If you are talking about using the antibodies as the affinity matrix (such as in immunoaffinity chromatography), I would suggest that the way to avoid damage to the antibodies from proteases in a crude extract is to save the immunoaffinity chromatography for the last step in the purification, at which point the protease activity from the crude extract should be greatly reduced. Begin the purification with ion exchange, hydrophobic interaction, and/or gel filtration chromatographies. Use a protease inhibitor cocktail when making the crude extract.

Adam B Shapiro, Thank you for your response.

Yes, I mean immunoaffinity chromatography, I am trying to find a way to purify hyaluronidase from hepatopancreas of red king crab (using polyclonal antibodies). In this case, inhibitors like EDTA, Benzamidine, PMSF don't prevent proteolysis.

So, I was seeking a way to overcome proteolysis without having to use inhibitors at any step.

I read that it could be done through modifications to the antibodies, like unnatural amino acids or mPEG. But, I don't think this is the best way

Chemically modifying the antibodies with PEG would run the risk of the antibodies no longer recognizing the antigen.

1) How do you know that the immobilised antibody has been digested by proteolysis?

2) Immunoglobulins are very resistant to proteolysis. Even strong enzymes like pronase and proteniase K with prolonged treatment (many hours) will still give you biologically active Fab or Fab2 fragments. Carrying out purification at 4oC can minimise proteolytic effect.

3) coupling antibodies to Sepharose or agarose gel matrix usually confer additional resistance to proteolysis.

4) Is the immobilised polyclonal antibody the IgG fraction or actual purified antibody. If it the former then there is a possibility that you do not have high proportion of the IgG fraction as the real anti-analyte antibody

i'm interested in.