I have been performing antibody stability test at different solvents and temperature. My solvents were glycerol, DMSO, Tween 20, PEG. I stored the antibody solution with these solvents in room temperature and 4'C. After running ELISA in day 0, 7, and 14, the result shows no major differences.
Has anyone done similar test and got the same result? only day 0 had slightly higher absorbance. Is this something expected?
It depends on your concentrations, and I have never tested DMSO. However, I have tested the other 3, PEG(0.1-5%), tween20(0.1-10%) and glycerol (25-50%). I have not seen any difference in these three chemicals after 1-3 months. I assume you are aware that PEG, tween and glycerol are all commonly used antibody stabilization reagents? As such, you would not expect to see reactivity differences between these reagents. If you goal is to find differences try waiting 6-12 months, that when you start noticing differences between stabilization buffers. You could also try freezing, that sometimes speeds up Ab degradation.
Thank you Daniel.
I wasn't expecting degradation, this was just a test for preliminary data to show the antibody stability. My concentrations were 20% PEG, 20% DMSO, 10% Glycerol, and 5% tween 20. I expected to see difference in antibody reactivity at two different temperature storage. However there was no major difference in between room temperature and 4C storage.
Hanif, is this a specified protocol you are following? Industry usually is simulating aging of samples by storage at elevated temperatures (37°C, 56°C)
Hi Wolfgang
I designed this stability test my self, there was no specificied protocol.
How big are your proteins? Small proteins tend to be more stable and behave in similar ways in different solvents. You are not changing the charges of your solvents (the polarity) very much by using a non ionic detergent, a sulfoxide and alcohols (PEG and Glycerol) and therefore I would expect your proteins to behave in similar ways in those solvents. I hope this helps.
Hi Hanif,
what was the goal of your stability tests? Would you like obtain long-term stability data?
There are some really nice application notes on antibody stability analysis with nanoDSF and the Prometheus instrument. The method can be used to screen different solvent and formulation conditions in a label-free setting. The ultra-high resolution allows to distinguish multiple unfolding transitions which can then be assigned to individual antibody domains.
I attached the ones that fit best to your question. I hope this helps!
In ELISA, the incubation temperature is always 37°C, the progress when you add antibody to the well which has your protein. But too many factors may influence the results of ELISA.
For stability testing, I have put my protein in incubator at 37°C for 0,10min,0.5h,1h,4h,8h,24h. At different time, I pick it to test its cell killing effect to determine the stability.
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