Recently I work with some antibodies, trying to measure the KD of antibodies. I used CM5 chip and running buffer was HBS EP+ (GE). I immobilized the target antigen as a ligand and the RU for this step was 60. After that, I flow the antibody which has a concentration of 333.33nM to 0.053nM at 50ul/min. I inject the antibody for 60 sec and dissociation for 2400 sec BUT still can not see the dissociation phase that's why the Biacore couldn't calculate the KD.
So what should I do to make this antibody dissociate from the antigen?
Do you have any suggested buffer instead of HBS EP+?
@thittayaaudomsun your sensorgram is typically observed when you have an antibody in solution. That's something completely normal! Your antibody has 2 binding sites and it generates an avidity-based binding, i.e.your dissociation phase is almost flat.
If you want to determine a KD with correct kinetic parameters and minimizing the avidity, you need to immobilize your antibody (sensor chip CM5 +capture kit IgG (cytiva) or a protein A sensorchip for example.
If you want to keep your antibody in solution, you should calculate a kd with a steady state affinity model. For that, you need to immobilize less ligand ( theoretical rmax expected between 10 and 50 RU), to work in the correct range of KD ( 10times of KD for the top concentration). Finally, you need to inject at least 600seconds (at lower flow rate, you don't need it to reach a plateau at each concentration, I recommand you 10 ul min).
With a steady state model you have a KD value (and no kinetic values koń and koff).
Good luck.
Vanessa
Vanessa Porkolab Thank you so much for your suggestion. I'm going to try that.
As Vanessa Porkolab already explained correctly, you are observing avidity-based binding of bivalent antibodies. To overcome this problem, there are at least two more options:
1. You reduce the density of your antigen on the surface.
2. You cleave the antibody enzymatically in monovalent Fab fragments.
Thittaya Audomsun You can also try the suggestion by Arnoud from SPR pages
https://www.sprpages.nl/kunena/new-forum/580-antibody-antigen-slow-dissociation
increasing the flow rate of varying the temp.
The T-200 has a max flow rate of 100 ul/min.
Just asking: are you using mono or polyclonal?
Take a look at this paper by Quinn et al at Genentech: Article Determination of Affinity and Residence Time of Potent Drug-...
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