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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   

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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)

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.

简介

循环抗心磷脂抗体是抗磷脂综合征的标志。 它也出现在许多自身免疫小鼠模型中,并且指示对自身抗原的耐受性的破坏。 该协议描述了一种可靠的方法来确定抗心磷脂在自身免疫小鼠模型中的相对血清滴度。

关键字:鼠标, ELISA, 自身免疫性, 抗心磷脂抗体

材料和试剂

  1. 心磷脂(Sigma-Aldrich,目录号:C0563)
  2. 乙醇
  3. 磷酸盐缓冲盐水(PBS)
  4. 吐温20
  5. Na 2 HPO 4(无水)
  6. NaH 2 PO 4(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)]
  11. ABTS过氧化物酶底物溶液A和B(Kirkegaard& Perry Laboratories,目录号:50-62-01)
  12. ABTS过氧化物酶终止液(Kirkegaard& Perry Laboratories,目录号:50-85-01)
  13. 10x PBS-Tween 20(参见配方)
  14. 阻止解决方案(参见配方)

设备

  1. 标准台式离心机
  2. Immulon 2HB板(Fisher Scientific,目录号:14-245-61)
  3. ELISA读数器

程序

  1. 向Immunlon 2HB板中加入100μl/孔的75μg/ml卡波醇的乙醇溶液,并使其在室温下干燥。
  2. 每孔加入100μl封闭溶液,在室温(RT)封闭平板90分钟 注意:FBS是beta糖蛋白I的来源。
  3. 弃去封闭溶液,用120μl/孔PBS洗板四次 注意:可以用ELISA板清洗机或用手动移液器进行PBS洗涤。
  4. 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平板中。
    注意:1:500稀释通常给予我们在12-22周龄的雄性和雌性NZW×BXSB F1小鼠中抗心磷脂血清水平的最佳结果。 建议进行滴定以达到最佳检测。
  5. 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
  6. 将板在37℃孵育2小时
  7. 弃去稀释的血清,用1×PBS-Tween洗涤板10次
  8. 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。
  9. 用1×PBS-Tween洗涤板10次
  10. 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。
  11. 在室温下在黑暗中显影板。 孵育时间取决于您的测定。
  12. 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。
  13. 使用波长为410 nm的ELISA读数器读取板。
  14. 使用高滴度血清样品的系列稀释确定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. Kahn,P.,Ramanujam,M.,Bethunaickan,R.,Huang,W.,Tao,H.,Madaio,MP,Factor,SM和Davidson,A。(2008)。通过BAFF阻断预防鼠抗磷脂综合征 关节炎风湿 58(9):2824-2834 。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用: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.
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