3 replies to this topic

[Michael Starr]

I am doing some affinity purification on a batch of antibodies to purify out antigen-specific antibodies. Is there a way to further enrich the eluted fraction for higher binding strength antigen-specific antibodies? My initial thoughts were that a gradient elution would be the go-to, but for some reason my boss(es) didn't think that would be reproducible, which makes no sense to me--an FPLC is a precise liquid handling system and the consistency of the gradient would be high inter-run! Ugh.

[Missle]

What is your gradient?  pH?  Gradient purification is the first thing that comes to mind as the highest affinity antibodies will be the last to come off but they also will undergo the harshest elution.  You could try to modify your binding conditions so that it favors higher affinity antibodies as low affinity ones won't have time to bind effectively.  This could be done by altering buffer conditions or speeding up the flow rate...

[Michael Starr]

What sort of loading buffer modifications do you propose? I don't know if we can speed up flow; our affinity column is 1 mL capacity and the manufacturer recommends not exceeding 1 mL/min, which we are already running at.

 

My supervisors recommended against a gradient elution. We are eluting using pH. They think the method wouldn't be reproducible, which I disagree with but yielded to their experience.

Edited by Michael Starr, 18 March 2016 - 08:16 AM.

[Missle]

We routinely (with reproducible results) elute with a gradient pH elution.  However, the vast majority of my experience is with Protein A/G antibody purification.  I have much less experience with an antibody affinity column.  Unfortunately, it seems like trying to selectively purify high affinity antibodies is a variable project to begin with.

 

Buffer modifications could include changing pH or adding a little glycerol to prevent weaker interactions.  Full disclosure, altering binding buffer is a last resort for me so I don't have much experience.  Good luck!