Posted 10 February 2012 - 06:10 PM
Capture reagent is a species specific polyclonal anti IgG heavy and light and detection is via a species specific monoclonal anti Fc.
During stability assessments, recovery of the analyte in the sample is 85% after overnight at 4 degrees, 70% overnight at 20 degrees and 60% overnight at 37 degrees. This is consistent from experiment to experiment.
The stability problem is present in serum, plasma, and a non-complex diluent.
The stabilty problem is not removed by inclusion of 10 mM mercaptoethanol, 1 M NaCl, 0.8 M Tris Base (high pH) or broad spectrum protease inhibitor in the sample. 200 mM acetic acid in the sample stops degradation, but does not reverse it.
What is happening at the molecular level?
Interested to know others' thoughts!
Posted 13 February 2012 - 12:07 PM
If the analyte is stored in the original tissue culture medium is it stable?
With the Elisa you are not testing stability of the analyte only that it is still recognized by the abs in the system. It could be that the analyte is still functional.
Ab could be undergoing conformational change and not recognized by abs in the system.
For storage you could try diluting in commercial stabilizing agents such as those from Surmodics.
Posted 13 February 2012 - 06:59 PM
The test matrix is serum or plasma. The analyte is spiked in, and aliquots frozen. Each aliquot is then subjected to the stability treatment and compared to a control aliquot which is thawed immediately before the assay.
The antibody itself is GMP prepared and undergoes stability testing using a process not related to this assay (HPLC etc)
Yes, I agree, the effect is likely not going to affect biological activity and I suspect that the epitope on the Fc to which the monoclonal detection reagent binds is in some way being modified, just want to know what the modification is so that i can attempt to reverse it. Doesn't appear to be proteolytic or oxidative but is pH dependent. I will try a polyclonal anti-Fc as a detection reagent which might avoid the problem.
I'll let you know how this pans out...
Posted 14 February 2012 - 03:12 AM
Posted 14 February 2012 - 04:06 PM
I would love to know the nature of the pH dependent degradation!
I'll keep you posted.
Posted 15 February 2012 - 03:23 AM
You are right IgG is very stable. Another question....I am assuming the therapeutic is also monoclonal...could the IgG after thawing aggregate and thus show lower concentrations? High concentrations of purified Mab are known to precipitate.
I am also going to assume eventually you would be measuring the therapeutic in human samples and are trying to get some baseline info.
Posted 15 February 2012 - 04:39 PM
I was convinced it was sulphydril oxidation, but the mercaptoethanol would negate that. Maybe it's pH dependent phosphorylation, but I'm not a chemist so not too clear on this.
The other option is simple sticking to plastic due to charge, which is consistent with the pH dependency, but this is typically a rapid process and would not be time dependent, also, there should be a vast excess of endogenous IgG in these samples which should block the sites.
I am hopeful for the poly's performance.
Thanks for your suggestions!
Posted 16 February 2012 - 03:33 AM
Posted 28 March 2012 - 05:13 PM
Thanks for your input!