Welcome guest, Sign in

Home

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Total Murine Immunoglobulin

Immunology > Antibody analysis > Antibody detection
Author: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, 2/5/2011, 6973 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.33

[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, Mouse, Immunoglobulin, Serum

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.


How to cite this protocol: 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; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register