Competitive SPR with serum antibodies

I'm setting up a competitive SPR assay to be able to characterize epitopes targeted by vaccine-induced antibodies. I immobilize my antigen on the sensor chip surface, then test my monoclonal antibodies individually to determine their binding level. Then for my experiment I inject purified serum IgG from vaccinated individuals over the sensor chip surface, followed by a monoclonal antibody (of course at the same concentration as when injected individually). Then I can compare the binding of the mAb with and without serum IgG to determine if the serum IgG are targeting the same epitope as the mAb.

- How much serum IgG needs to bind to the immobilized antigen in order provide enough competition that I can confidently say if the mAb is competing for the same epitopes? How do I calculate it?

Thanks!

Hi Jena,

What you are doing is a kind of epitope tagging. I looked for a comparable publication but did not find one.
Theoretically I would say that you should try to saturate your ligand with serum IgG to be sure. On the other hand, you know the binding level of the individual monoclonal antibodies. Thus when the binding level decreases significantly, I would conclude that the serum IgG and the monoclonal compete for the same epitope or epitopes close together.
Not much of an answer but I hope you can get some results. In addition we are interested in how you solved this question.

Kind regards

Arnoud

Thanks! I've now run some assays and was able to optimize the protocol by reducing the amount of immobilized antigen, and increase the amount of serum IgG (but still be within reason). I also found a good publication that could be of help to others: Whitbeck 2014, Repertoire of epitopes recognized by serum IgG from humans vaccinated with herpes simplex virus 2 glycoprotein D (Journal of Virology, 8:14, 7786-7795). Hope this can help someone else!