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The circulating anticardiolipin antibody is a hallmark of antiphospholipid syndrome. It also appears in a number of autoimmune mouse models and is indicative of the break of tolerance against self antigens. This protocol describes a reliable method to determine the relative serum titer of anticardiolipin in autoimmune mouse models.

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Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Murine Anti-cardiolipin Antibodies
酶联免疫吸附法(ELISA)定量检测鼠抗心磷脂抗体

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
Vol 2, Iss 12, 6/20/2012, 6202 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.227

[Abstract] The circulating anticardiolipin antibody is a hallmark of antiphospholipid syndrome. It also appears in a number of autoimmune mouse models and is indicative of the break of tolerance against self antigens. This protocol describes a reliable method to determine the relative serum titer of anticardiolipin in autoimmune mouse models.
Keywords: Mouse(鼠标), ELISA(ELISA), Autoimmune(自身免疫性), Anti-cardiolipin antibody(抗心磷脂抗体)

[Abstract] 抗心磷脂抗体循环是抗磷脂综合症的标志。它也表现在一些自身免疫鼠模型中并且显示自身抗原耐受的崩溃。这个规程描述了一个可靠的在自身免疫鼠模型中检测抗心磷脂相关血清滴度的方法。

Materials and Reagents

  1. Cardiolipin (Sigma-Aldrich, catalog number: C0563 )
  2. Ethanol
  3. Phosphate buffered saline (PBS)
  4. Tween 20
  5. Na2HPO4 (anhydrous)
  6. NaH2PO4 (anhydrous)
  7. NaCl
  8. Fetal bovine serum (FBS) (Hyclone)
  9. Bovine serum albumin (BSA)
  10. Horseradish peroxidase (HRP) conjugated goat anti-mouse isotype specific antibodies [Southern Biotech, catalog number: 1040-05 (IgA); 1030-05 (IgG); 1021-05 (IgM)]
  11. ABTS Peroxidase Substrate Solution A and B (Kirkegaard & Perry Laboratories, catalog number: 50-62-01 )
  12. ABTS Peroxidase Stop Solution (Kirkegaard & Perry Laboratories, catalog number: 50-85-01 )
  13. 10x PBS-Tween 20 (see Recipes)
  14. Blocking solution (see Recipes)

Equipment

  1. Standard bench-top centrifuge
  2. Immulon 2HB plates (Fisher Scientific, catalog number: 14-245-61 )
  3. ELISA reader

Procedure

  1. Add 100 μl/well of 75 μg/ml cartiolipin in ethanol to an Immunlon 2HB plate and allow it to dry at room temperature.
  2. Add 100 μl of blocking solution per well and block the plate at room temperature (RT) for 90 min.
    Note: FBS is the source of beta glycoprotein I.
  3. Discard the blocking solution and wash the plate four times with 120 μl/well PBS.
    Note: Washes can be done with an ELISA plate washer or by manually pipeting in and out PBS.
  4. Dilute the mouse serum in 1% BSA in PBS and add 100 μl/well in duplicates or triplicates to the plate.
    Note: A 1:500 dilution generally gave us optimal results of serum levels of anti-cardiolipin in 12-22 week old male and female NZW x BXSB F1 mice. Titration is recommended to achieve optimal detection.
  5. Make serial dilutions of a high titer serum sample and add the serial dilution to the plate.
  6. Incubate the plate at 37 °C for 2 h.
  7. Discard the diluted serum and wash the plate with 1x PBS-Tween 10 times.
  8. 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.
  9. Wash the plate with 1x PBS-Tween 10 times.
  10. Add 100 μl/well of 1:1 mix of ABTS Peroxidase Substrate Solution A and B to the plate.
  11. Develop the plate at RT in dark. Incubation times will vary depending on your assay.
  12. 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.
  13. Read the plate using an ELISA reader with a wavelength of 410 nm.
  14. 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. Kahn, P., Ramanujam, M., Bethunaickan, R., Huang, W., Tao, H., Madaio, M. P., Factor, S. M. and Davidson, A. (2008). Prevention of murine antiphospholipid syndrome by BAFF blockade. Arthritis Rheum 58(9): 2824-2834.

材料与试剂

          

1.         心磷脂(Sigma 公司,货号#C0563)

2.         乙醇

3.         PBS

4.         吐温 20

5.         Na2HPO4 (无水)

6.         NaH2PO4 (无水)

7.         NaCl

8.         胎牛血清 (FBS, Hyclone)

9.         牛血清白蛋白 (BSA)

10.     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)

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

12.     ABTS 过氧化物酶终止溶液(货号# 50-85-01)

 

设备

 

1.         离心机

2.         联反应2HB (Fisher 科学,货号# 14-245-61)

3.         ELISA 酶标仪

 

程序

 

1.         在一个 Immunlon 2HB 板的每孔加入75 μg/ml 乙醇溶解的心磷脂溶液100 μl 并让它在室温下干燥。

2.         每孔中加入100 μl 封闭溶液,于室温下封闭板90分钟。

注意: 胎牛血清是β糖蛋白的来源。

3.         弃封闭液,每孔120 μl PBS 洗板4次。

注意: 洗板可采用ELISA 板洗板器洗或是手动使用移液管来回吸取PBS洗板。

4.         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。

注意: 1:500 稀释一般可得到12-22 周龄的雄性和雌性NZW x BXSB F1 鼠抗心磷脂血清水平的结果. 滴定检测最好达到最佳检测条件。

5.         高滴度血清样品做系列稀释并加入板中。

6.         37 oC 孵育板 2小时。

7.         弃稀释的血清, 并用 1X PBS-吐温洗板10 次。

8.         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/PBS1:4000稀释) 并在37oC 孵育 1小时。

9.          1X PBS-吐温洗板 10 次。

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

11.     室温下暗处进行板显色。孵育时间依据你的检测要求。

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

注意: 一旦反应终止后板必须在30分钟内读完。

13.     读板在ELISA酶标仪中读取数值,波长为410nm.

14.     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.         Kahn P., Ramanujam M., Bethunaickan R., Huang W., Tao H., Madaio M.P., Factor S.M., Davidson A. (2008). Prevention of murine antiphospholipid syndrome by BAFF blockade. Arthritis and Rheumatism 58(9): 2824-34.

 

 

 

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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-cardiolipin Antibodies. Bio-protocol 2(12): e227. DOI: 10.21769/BioProtoc.227; Full Text



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