Antibody-antigen SPR help

Hi All,

I am new to SPR and would like some help. I am doing antibody-antigen interaction in single step cycle. I am using protein G chip on T200 biacore machine. There are some issues that I was wondering if you can help me.

1. In FC1, I can see that my antigen is binding at low level, and I am not sure why. The antibody and antigen were purified and ran for QC.
2. Some antibodies, after coupled to the chip, the RU slowly drifted down, suggesting that the antibodies is falling off the chip

To overcome the antigen binding to FC1, will I believe more of the result from analysis for heterogenous binding than 1:1 binding? The result from both analysis is off by 3 decimal points, which I think is quite significant difference.

The graph looks decent for some antibodies that fall off the chip, and looks bad for others. I am showing an example of one that looks pretty bad (and also our most important antibody!) I tried to run analysis for 1:1 binding and heterogenous binding, and the numbers looked close. Do I believe this number though?

If I need to repeat the experiment, how would you suggest me to proceed? Especially to circumvent the problem of the antibody falling off the chip.

Thank you very much for your assistance!

Hi,
In figure 1 the 'binding' seems more a bulk effect (sharp rise and fall) than actual binding. A minimal binding to the reference (protein G) can maybe blocked by binding a non-related antibody.
In figure 2 the fitting looks rather well except that the bulk effect seems not to be fitted.
In figure 3 the curves are not reference subtracted. This will give less accurate kinetic values. In addition, the heterogeneous model is not suited to compensate for antibody loss.
In figure 4 and 5 you show the antibody dissociation from the protein G. This can happen when the antibody is off certain origin or class (search for protein G and antibody class). One way to compensate for the antibody loss is by running a blank with antibody alone and buffer injections and use double referencing.
In figure 4 and 5 it looks like that there is a positive drift calculated and the dissociation is not correctly resolved. Try different starting values and add bulk effect after a better fit is reached.

I hope this answers bring you further.
Kind regards
Arnoud