Interest of EDTA in buffer for antibody-antigen binding analysis - Forum


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TOPIC: Interest of EDTA in buffer for antibody-antigen binding analysis

Interest of EDTA in buffer for antibody-antigen binding analysis 21 Oct 2019 17:24 #1

  • cmondiel
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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

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Interest of EDTA in buffer for antibody-antigen binding analysis 21 Oct 2019 20:51 #2

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 KD 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 KD than expected.
Can you verify the functionality of the ligand surface and analyte with another assay?

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Last Edit: by Arnoud.

Interest of EDTA in buffer for antibody-antigen binding analysis 22 Oct 2019 15:02 #3

  • cmondiel
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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 5┬ÁL/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,

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Interest of EDTA in buffer for antibody-antigen binding analysis 22 Oct 2019 19:48 #4

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

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