Interest of EDTA in buffer for antibody-antigen binding analysis

Hi everyone,

I am currently setting up SPR assays for a high affinity binding interaction between an immobilized antibody on protein G (5RUs immobilized) and a protein X.

I work in HBS-P buffer but I see that very often HBS-EP is preferred for antibody assays.

Is there any reason for that ? I know EDTA is a chelating agent but why removing metals ? Does it intefere with binding?

I actually have a quite low surface activity (around 25%) which is surprising because the interaction I study is supposed to be very high (Kd 0.1 nM) and optimized, I would expect a surface activity >50%.
I wondered if I could see a big change by adding EDTA in my running buffer.

Many thanks for your answers

Hi,
the HBS-EP was the first buffer promoted by Biacore and therefore stuck in the mind of the users. As far as I know, metal ions do not interfere with normal interactions on a CM-dextran chip and including EDTA also should not interfere.
But, we routinely leave out the EDTA since we require often calcium or zinc ions. So the message is: use the appropriate buffer for your interaction. There are no obligatory buffers.
With low surface activity you mean that the actual Rmax is only 25% of the theoretical Rmax based on the protein sizes and immobilization level of the ligand? Assuming that, the K_D is independent of the amount of functional ligand on the surface. You can see the Rmax as a kind of scaling factor for the curves (and the amount of functional ligand). However, when your analyte is not 100% functional, you think that you inject a 100 nM solution and it is only a 25 nM functional solution then you get a lower Rmax and K_D than expected.
Can you verify the functionality of the ligand surface and analyte with another assay?

Hi,

Thank you for your answer. The functionnality of the ligand is not a problem we performed a quality control of it. But I think the analyte might be the problem. I will run a SEC-MALS to check.

Another point, I try to perform both kinetic & steady state experiments. I manage to see a small dissociation and to reach only a start of plateau (maximum contact time of the apparatus used (with a flow rate of 5µL/min, 1h30 association & 5h dissociation)

In kinetics, I get a Kd of 0.2 nM with correct T-values etc..

In steady state, I shift from 1 log the Kd (I see 5 nM)

Is there any explanation in such differences between kinetics and steady state analysis?

Many thanks,

Hi,
The fact that the curves are not steady state will give you a lower KD value as you observed.
See www.sprpages.nl/kinetics/steady-state and www.sprpages.nl/sensorgram-tutorial/a-curve

Arnoud