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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.

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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

免疫学 > 抗体分析 > 抗体检测
作者: Huagang Zhang
Huagang ZhangAffiliation: Albert Einstein College of Medicine, Yeshiva University, New York City, USA
For correspondence: huagangzhang@gmail.com
Bio-protocol author page: a21
Vol 2, Iss 6, 3/20/2012, 8665 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.120

[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.

[Abstract] IFN-γ(干扰素-γ)主要产生于激活的T细胞和NK细胞。TH1型响应的特点是通过辅助T细胞和细胞毒T细胞产生IFN-γ,所以高水平IFN-γ的产生是由于寄主应对细胞内的病原体产生了有效的防御。酶联免疫斑点法通常用于评估抗原特异的T细胞的功效,即检测IFN-γ的释放。酶联免疫斑点法是一种非常灵敏的免疫检测方法,它可以在单细胞水平检测细胞因子的分泌。酶联免疫斑点法是最灵敏的细胞检测方法之一,它检测的灵敏度可以达到1:100000个细胞。依赖于化学物质的分析,酶联免疫斑点分析可以比传统的ELISA灵敏20到200倍。

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.

材料和试剂

 

I.   ELISpot Kits

1.       Mouse IFN-γ ELISpot kit (ALP) (Mabtech 3321-2A)

2.       Human IFN-γ ELISpot kit (ALP) (Mabtech 3420-2A)

注释:以上的ELISpot Kits可用性已经经过作者测试,用户也可以用按自己的需要换别的替代。

II.   其它材料

3.       SIGMAFASTTM  BCIP/NBT (Sigma B5655)

4.       1X Phosphate Buffered Saline (PBS)

5.       Wash Buffer: 1X PBS, 0.1% Tween-20 (Santa cruz biotechnology, sc-29113)

6.       Phorbol 12-myristate 13-acetate (PMA) (Sigma 79346)

7.       钙离子载体Ionomycin (Sigma I9657)

 

设备

 

1.       AID EliSpot Reader classic (Autoimmun Diagnostika GmbH)

 

步骤

 

1.       第一天:准备ELISpot板(无菌环境)

1)          用无菌的PBS pH 7.4,稀释包被抗体(AN18)(16μl 抗体稀释在8ml PBS, 1:500)。

2)          ELISpot板从包装里拿出来,每个孔加75μl抗体溶液,4-8过夜孵育。

2.       第二天:在板上孵育细胞(无菌环境)

1)            倒掉过多的抗体,用无菌的Wash Buffer (PBS-T)洗板5次,每个孔250μl

2)            每个孔加250μl的培养基,培养基中包含与用于悬浮细胞的培养液相同的10%的血清(通常是FBS),置于室温孵育至少2个小时。

3)            倒掉培养基,并加入含有诱导物的细胞悬浮液。一般要做如下几组:不含诱导物的细胞、混有抗原(蛋白或多肽)的细胞和含PMA(10ng/ml)和钙离子载体Ionomycin (500ng/ml)的细胞(阳性对照)。每个孔中的细胞数目决定于你的样品中IFN释放细胞的比例,通常是从2x1042.5x105。如果靶细胞是多肽脉冲T2T2 pulsed with peptide)或瘤细胞,那么T细胞/靶细胞的比例需要满足达到最佳的检测效果。

4)            把板置于37湿润的培养箱,CO2的浓度为5%,培养12-48小时。在这期间不要挪动板,并设法避免蒸发。

3.       第三天:在板上孵育细胞(无菌环境)

1)            倒空板上的细胞,每个孔加250μl的水,保持在冰上10分钟。

2)            Wash Buffer洗板5次,每个孔250μl

3)            稀释检测抗体(R4-6A2-biotin)至浓度为0.5-1μg/ml4μl抗体稀释在8ml PBS1:2000)。用加有0.5% FBSPBS稀释。每个孔加75μl37下孵育2个小时。

4)            Wash Buffer洗板5次,每个孔250μl

5)            PBS-0.5% FBS稀释Streptavidin-ALP (1:1000)每个孔加100μl,室温孵育1个小时。

6)            Wash Buffer洗板5次,每个孔250μl

7)            10ml ddH2O中溶解一片BCIP/NBT即配成底物溶液,每个孔加100μl

8)            把板放在黑暗中反应,直到有清楚的斑点显示出来。

9)            用大量的自来水清洗终止颜色反应。如果有必要,把板从盘里取出来漂洗干净底下的膜。

10)        晾干板,用ELISpot reader (AID EliSpot Reader classic)或在解剖镜下(不推荐)检测并统计斑点数。

11)        板在避光的条件下室温保存。

 

参考文献

 

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., 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. Proceedings of the National Academy of Sciences of the United States of America 97(22): 12198-203. 

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., Chen W.F. (2004). The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients. Clinical Cancer Research 10(20): 6946-55. 

 

 

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How to cite this protocol: Zhang, H. (2012). INF-gamma Release ELISpot Assay. Bio-protocol 2(6): e120. DOI: 10.21769/BioProtoc.120; Full Text



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but then you will have security iesuss and also it will reqire you to have a computer on all the time. also if its hosted wqith some one else they usualy will look after the computer maintanance and will makesure its upp 99.9% of the timebut i agree personal servers is? the best (dedicated servers for the win!)

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