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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, 5822 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.

材料和试剂

  1. 来自小牛胸腺的组蛋白(Roche Diagnostics,目录号:10223565001)
  2. 磷酸盐缓冲盐水(PBS)
  3. 吐温20
  4. Na 2 HPO 4(无水)
  5. NaH 2 PO 4(无水)
  6. NaCl
  7. 胎牛血清(FBS)(Hyclone)
  8. 牛血清白蛋白(BSA)
  9. 辣根过氧化物酶(HRP)缀合的山羊抗小鼠同种型特异性抗体[Southern Biotech,目录号:1040-05(IgA); 1030-05(IgG); 1021-05(IgM)]
  10. ABTS过氧化物酶底物溶液A和B(Kirkegaard& Perry Laboratories,目录号:50-62-01)
  11. ABTS过氧化物酶终止液(Kirkegaard& Perry Laboratories,目录号:50-85-01)
  12. 10x PBS-Tween 20(参见配方)
  13. 阻止解决方案(参见配方)

设备

  1. 标准台式离心机
  2. Falcon 96孔ELISA板(BD Biosciences,目录号:35-3915)
  3. ELISA读数器
  4. parafilm

程序

  1. 向Falcon板中加入100μl/孔的10μg/ml组蛋白的PBS溶液
  2. 用石蜡膜密封该板,并在4℃下孵育过夜。
  3. 弃去组蛋白溶液,用1x PBS-Tween洗板5次。 在纸巾上擦干。
    注意:可以使用ELISA板清洗机或通过手动移入和移出1x PBS-Tween进行洗涤。
  4. 每孔加入100μl封闭溶液,在室温(RT)封闭平板90分钟
  5. 弃去封闭溶液,用1x PBS-Tween洗涤板4次,每次10次
  6. 在PBS中的1%BSA中稀释小鼠血清,并以100μl/孔一式两份或一式三份加入板中。 建议滴定以确定最佳稀释度。
  7. 制备高滴度血清样品的系列稀释液,并将连续稀释液加入板中
  8. 将板在37℃孵育1小时
  9. 弃去稀释的血清,用1×PBS-Tween洗涤板10次
  10. 向板中加入100μl/孔的HRP缀合的山羊抗小鼠同种型特异性抗体(1/4,000在1%BSA/PBS中),并在37℃孵育1小时。
  11. 用1×PBS-Tween洗涤板10次
  12. 向板中加入100μl/孔的ABTS过氧化物酶底物溶液A和B的1:1混合物。
  13. 在室温下在黑暗中显影板。 孵育时间取决于您的测定。
  14. 通过加入100μl/孔的ABTS过氧化物酶终止溶液停止反应。
    注意:反应停止后,需要在30分钟内读取反应板。
  15. 使用波长为410 nm的ELISA读数器读取板。
  16. 使用高滴度血清样品的系列稀释液建立的标准曲线计算血清样品的浓度

食谱

  1. 10x PBS-Tween 20 [0.1M PBS,0.5%Tween 20(pH 7.4)]
    Na 2 HPO 4(无水)
    10.9克
    NaH 2 PO 4(无水)
    3.2克
    NaCl  
    90克
    蒸馏水
    1000 ml
    混合溶解并调节pH至7.4,然后加入5ml吐温20,将该溶液在室温下储存。 在使用前用蒸馏水稀释1:10,必要时调节pH值
  2. 封锁解决方案
    5%FBS和3%BSA的PBS溶液中

致谢

该方案在Anne Davidson博士在Feinstein Institute for Medical Research,NY,USA的实验室中开发或修改。 这项工作得到NY SLE基金会(RB),Rheuminations,NIH AI082037和AR 049938-01,NIH(PO1 AI51392和PO1 AI51392的流式细胞术和蛋白质表达和四聚体核)的赠予的支持。

参考文献

  1. Martin,D.A.,Zhang,K.,Kenkel,J.,Hughes,G.,Clark,E.,Davidson,A.and Elkon,K.B。(2007)。 过继转移的树突状细胞刺激的自身免疫由alphabeta和gammadelta T细胞启动,但不需要MyD88 信号传导。 79(9):5819-5828。
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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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