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.
The interaction is so strong that it is really hard to observe dissociation. I had the idea to decrease flow rate to 5 µL/min to increase contact times to the maximum. With this I get a start of dissociation. However, the main disadvantage is that I created mass transport limitation & re-binding event, and moreover the off rate is out of the limit of the instrument. Thus, I cannot exploit datas.
We discussed about several options but I am not sure which one is the best:
1. Set-up protein G modified
- Capture the antibody Fc2 = 5 RU Fc4 = 15 RU at 10 µL/min
- Injection of protein X in SCK at 80 µL/min at 37°C ( Asso 260s / Disso 2h)
SCK removes regeneration & capture unstability over injections
High flow rate should remove MTL & re-binding events
Increasing temperature should increase kinetic constants (Arrhenius law, activation energy)
2. End-point measurement
Amine coupling of protein X (50 RU immobilized Rmax mAb 40 RU) with pH scouting & pre-concentration on Fc2 & Fc3
mAb injection on Fc1 & Fc2, 120s binding should be observed
Washing step during 4h to see if some mAb goes out
mAb injection on Fc1, Fc2 & Fc3
Compare Fc3 & Fc2
This should give a first timescale of the off-rate, e.g. 25% dissociation after a given time point (e.g. 4h)
What do you think of this?
Do you have any experience in increasing the temperature of the IFC?
Decreasing the flow rate to increase the contact time, only works for the association. Increasing the contact time will increase the binding response and hopefully give the association curve more curvature. For the dissociation a high flow rate is favourable as you mention to decrease rebinding.
Increasing the temperature will alter the kinetics (in general the association and dissociation will be faster). It is a good idea to degas the flow buffer when using the 37 C even when there is a degasser in the system. Also degas the buffer for the dilution of your sample. Changing the temperature of the IFC is normally done when we want to investigate the thermodynamics of the interaction.
Your main problem is the slow dissociation. If you have a higher response at the end of the association then it is more easy to get some decay of the complex to fit the dissociation. This will be a balance between response, dissociation time and possible rebinding. To lower the rebinding you can inject the ligand during dissociation but this can be expensive.