Discrimination against denatured proteins?! - Can ELISA discriminate agains denatured proteins? (Jul/18/2011 )
Hello forum users,
My question for you is; can an ELISA discriminate against denatured proteins. For example, if I were to have an antibody against the globular protein BSA (bovine serum albumin), would I receive a signal if heat denatured (or Urea, ect.) BSA were added instead? Honestly I hope that this technique is sensitive enough to determine the difference, but I am new to it and in need of your advice with regards to it.
Thank you for your advice!
Best,
Feldman
Dear Feldman,
the answer to your question is a "yes" or "no" depending on the antibody you are using to capture your protein of interest. The antibodies always recognize the conformation of the epitopes, but these can be linear or spatial conformations. Hence, if you use a monoclonal antibody to capture your protein and this antibody binds a linear epitope, then it will most probably bind both native and denatured protein equally well (i.e. no difference in signal in your ELISA). On the other hand, if your monoclonal antibody binds a structured epitope, then you will clearly see the difference in signal when your protein is native or denatured. To note, many ELISA tests contain a polyclonal antibody for capture and use a labelled monoclonal for the detection. In that case, you have so many recognized conformations that -most probably- you won´t see any difference upon conformation.
I have run projects to develop ELISA tests to distinguish globular vs. fibrillar states of a protein. That is why I am confident that you will be able to use this technique for your purposes. The key, once more, is to use the correct monoclonal antibody.
Chelo on Tue Jul 19 06:56:07 2011 said:
Dear Feldman,
the answer to your question is a "yes" or "no" depending on the antibody you are using to capture your protein of interest. The antibodies always recognize the conformation of the epitopes, but these can be linear or spatial conformations. Hence, if you use a monoclonal antibody to capture your protein and this antibody binds a linear epitope, then it will most probably bind both native and denatured protein equally well (i.e. no difference in signal in your ELISA). On the other hand, if your monoclonal antibody binds a structured epitope, then you will clearly see the difference in signal when your protein is native or denatured. To note, many ELISA tests contain a polyclonal antibody for capture and use a labelled monoclonal for the detection. In that case, you have so many recognized conformations that -most probably- you won´t see any difference upon conformation.
I have run projects to develop ELISA tests to distinguish globular vs. fibrillar states of a protein. That is why I am confident that you will be able to use this technique for your purposes. The key, once more, is to use the correct monoclonal antibody.
Chelo,
If you are purchasing an antibody, in my case I would like to use one against BSA, how do you know if it binds a structured epitope or not? Is there a way to tell? Do you have a recommendation regarding where I could find one?
Thank you,
Feldman