I want to perform an ELISA test and I want to know what kind of buffer I should use to dissolve and dilute my antibody and antigen. The buffer for the antibody/antigen should enable them to conjugate along antigen-antibody binding.
1. For coating antigen to plate by yourself, use phosphate-buffered saline (PBS), pH 7.5.
2. If the antigen has been coated on the plate (like commercially available plate), either PBS or 50 mM Tris-buffer saline (TBS), pH 7.5, are fine for antigen-antibody reaction. To prevent degradation of both antigen and antibodies, I added 0.1%-5% BSA, goat or human serum to the buffer. Rabbit and horse serum yields more background.
thank you Kuan-Chih Chow for you reply. Could you let me know the buffer for capturing antibody and should be compatible with the buffer of antigen.
1. What do you mean? To coating antibodies onto the ELISA plate? For that, dissolving antibodies (1-5 ng/microliter) in PBS is fine.
2. Otherwise, as indicated in the previous answer 2, PBS or 50 mM Tris-buffer saline (TBS), pH 7.5, with 0.1% BSA, goat or human serum, is fine for antigen-antibody reaction.
3. To capture antibody in solution? Then you have to coat your antigens onto solid vessels, such as beads, and follow immunoprecipitation protocol.
Dear Kuan-Chin Chow
Yes, I want to coat antibody onto ELISA plate and then antigen and labelled antibody manually.
Some protocols are in attached publication
Article Determination of antibodies against human growth hormone usi...
Om,
It all depends on the proteins, antibodies and antigens, that you will use for your ELISA. Some proteins are stable in PBS and some are not. By being stable, this means that they do not precipitate or lose their active conformation in the buffer. The best way to evaluate this is to read the product specification very carefully. The protein manufacturer or production company have performed the necessary stability evaluations already before these were released for commercialization. So, follow their recommendations and use their suggested buffer. Some will recommend PBS, or HEPES, or MES or Tris buffer at certain pH and concentration. Follow their recommendations very strictly.
Additionally, you also need to consider the stability and activity of the enzyme in your ELISA. Some enzymes are inhibited by some buffers and by-products in the assay. You need to evaluate and watch this out as well.
Overall, the best thing for you to do is to study the buffer needs of each of the proteins in your ELISA. Consult with the manufacturer if you have any doubts or trouble making the assay work.
Some more options in attachement.
Article Magnetic Gold Nanoparticles in SERS-Based Sandwich Immunoass...
For coating antigen _ NaHCO3 buffer
For ELISA test PBS or TBS buffer.
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