. 用洗涤缓冲液(250μl/孔)洗板5次
  3. 在含有0.5%FBS的PBS中稀释检测抗体(R4-6A2-生物素)至0.5-1μg/ml(4ml抗体在8ml PBS中,1:2000)。 加入75μl/孔,并在37℃下孵育2小时
  4. 用洗涤缓冲液洗涤平板5次,250μl/孔
  5. 在PBS-0.5%FBS中稀释链霉抗生物素蛋白-ALP(1:1,000),并加入100μl/孔。 在RT孵育1小时。
  6. 用洗涤缓冲液洗涤平板5次,250μl/孔
  7. 通过将一个BCIP/NBT片剂溶解在10ml ddH 2 O中并加入100μl/孔来制备底物溶液。
  8. 在黑暗中显影板,直到出现明显的斑点
  9. 通过在自来水中大量洗涤来停止显色。 如果需要,请从托盘或内衬中取出板,并冲洗膜的下侧
  10. 让板干燥。 检查和计数ELISpot阅读器或解剖显微镜中的斑点(不推荐)。
  11. 将板在室温下置于黑暗中。

食谱

  1. 洗涤缓冲液
    1x PBS
    0.1%Tween-20

致谢

该协议以前在Jager等人(2000)和Shang等人(2004)中使用。

参考文献

  1. Jager,E.,Gnjatic,S.,Nagata,Y.,Stockert,E.,Jager,D.,Karbach,J.,Neumann,A.,Rieckenberg,J.,Chen,YT,Ritter,G.,Hoffman ,E.,Arand,M.,Old,LJand Knuth,A。(2000)。 主要NY-ESO-1免疫的诱导:肽接种患者中的CD8 + T淋巴细胞和抗体反应 with NY-ESO-1 + cancer。 Proc Natl Acad Sci USA 97(22):12198-12203。
  2. G,G,G,G,G,G,G,G,G,G G,G G G G G G G G G G G G G G G G G G G G G G G G 。,Simpson,AJ,Old,LJand Chen,WF(2004)。 肝细胞中HLA-A2限制性NY-ESO-1b肽的自发性CD8 + T细胞应答 癌症患者。 Clin Cancer Res 10(20):6946-6955
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引用:Zhang, H. (2012). INF-gamma Release ELISpot Assay. Bio-protocol 2(6): e120. DOI: 10.21769/BioProtoc.120.
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