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[Bio101] Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Total Murine Immunoglobulin
[Bio101] 定量酶联免疫吸附法 (ELISA) 检测鼠血清中的免疫球蛋白浓度   

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Abstract

ELISA is an easy and relatively sensitive way to measure protein concentration. This protocol describes how to measure serum levels of murine total immunoglobulin of various isotypes. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.

Keywords: ELISA(ELISA), Mouse(老鼠), Immunoglobulin(免疫球蛋白), Serum(血清)

Materials and Reagents

  1. Coating antibody: goat anti-mouse IgX* unlabeled (Southern Biotech)
    Note: IgX*: the isotype of the Ig you wish to detect.
  2. Murine immunoglobulin of the desired isotype (Sigma- Aldrich, catalog number: M5284 )
  3. Horseradish peroxidase (HRP) conjugated goat anti-mouse isotype specific antibodies (Southern Biotech)
  4. 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) Peroxidase Substrate Solution A (Kirkegaard & Perry Laboratories, catalog number: 50-64-00 )
  5. ABTS Peroxidase Substrate Solution B (Kirkegaard & Perry Laboratories, catalog number: 50-65-00 )
  6. ABTS Peroxidase Stop Solution (Kirkegaard & Perry Laboratories, catalog number: 50-85-01 )
  7. 10x PBS-Tween 20 (see Recipes)
  8. Blocking solution (see Recipes)

Equipment

  1. Clear 96-well Microtest polystyrene assay plate (BD Biosciences)

Procedure

  1. Add 100 μl/well of coating antibody (1 μg/ml in PBS) to a 96-well Microtest assay plate.
  2. Wrap the plate with plastic wrap and incubate at 4 °C for overnight.
  3. Discard the coating antibody solution and wash the plate with 1x PBS-Tween 6 times.
  4. Dry the plate and add 100 μg of blocking solution per well to the plate.
  5. Incubate the plate at room temperature (RT) for 1.5 h.
  6. Discard the blocking solution and wash the plate with 1x PBS-Tween 5 times.
  7. Dilute the mouse serum in 1% BSA in PBS.
  8. Add 100 μl/well of diluted serum in duplicates or triplicate to the plate. Serum should be diluted in 1% BSA in PBS and titration is required to achieve optimal detection.
  9. Add 100 μl/well of serial dilutions of standard murine Ig (starting at 100 ng/ml) to the plate.
  10. Incubate the plate at 37 °C for 1 h.
  11. Discard the diluted serum and wash the plate with 1x PBS-Tween 10 times.
  12. Add 100 μl/well of HRP conjugated goat anti-mouse isotype specific antibodies (1/4,000 in PBS/1% BSA) to the plate and incubate at 37 °C for 1 h.
  13. Discard the diluted serum and wash the plate 10 times.
  14. Add 100 μl/well of 1:1 mix of ABTS Peroxidase Substrate Solution A and B to the plate.
  15. Develop the plate at RT in dark. Incubation times will vary depending on your assay.
  16. Stop the reaction by adding 100 μl /well of ABTS Peroxidase Stop Solution.
  17. Read the plate using an ELISA reader with a wavelength of 410 nm.
  18. Calculate the concentration of the serum samples using the standard curve established with the serial dilutions of standard murine Ig.

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. Mihara, M., Tan, I., Chuzhin, Y., Reddy, B., Budhai, L., Holzer, A., Gu, Y. and Davidson, A. (2000). CTLA4Ig inhibits T cell-dependent B-cell maturation in murine systemic lupus erythematosus. J Clin Invest 106(1): 91-101.

简介

ELISA是测量蛋白质浓度的简单且相对灵敏的方法。 该协议描述如何测量各种同种型的小鼠总免疫球蛋白的血清水平。 这个协议是在费恩斯坦医学研究所的Anne Davidson博士的实验室中开发或修改的。

关键字:ELISA, 老鼠, 免疫球蛋白, 血清

材料和试剂

  1. 包被抗体:山羊抗小鼠IgX *未标记(Southern Biotech)
    注意:IgX *:您希望检测的Ig的同种型。
  2. 所需同种型的鼠免疫球蛋白(Sigma-Aldrich,目录号:M5284)
  3. 辣根过氧化物酶(HRP)结合的山羊抗小鼠同种型特异性抗体(Southern Biotech)
  4. 2,2'-亚氨基双(3-乙基苯并噻唑啉-6-磺酸酯)(ABTS)过氧化物酶底物溶液A(Kirkegaard& Perry Laboratories,目录号:50-64-00)
  5. ABTS过氧化物酶底物溶液B(Kirkegaard& Perry Laboratories,目录号:50-65-00)
  6. ABTS过氧化物酶终止液(Kirkegaard& Perry Laboratories,目录号:50-85-01)
  7. 10x PBS-Tween 20(参见配方)
  8. 阻止解决方案(参见配方)

设备

  1. 清除96孔微量聚苯乙烯测定板(BD Biosciences)

程序

  1. 向96孔Microtest测定板中加入100μl/孔的包被抗体(1μg/ml PBS中)。
  2. 用塑料包裹包装板,并在4℃孵育过夜
  3. 弃去涂层抗体溶液,并用1×PBS-Tween洗板6次
  4. 干燥平板,每孔加入100微克封闭溶液到平板上
  5. 在室温(RT)下孵育平板1.5小时
  6. 弃去封闭液,用1×PBS-Tween洗板5次
  7. 在1%BSA的PBS溶液中稀释小鼠血清
  8. 加入100μl/孔稀释的血清一式两份或一式三份到板。 血清应在PBS中的1%BSA中稀释,需要滴定以达到最佳检测。
  9. 将100μl/孔的标准鼠Ig(以100ng/ml开始)的连续稀释液加入板中
  10. 将板在37℃孵育1小时
  11. 弃去稀释的血清,用1×PBS-Tween洗涤板10次
  12. 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PBS/1%BSA中),并在37℃孵育1小时。
  13. 弃去稀释的血清,洗板10次
  14. 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。
  15. 在室温下在黑暗中显影板。 孵育时间取决于您的测定。
  16. 通过加入100μl/孔的ABTS过氧化物酶终止溶液停止反应。
  17. 使用波长为410 nm的ELISA读数器读取板。
  18. 使用标准鼠Ig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
    混合以溶解并将pH调节至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。 这项工作得到了NY SLE基金会(RB),Rheuminations,NIH AI082037和AR的资助 049938-01,NIH(PO1AI51392和PO1 AI51392的流式细胞术和蛋白质表达和四聚体核心)。

参考文献

  1. Mihara,M.,Tan,I.,Chuzhin,Y.,Reddy,B.,Budhai,L.,Holzer,A.,Gu,Y.and Davidson,A。(2000)。 CTLA4Ig抑制小鼠系统性红斑狼疮中的T细胞依赖性B细胞成熟。 em Clin J Clin Invest 106(1):91-101。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, Z. (2011). Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Total Murine Immunoglobulin. Bio-protocol Bio101: e33. DOI: 10.21769/BioProtoc.33;
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