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Autoimmune disease is characterized with the break of tolerance against self antigens. Anti-histone antibodies can be found in the majority of patients with system lupus erythematosus (SLE) and a number of lupus-prone mouse strains. This protocol describes a reliable method to determine the relative serum titers of anti-histone antibodies in these mice.

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[Bio101] Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Murine Anti-histone Antibodies
[Bio101] 酶联免疫吸附法(ELISA)定量检测鼠抗组蛋白抗体的血清水平

免疫学 > 抗体分析 > 抗体检测
作者: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
6/20/2012, 5466 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.228

[Abstract] Autoimmune disease is characterized with the break of tolerance against self antigens. Anti-histone antibodies can be found in the majority of patients with system lupus erythematosus (SLE) and a number of lupus-prone mouse strains. This protocol describes a reliable method to determine the relative serum titers of anti-histone antibodies in these mice.
Keywords: ELISA(ELISA), Mouse(鼠标), Lupus(系统性红斑狼疮), Autoimmune(自身免疫性), Anti-histone antibody(抗组蛋白抗体)

[Abstract] 自身免疫疾病是以自身抗原耐受的崩溃为特征。在大多数系统性红斑狼疮(SLE)病人和一些狼疮易感鼠中能找到抗组蛋白抗体,这个规程描述了一个可靠的在这些鼠中检测相关的抗组蛋白抗体血清滴度的方法。

Materials and Reagents

  1. Histone from calf thymus (Roche Diagnostics, catalog number: 10223565001 )
  2. Phosphate buffered saline (PBS)
  3. Tween 20
  4. Na2HPO4 (anhydrous)
  5. NaH2PO4 (anhydrous)
  6. NaCl
  7. Fetal bovine serum (FBS) (Hyclone)
  8. Bovine serum albumin (BSA)
  9. Horseradish peroxidase (HRP) conjugated goat anti-mouse isotype specific antibodies [Southern Biotech, catalog number: 1040-05 (IgA); 1030-05 (IgG); 1021-05 (IgM)]
  10. ABTS Peroxidase Substrate Solution A and B (Kirkegaard & Perry Laboratories, catalog number: 50-62-01 )
  11. ABTS Peroxidase Stop Solution (Kirkegaard & Perry Laboratories, catalog number: 50-85-01 )
  12. 10x PBS-Tween 20 (see Recipes)
  13. Blocking solution (see Recipes)

Equipment

  1. Standard bench-top centrifuge
  2. Falcon 96-well ELISA plates (BD Biosciences, catalog number: 35-3915 )
  3. ELISA reader
  4. Parafilm

Procedure

  1. Add 100 μl/well of 10 μg/ml histone in PBS to a Falcon plate.
  2. Seal the plate with Parafilm and incubate at 4 °C overnight.
  3. Discard histone solution and wash the plate 5 times with 1x PBS-Tween. Dry the plate on paper towel.
    Note: Washes can be done with an ELISA plate washer or by manually pipeting in and out 1x PBS-Tween.
  4. Add 100 μl of blocking solution per well and block the plate at room temperature (RT) for 90 min.
  5. Discard the blocking solution and wash the plate four times with 1x PBS-Tween 10 times.
  6. Dilute the mouse serum in 1% BSA in PBS and add 100 μl/well in duplicates or triplicates to the plate. Titration is recommended to determine the optimal dilution.
  7. Make serial dilutions of a high titer serum sample and add the serial dilution to the plate.
  8. Incubate the plate at 37 °C for 1 h.
  9. Discard the diluted serum and wash the plate with 1x PBS-Tween 10 times.
  10. Add 100 μl/well of HRP conjugated goat anti-mouse isotype specific antibodies (1/4,000 in 1% BSA/PBS) to the plate and incubate at 37 °C for 1 h.
  11. Wash the plate with 1x PBS-Tween 10 times.
  12. Add 100 μl/well of 1:1 mix of ABTS Peroxidase Substrate Solution A and B to the plate.
  13. Develop the plate at RT in dark. Incubation times will vary depending on your assay.
  14. Stop the reaction by adding 100 μl/well of ABTS Peroxidase Stop Solution.
    Note: The plate needs to be read within 30 min once the reaction is stopped.
  15. Read the plate using an ELISA reader with a wavelength of 410 nm.
  16. Calculate the concentration of the serum samples using the standard curve established with the serial dilutions of the high titer serum sample.

Recipes

  1. 10x PBS-Tween 20 [0.1 M PBS, 0.5% Tween 20 (pH 7.4)]
    Na2HPO4 (anhydrous)
    10.9 g
    NaH2PO4 (anhydrous)
    3.2 g
    NaCl  
    90 g
    Distilled water
    1,000 ml
    Mix to dissolve and adjust pH to 7.4 and then add 5 ml of Tween 20, store this solution at RT. Dilute 1:10 with distilled water before use and adjust pH if necessary.
  2. Blocking solution
    5% FBS and 3% BSA in PBS

Acknowledgments

This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).

References

  1. Martin, D. A., Zhang, K., Kenkel, J., Hughes, G., Clark, E., Davidson, A. and Elkon, K. B. (2007). Autoimmunity stimulated by adoptively transferred dendritic cells is initiated by both alphabeta and gammadelta T cells but does not require MyD88 signaling. J Immunol 179(9): 5819-5828.

材料与试剂

          

1.         来自小牛胸腺的组蛋白 (Roche Applied Sciences 公司,货号#10223565001)

2.         PBS

3.         吐温 20

4.         Na2HPO4 (无水)

5.         NaH2PO4 (无水)

6.         NaCl

7.         胎牛血清 (FBS, Hyclone)

8.         牛血清白蛋白 (BSA)

9.         辣根过氧化物 (HRP) 连接的山羊抗鼠同型特异性抗体 (Southern Biotech公司, IgA 货号#1040-05, IgG货号#1030-05, IgM货号#1021-05)

10.     ABTS 过氧化物酶底物溶液AB (货号# 50-62-01)

11.     ABTS 过氧化物酶终止溶液(货号# 50-85-01)

 

设备

 

1.         离心机

2.         Falcon 96- ELISA  (BD 公司,货号#35-3915)

3.         ELISA 酶标仪

4.         石蜡封口膜

 

程序

 

1.         Falcon 板中每孔加入10 μg/ml PBS溶解的组蛋白100 μl

2.         用封口膜封闭板,4℃孵育过夜。

3.         弃组蛋白溶液,用1X PBS-吐温溶液洗板5次,用纸巾干燥。

注意: 可用ELISA洗板机洗板或是用移液管来回吸取1X PBS-吐温手动洗板。

4.         每孔加入100 μl 封闭液,室温下板封闭90分钟。

5.         弃封闭液并且使用1X PBS-吐温溶液洗板10 times.

6.         使用PBS配制的1% BSA 稀释鼠血清并且每孔加入 100 μl,每个板上做两到三个重复。滴定采用最佳稀释条件进行检测。

7.         高滴度血清样品做系列稀释并将系列稀释液加入到板中。

8.         37oC 孵育板1小时。

9.         弃稀释血清,用1X PBS-吐温溶液洗板10次。

10.     板中每孔加入辣根过氧化物 (HRP) 连接的 山羊抗鼠同型特异性抗体100 μl (1% BSA/PBS 1:4000稀释) 并在37oC 孵育 1小时。

11.      1X PBS-吐温溶液洗板 10 次。

12.     板中每孔加入1:1 混合的 ABTS过氧化物酶底物 A  B 100 μl 

13.     室温下暗处进行板显色。孵育时间依据你的检测要求。

14.     每孔加入 ABTS 过氧化物酶终止溶液100 μl终止反应。

注意: 一旦反应终止,板必须在30分钟内读完。

15.     读板在ELISA酶标仪中读取数值,波长为410nm.

16.     依据高滴度血清样品系列稀释建立的标准曲线计算血清样品浓度。

 

配制溶液

 

1.         10X PBS-吐温 20 (0.1M PBS, 0.5% 吐温20, pH 7.4):

Na2HPO4 (无水) ------------------------ 10.9 g

NaH2PO4 (无水) ------------------------ 3.2 g

氯化钠 -------------------------------------- 90 g

蒸馏水 ----------------------------------- 1000 ml

混合溶解并调节pH  7.4 ,然后加入5 ml 吐温 20

溶液室温保存。使用前1:10蒸馏水稀释,必要的话还要调整PH

2.         封闭液: PBS配制的5% 胎牛血清 (FBS, Hyclone)  3% 牛血清白蛋白(BSA) 

 

参考文献

 

1.         Martin D.A., Zhang K., Kenkel J., Hughes G., Clark E., Davidson A., Elkon K.B. (2007). Autoimmunity stimulated by adoptively transferred dendritic cells is initiated by both alphabeta and gammadelta t cells but does not require myd88 signaling. Journal of Immunology 179(9): 5819-28. 

 

 

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How to cite this protocol: Liu, Z. (2012). Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Murine Anti-histone Antibodies. Bio-protocol Bio101: e228. DOI: 10.21769/BioProtoc.228; Full Text



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